Malcolm R. Brandon

Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons

Abstract:  Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement‐mediated rejection, as shown here using non‐immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high‐level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement‐mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti‐Galα(1,3)Gal epitope (anti‐GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre‐sensitized with antibody were highly resistant to human complement‐mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non‐immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at ∼24‐h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti‐GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced ∼8‐fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.

Inflammatory response in the pig uterus induced by seminal plasma

The immunological and physiological influence of seminal plasma on the local uterine environment was investigated by immunohistochemical and flow cytometrical studies on uterine tissues and lymph nodes taken from gilts after mating with a vasectomised boar and from control, unmated gilts. These studies revealed that mating with a vasectomised boar induces an acute transient inflammatory response in the endometrium resulting in marked changes in the presence and distribution of leukocytes and extensive proliferation of the endometrial glands. At the same time there was an increase in CD8L and sIg+ cells and an up-regulation of MHC class II and IL-2 receptor expression in the uterine lymph nodes of mated pigs. This would suggest that seminal plasma deposited in the uterus can activate cells in the local draining lymph nodes. Together, these results demonstrate in utero that pronounced immunological and physiological changes are induced in vivo by seminal plasma.

Cellular responses during liver fluke infection in sheep and its evasion by the parasite

The cellular immune response in sheep to an acute and chronic primary and an acute secondary liver fluke infection were examined by immunohistology of liver tissue and flowcytometry of lymphocytes from the draining hepatic lymph nodes. Ten days after primary infection, portal tract areas surrounding migratory tunnels were infiltrated with CD4+ and CD8+ lymphocytes with fewer B cells and T19+ T cells. Micro abscesses were distributed sporadically in the liver parenchyma and young flukes could be easily observed in the liver tissue free from inflammatory cells. More intensive infiltration of the portal tract areas was observed during a secondary liver fluke infection characterized by a pronounced increase in eosinophils, B cells and CD4+ T cells. In addition, there was an increase in MHC class II+ fibroblastic‐like cells surrounding the migratory tracts. In contrast to the primary infection, no young flukes were observed in the same tissue areas during the secondary infection. Chronic primary infections were characterized by perilobular fibrosis and a predominance of CD8+ and γδ‐TCR+ T19‐ T cells distributed within fibrotic strands. Distinct B cell follicles were observed in the fibrotic strands and near major bile ducts and necrotic patches. Pronounced lymphocyte infiltration could occasionally be observed surrounding liver fluke eggs lodged in liver tissue. A progressive increase in lymph node weight, cell number and CD4/CD8 ratio was observed in the acute and chronic primary infections. The role of the infiltrating cell populations and possible mechanisms of immune evasion by the parasite are discussed.

Cellular immune responses in the pig uterus during pregnancy

In pigs, little is known about the role of the uterine immune system during pregnancy. Immunohistochemical studies were conducted using a panel of monoclonal antibodies to pig leukocytes on uterine tissues taken from gilts after fertile mating and at different stages of pregnancy. Acute inflammation in the endometrium in response to fertile mating which included marked changes in the tissue and immune cell components of the endometrium was observed. Throughout pregnancy the pig uterus contained a substantial population of leukocytes. MHC class II staining was prominent in the endometrium at all stages examined and included macrophages, dendritic and fibroblast-like cells, lymphocytes and the endothelial lining of many uterine blood vessels. The majority of lymphoid cells were CD2+, indicating the prevalence of T cells. In early pregnancy specific changes were seen in the tissue distribution of uterine immune cells. Following placentation distinct cellular changes in the local immune cell environment of the uterus were also observed despite the non-invasive nature of the pig placenta. There appears to be suppression and activation of various immune cell components in the uteri of pregnant pigs. This phenomenon is presumably in response to foetal or trophoblast antigens, suggesting that the local immune system is involved in the uterine response to pregnancy.

Quantitative and Qualitative Changes in the Intraepithelial Lymphocyte Population in the Uterus of Nonpregnant and Pregnant Sheep

PROBLEM: Previous studies have revealed the presence of a unique population of CD45R + granulated cells in the sheep uterine epithelium. In the present study, dramatic changes in this cell population and in the nongranulated lymphocytes in the uterine and endometrial glandular epithelium of non‐cycling, cycling, pregnant, and postparturient sheep are described. In noncycling and cycling sheep, the granules in the granulated intraepithelial cells were small. From days 55 to 134 of pregnancy, the granules in these cells were large, and there was a significant increase (P < 0.01) in the proportion of this cell population in the uterine epithelium but not in the endometrial glandular epithelium located in the deeper region of the stroma. The number of these cells declined dramatically (P < 0.01) from 2 to 15 days after parturition. Both the tissue distribution and the time of activation of these cells suggests they are different from the granulated lymphocytes described in placentae of mice and man.

Genetic organization of the ovine MHC class II region.

To study the class II genes of the major histocompatibility region of the sheep genome, human HLA class II genes corresponding to the known subregions in man (DR, DQ, DP, DO, and DZ) were used for Southern hybridization analysis of sheep DNA and to probe a sheep genomic library. Hybridizing bands were noted for all probes except DPα. DQ α and β and DRβ appear to be present as multicopy genes, while DRα-, DZα-, , and DOβ-like genes appear to be single copy. All bands detected with the DPβ probe were also detectable with other β chain probes. From eight λ-bacteriophage clones of a sheep genomic library nine distinct class II genes were identified. These genes were characterized by differential hybridization analysis and restriction mapping. Two genes were DRβ-like, three DQα-like and four DQβ-like. The extensive cross-hybridization observed with β chain probes was not seen with α chain probes. The results of this study suggest that the major histocompatibility complex class II region of the sheep has a similar genetic organization to that of man, with the provisional exception of the DP subregion.

Studies on the distribution of immune cells in the uteri of prepubertal and cycling gilts

To establish the cellular basis for the local immune response in the porcine uterus, immunohistochemical studies using a panel of monoclonal antibodies to pig leukocytes were conducted on uterine tissues from prepubertal and cycling gilts. In prepubertal uteri, neutrophils were the most predominant cell type, while MHC class II+ cells and CD2+ T lymphocytes were also common. At the early-stage of the oestrous cycle, CD2+ T cells were numerous in the endometrium, particularly in the uterine epithelium and subepithelial regions. However, by the mid-stage of the cycle there was a significant and dramatic fall in CD2+ T cells and other lymphocytes expressing the CD4, CD8 and CD1 phenotypes, MHC class II+ cells were predominant throughout the endometrium. During late oestrus there was a dramatic infiltration of neutrophils into the subepithelial stroma. A distinct increase in the CD2+ intraepithelial T lymphocyte population was also observed at this stage of the cycle. It was concluded that in the healthy, non-pregnant pig uterus T lymphocytes, macrophages and neutrophils were the prominent leukocyte cell types and their migration and distribution in the uterus was strongly influenced by the oestrous cycle. These immune cells may play an important interactive role in the cyclic cellular changes in both the structure and function of the endometrium. Furthermore, the leukocyte phenotypes found in the porcine endometrium indicate that a local cellular immune response could be elicited.

Molecular cloning, expression and characterization of ovine TNFα

Tumour necrosis factor α (TNFα) is a cytokine with a wide range of effects on both lymphoid and non‐lymphoid cell types. By hybridization with a human TNFα cDNA probe the corresponding ovine cDNA was isolated from a lipopolysaccharide (LPS) stimulated alveolar macrophage cDNA library. The sequence of the cDNA clone showed that ovine TNFα encodes a polypeptide of 234 amino acids that, based on analysis of human TNFα is processed to a protein of 157 amino acids. The nucleotide and amino acid sequences showed a high degree of homology to the equivalent human and mouse molecules. In a mammalian COS cell expression system the ovine cDNA was found to encode a protein which was able to lyse actinomycin‐D treated WEHI‐164 cells and induce COS cells to produce and secrete interleukin 6 (IL‐6). Further experiments demonstrated the importance of sequences within the 3’untranslated region of the cDNA in determining the level of expression of ovine TNFα Northern blot analysis was used to analyse the kinetics of induction of ovine TNFα mRNA in alveolar macrophages stimulated with a variety of mitogens. Addition of LPS increased mRNA encoding TNFα at 1 hand 5 h but not 24 h post stimulation. In contrast, addition of phorbol myristic acid (PMA) led to increased TNFα mRNA at 5 h while the combination of PMA and ionomycin increased the level of specific mRNA detected at 1 h, 5 h and 24 h. From genomic analysis ovine TNFα appears to exist as a single copy.

Nucleotide sequence, polymorphism, and evolution of ovine MHC class II DQA genes

The nucleotide sequence of all exons and introns, excluding exon 1, of the ovine major histocompatibility complex (MhcOvar) genes analogous to the HLA-DQA1 and -DQA2 genes has been determined and the gene structure found to be similar to that reported for other species. The predicted amino acid sequences of the Ovar-DQA genes have been compared with the equivalent DQA genes in man, mouse, rat, rabbit, and cattle and used to determine the evolutionary relationships of the sheep class II genes to these other species. Northern blot analysis of sheep mRNA using exon specific probes for each of the two Ovar-DQA genes show that both genes are transcribed, whereas in humans there is no evidence that HLA-DQA2 is transcriptionally active. Restriction fragment length polymorphisms (RFLPs) have been used to define a polymorphic series of alleles in both Ovar-DQA genes and have indicated that the number of DQA genes is not constant in sheep as it is in humans, but varies with the haplotype.

Continuous antigen delivery from controlled release implants induces significant and anamnestic immune responses.

Two continuous delivery injectable silicone implants were tested to determine if they were capable of delivering vaccines in a single shot. The Type A implant delivers antigen in vitro over a 1-month-period and the Type B over several months. Vaccination studies in sheep were designed to compare the responses induced by the Type A and B implants, Alzet mini-osmotic pumps and conventional antigen delivery. A model antigen, avidin, was used along with IL-1beta or alum as adjuvants. Sheep were immunised with various formulations and the titre and isotype of the antigen specific antibodies monitored. The Type B implant induced antibody (Ab) titres of greater magnitude and duration than soluble vaccines or the Type A implant with adjuvant, but only if IL-1beta was included in the formulation. Both implants induced antibodies of IgG1 and IgG2 isotype. A memory response to soluble antigen challenge was induced by the Type B+IL-1beta implant, which was predominantly of an IgG1 isotype.