Pig Lymphocytes Research Articles

INHIBITION OF HUMAN ANTI-PIG T CELL RESPONSES BY MONOCLONAL ANTIBODIES TO PORCINE CD45 MOLECULES

P1057 Background: Targeting of pig cell surface molecules by monoclonal antibodies (mAb) offers the possibility of an organ-specific immunomodulation after pig to human xenotransplantation. We have previously shown that mAb directed to porcine CD45 molecules inhibit human anti-pig T cell responses in vitro without affecting T cell activation induced by alloantigen or mitogens. Aims: In the present study we analyzed the immunomodulatory potential of mAb directed to different isoforms of the porcine CD45 molecule. Furthermore, to elucidate the underlying mechanisms of CD45-mediated immunomodulation cellular and biochemical consequences were analyzed after binding of CD45 mAb to pig cells. Results: In the absence of CD45 mAb porcine stimulatory cells induced significant activation of human T cells as shown by proliferation, upregulation of the IL-2 receptor (CD25), and cytokine production. In the presence of mAb to porcine CD45RA isoforms human T cell responses were reduced by 40 - 70 %. The inhibitory effects were less pronounced when CD45 common mAb were used (10 – 25 %). MAb directed to CD45RC isoforms did not significantly influence the capacity of pig lymphocytes to induce human T cell activation. Binding of CD45 mAb changed the tyrosine phosphorylation pattern as detected by Western-Blot analysis of whole cell lysates of porcine stimulatory cells. Furthermore, treatment of porcine stimulatory cells with CD45 mAb induced strong cellular aggregation. Conclusions: Inhibition of human anti-pig T cell responses by porcine CD45 mAb may result from the induction of signals which change the adhesion properties of pig stimulatory cells. Alterations in adhesion could influence their capacity to interact with human T cells. Since CD45 molecules are not expressed on endothelial cells the usage of anti-pig CD45 mAb might be an approach to block the direct pathway of T cell activation initiated by porcine APC without affecting the endothelium of a graft. Supported by the Deutsche Forschungsgemeinschaft, FOR 535.

Localization of fragile sites in the karyotype of Sus scrofa domestica: present status.

Fragile sites were induced to expression in pig lymphocyte chromosomes by in vitro exposure to folate antagonists and BUdR-Hoechst 33258. Expressed fragility was localized according to the presented idiograms and compared with the location of known spontaneous translocations.

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.

The normalization of guinea pig leukocyte fractions and lymphocyte subsets in blood and lymphoid tissues using a flow cytometric procedure.

Many hematological and immunological parameters remain unclear in the study of the guinea pig. In this study, we established the mean values of blood counts, the percentage of leukocyte fractions and lymphocyte subsets in blood and various lymphoid tissues of the guinea pig with a flow cytometric procedure using MIL4/SSC. The mean counts of WBC and RBC in the blood were lower, and MCV and MCH were higher than those of other rodents, resembling those of humans. Furthermore, the mean percentages of blood lymphocytes were smaller and that of granulocyte was larger than those of other rodents, resembling those of humans. We further established a flow cytometric procedure for lymphocyte subsets and clarified the mean percentages of T- and B-cells, CD4(+)-, CD8(+)- and MHC Class II(+)- T-cells, and CD4(-)CD8 (-) T-cells. The latter were morphologically larger in cell size and cytoplasm than CD4(+)- plus CD8(+) T-cells, and this subset had a significantly higher percentage in newborn animals. Furthermore, the appearance of the MHC Class II(+) T-cell subset was suggested to be a marker of hyper-activation of T-cells in BCG-immunized animals. Thus, both the novel flow cytometric procedure for leukocyte fractions and lymphocyte subsets, and the established normal values will be useful tools in studying guinea pigs as models of various diseases and biological phenomena.

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

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-Galalpha(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 approximately 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 approximately 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.

Cross-reactivity between swine leukocyte antigen and human anti-HLA-specific antibodies in sensitized patients awaiting renal transplantation.

Xenotransplantation is increasingly viewed as a promising way to alleviate the problem of patients who have alloreactive lymphocytotoxic antibodies and therefore tend to accumulate on the waiting list for renal transplantation. One barrier to xenotransplantation in these patients could be the hyperacute or acute vascular rejection as a result of preexisting anti-HLA antibodies that recognize swine leukocyte antigens. The cross-reactivity of sera from 98 patients with pig lymphocytes was studied by flow cytometry. After absorption of xenoreactive natural antibodies (XNA), isotype, class, and antibody specificity causing a positive cross-match (XM) were determined. For nonsensitized patients, all of the antibody binding to pig lymphocytes was due to XNA, which were removed by pig red blood cells absorption. In contrast, in sensitized patients, after removal of XNA, pig lymphocyte XM remained positive. There was no correlation between antibody binding to pig lymphocytes and Ig isotype (IgG or IgM) or HLA class-specific antibodies. For testing evidence that class II-specific antibodies were responsible for antibody binding to pig lymphocytes, HLA class I-specific antibodies were absorbed with pooled human platelets. It was confirmed that HLA class II-specific antibodies were responsible for the positive pig XM, but the strength of the positive XM was weaker than the strength caused by HLA class I-specific antibodies. Sera with multiple specificities (plurispecific sera) displayed a greater frequency of cross-reactivity with swine leukocyte antigens (P < 0.05). Seven of 11 highly immunized patients without cross-reactivity IgG with porcine lymphocytes showed positive XM before an IgM was used. The results demonstrate the cross-reactive nature of HLA antibodies and therefore point out the need to perform a prospective XM after absorption of XNA in presensitized individuals.

Release of pig leukocytes during pig kidney perfusion and characterization of pig lymphocyte carbohydrate xenoantigens.

The Galalpha1-3Gal (alphaGal) antigen is considered the main xenoantigen in the pig to human species combination but other porcine antigens have to be considered such as the swine lymphocyte antigen (SLA), the blood group A/O and the Hanganutziu-Deicher (H-D) antigens. The H-D antigens are N-glycolyl-neuraminic acid (NeuGc) terminated gangliosides that are widely distributed in mammalian species but absent in humans. Upon exposure to a vascularized pig organ, the human recipient can be immunized by direct interaction with the pig tissue or/and by transfer of tissue/cells from the organ into the recipient. In the present work, we describe the release of cells from porcine kidneys upon perfusion and the expression of glycolipid based alphaGal, blood group A/O and H-D antigens in pig lymphocytes. Pig kidneys were flushed with 20 ml of NaCl or Lidocain containing 5000 U heparin, and thereafter perfused with 3000-ml perfusion solution and the cells released were counted and examined microscopically. Neutral glycolipid and ganglioside fractions were extracted from purified pig lymphocytes. The extracted components were characterized by thin layer chromatography, degradation and mass spectrometry. The expression of alphaGal and H-D epitopes on cells released from pig kidneys and purified pig lymphocytes were studied by immune electron microscopy. A total amount of about 300 x 106 leukocytes, mainly lymphocytes were released in the perfusate from the kidneys, of which about 100 x 106 cells were eluated in the 600 to 2400 ml perfusate fraction. Immunelectron microscopical analysis with Griffonia simplicifolia isolectin B4 showed staining of pig leukocytes and other cells, morphologically similar to endothelial cells, released in the perfusate. The purified porcine lymphocytes contained 930 microg neutral glycolipid (4.2 microg/mg cell protein) of which 95% was glycolipids with one to four sugar residues. Immunostaining of the neutral glycolipid fractions revealed alphaGal terminated compounds migrating in the five and 10 to 12 sugar regions and blood group A compounds in the six and eight sugar regions. Two major gangliosides NeuGc-GM3 and NeuGc-GD3 were found in the pig lymphocytes. In a patient extracorporeally xenoperfused with a pig kidney, an increased staining of both alphaGal terminated structures as well as the H-D reactive gangliosides were found in the post-perfusion serum samples. In summary, leukocytes, mainly lymphocytes are released from pig kidneys during perfusion which may contribute to immunization of human xenograft recipients.

Gradual development of the interferon-γ response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination

Infection of swine with virulent porcine reproductive and respiratory syndrome (PRRS) virus induced a rapid, robust antibody response that comprised predominantly nonneutralizing antibodies and waned after approximately 3 months. In contrast, the initial onset of virus-specific interferon (IFN)-γ-secreting cells (SC) in the pig lymphocyte population remained at a fairly low level during this period and then increased gradually in frequency, plateauing at 6 months postinfection. A similar polarization of the host humoral and cellular immune responses was also observed in pigs immunized with a PRRS-modified live virus (MLV) vaccine. Even coadministration of an adjuvant that enhanced the immune response to a pseudorabies (PR) MLV vaccine failed to alter the induction of PRRS virus-specific IFN-γ SC (comprising predominately CD4/CD8α double positive memory T cells with a minority being typical CD4 −/CD8αβ + T cells) and the generation of neutralizing antibodies. Moreover, unlike inactivated PR virus, nonviable PRRS virus did not elicit virus-neutralizing antibody production. Presumably, an intrinsic property of this pathogen delays the development of the host IFN-γ response and preferentially stimulates the synthesis of antibodies incapable of neutralization.

Chromium incorporated in RNA and DNA.

The objective of this study was to examine the effect of Cr(III) (chromium chloride) and Cr(VI) (potassium dichromate) on RNA and DNA-chromium adducts formation in isolated nucleic acids and isolated pig lymphocytes. The incubation of cells with potassium dichromate and chromium chloride at concentrations of 10 and 100 microM results in binding of a 1.2-1.9 fold greater number of chromium atoms to nuclear DNA than to total cellular RNA. The incubation of total cellular RNA and nuclear DNA isolated from lymphocytes with CrCl3 and K2Cr2O7 yielded a binding of 1.1-1.6 fold more of Cr atoms to RNA than to DNA. The number of chromium atoms bound to nucleic acids is higher after incubation with K2Cr2O7 than with CrCl3 in both experimental systems.

A sub-population of circulating porcine γδ T cells can act as professional antigen presenting cells

A sub-population of circulating porcine γδ T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by γδ T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When γδ T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with γδ T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of γδ T cells with chloroquine. These results indicate that a sub-population of circulating porcine γδ T cells act as APCs and present antigen via MHC class II.

Blocking of human anti-pig xenoantibodies by soluble GAL alpha 1-3Gal and Gal alpha 1-2Gal disaccharides; studies in a pig kidney in vitro perfusion model.

Depletion of anti-pig xenoantibodies reduces cell cytotoxicity of human serum to pig endothelial cells and lymphocytes. The aim of this study was to test, in a pig kidney xenoperfusion model, the ability of soluble alpha Gal terminated disaccharides to prevent the hyperacute rejection process in an organ. Porcine kidneys were perfused with whole human blood lacking saccharide and blood supplemented with Gal alpha 1-3GAL, Gal alpha 1-2Gal and lactose. Parameters evaluated were, urine production, renal blood flow, vascular resistance, renal clearance, blood cell counts, xenoantibody titers, complement activation and histopathology. The blood flow was higher in the Gal alpha 1-3Gal (155 +/- 31 ml/min x 100 g-1 kidney tissue) group compared to Gal alpha 1-2Gal (138 +/- 16), lactose (92 +/- 78) and controls (69 +/- 16), When calculated as percent of the blood flow value at 1 min, the blood flow at 30 min was 157% for the Gal alpha 1-3Gal and for 187% the Gal alpha 1-2Gal. The corresponding values for the lactose and control groups were 102% and 74%, respectively. Urine production in the lactose/control groups was lower (0.7 ml/min x 100 g-1 kidney tissue) compared to Gal alpha 1-3Gal (3.0) and Gal alpha 1-2Gal (3.7). Urine sodium excretion was reduced in the lactose/control groups, compared to the Gal alpha alpha 1-groups during the perfusions. An increase in urine potassium excretion was found in the Gal alpha alpha 1-groups while a reduction occurred in the lactose/control experiments. An initial 40-50% reduction in platelet count was observed in all groups while the leukocyte count showed a continuous decrease. Immunohistochemistry revealed less deposition of IgM, IgG, C3 and C1 q in the Gal alpha alpha 1-saccharide groups compared to the lactose/control groups. Soluble Gal alpha a1-disaccharides improved both functional and histological parameters. However, significant pathological changes were still present indicating that this approach to inhibit HAR must be used in combination with additional therapeutic approaches such as solid phase xenoantibody immunoadsorption and blocking of complement activation.

The effective antigen presentation of human MHC on the lymphocytes of HLA DPW0401 transgenic pigs: examination with xenogenic mixed lymphocyte culture and primed lymphocyte tests

The effective antigen presentation of human MHC on the lymphocytes of HLA DPW0401 transgenic pigs: examination with xenogenic mixed lymphocyte culture and primed lymphocyte tests

Ganglioside xenoantigens in pig lymphocytes and aorta

THE main xenoantigen in the pig-to-human species combination is the carbohydrate antigen epitope Gala1-3Gal, present on both glycoproteins and glycolipids on the cell surface. All humans have preformed antibodies reacting with this antigen epitope, and these antibodies are responsible for initiating the hyperacute rejection when pig organs are transplanted to human recipients. It is very likely that other pig xenoantigen epitopes exist that may elicit an immune response in humans upon xenografting. Examples of such epitopes are the blood group A/O antigens and the Hanganutziu-Deicher (H-D) antigen. The latter is an N-glycolyl-neuraminic acid (NeuGc) terminated ganglioside present in animals such as pig and sheep but absent in humans. Upon exposure to a pig organ, a human recipient can be immunized owing to a direct interaction with the pig tissue or from the transfer of tissue/cells from the organ into the recipient. Two patients extracorporeally connected to pig kidneys reacted with a strong humoral immune response to a short time (65 and 15 minutes, respectively) exposure to the kidney tissue. The main part of the antibodies reacted with the Gala1-3Gal xenoantigen. In addition, glycolipid binding, which could not be inhibited by soluble Gala1-3Galb1-4GlcNAc saccharide, was also detected, indicating the possibility of an immunization to other carbohydrate xenoantigens. Because exposure to the xenogenic organ was rather short, the cells/tissue responsible for the immunization might have been released from the kidneys and transported into the patients. Most likely candidates for this are damaged endothelial cells and passenger lymphocytes present in the organ. In this report we show that a considerable amount of passenger leukocytes are continuously released from pig kidneys upon perfusion with large volumes of perfusion solution. Glycolipids were isolated from pig lymphocytes and aortas (as an endothelial cell source) and a strong immunostaining to several of the ganglioside components was found in serum samples collected from one patient 3 to 4 weeks after an extracorporeal pig kidney xenoperfusion.

The cellular immune recognition of proteins expressed by an African swine fever virus random genomic library

The cellular immune recognition of peptides expressed by an African swine fever virus (ASFV) random genomic library has been studied. DNA from the Malawi (LIL20/1) ASFV isolate was randomly sheared by sonication, cloned into a plasmid vector downstream of a bacteriophage T7 promoter, and 72 recombinant plasmids were arbitrarily selected. These plasmids were transiently expressed following transfection into major histocompatibility complex (MHC) class I + class II − matched pig skin cells, which had been co-infected with vTF7-3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. Such cells served as antigen presenting cells and each recombinant plasmid was screened in a proliferation assay for recognition by CD8 + lymphocytes from inbred pigs previously exposed to ASFV. This assay was demonstrated to measure CD8 + T cell proliferation, as predicted by the phenotype of the antigen presenting cell. Of the 72 randomly selected clones, 14 were reproducibly recognised by immune pig lymphocytes and 10 corresponded to non-overlapping and distinct nucleic acid sequences. This high frequency of ASFV encoded antigenic epitopes supports the concept that cellular immunity to the virus may play an important role in resistance to ASF.

High sequence homology between human and pig CD40 with conserved binding to human CD154.

Understanding the molecular interactions between pig tissues and human immune cells is fundamental to achieving long-term pig to human xenograft survival. CD40 has been shown to be central in the interaction of T cells with many antigen-presenting cells including B cells, and dendritic cells. It has been clearly shown in vitro that human T cells can effectively recognize pig major histocompatibility complex proteins, and that various accessory molecule interactions are compatible between these species, including human CD28 with pig B7 family members (CD80/CD86). The importance of CD40 in transplantation has been established using blocking antibodies to its ligand, CD154, which prolong allograft survival in mouse and primate models. Pig CD40 was cloned from a porcine spleen cDNA library and subsequently sequenced. Expression of pig CD40 was detected by flow cytometry using soluble human CD154 (hCD154-Ig). Results. Comparison of the derived amino acid sequence of pig with human shows 74% identity. Significantly, there is conservation between pig and human at 5 residues shown by mutagenesis studies to be essential for binding of human CD40 to CD154. hCD154Ckappa was shown to bind pig B cell lines and a proportion of human and pig lymphocytes and further confirmed by staining of COS cells transfected with pig CD40. Conclusions. Recipient human cells bearing CD154 will, therefore, be able to bind donor pig CD40, and these interactions might modulate effector functions and hence influence xenograft survival. Further investigation is necessary to ascertain the exact nature of these interactions and their implications for xenograft survival.

Identification of an anti-mycobacterial domain in NK-lysin and granulysin

NK-lysin and granulysin are homologous cationic anti-bacterial peptides produced by pig and human cytolytic lymphocytes, respectively. The solution structure of NK-lysin comprises five amphipathic alpha-helices. To investigate the properties of a helix-loop-helix region postulated to be a membrane-docking part of NK-lysin, we synthesized 22- and 29-residue peptides reproducing this region for both NK-lysin and granulysin. CD spectroscopy of the synthetic peptides in a liposomal solution showed spectra typical of alpha-helical peptides. The peptides were active against Gram-positive and Gram-negative bacteria, with the two NK-lysin peptides showing higher anti-bacterial activities than the two from granulysin. One NK-lysin peptide was active against Pseudomonas aeruginosa and Staphylococcus aureus, two organisms against which NK-lysin is inactive. Granulysin peptides were inactive against these bacteria, in contrast with granulysin, which is known to be active against them. Both NK-lysin and all synthetic analogues killed Mycobacterium tuberculosis and K562 tumour cells, but did not display haemolytic activity. These results identify a potent anti-mycobacterial domain in NK-lysin and granulysin consisting of a 22-residue (helix 3) sequence plus a disulphide-constrained loop.

Decreased Capacity of Recombinant 45/47-kDa Molecules (Apa) of Mycobacterium tuberculosis to Stimulate T Lymphocyte Responses Related to Changes in Their Mannosylation Pattern

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.

The influence of age and breeding conditions on the number and proliferation of intraepithelial lymphocytes in pigs.

The number and subset composition of intraepithelial lymphocytes (IEL) were studied in pigs in different age groups ranging from 1 day to 4.5 years. There were no major differences between the numbers of IEL in the jejunum and ileum. The postnatal increase of IEL largely depended on the breeding conditions: in germ-free animals there was a constant level, while in conventionally bred pigs the numbers increased more obviously than in specified pathogen-free (SPF) pigs. As the numbers of IEL can rise due to increased influx from other organs, decreased emigration, decreased apoptosis or local proliferation, the incorporation of the DNA precursor bromodeoxyuridine (BrdU) by IEL was studied after different labelling protocols. There were low but definite numbers of BrdU+ IEL 1 h after BrdU, indicating in-situ proliferation in conventional animals with a tendency to a higher index in the jejunum than the ileum. Repetitive labelling protocols for 14 days resulted in 12-20% BrdU+ IEL, which may be caused by local proliferation and immigration of lymphocytes produced in other lymphoid organs as documented for the pig. Future studies need to focus on the factors regulating local proliferation and the migration of IEL in different species.

Definition and characterization of chicken Gal alpha(1,3)Gal antibodies.

The Gal alpha(1,3)Gal epitope is of interest as, in pig-to-primate xenotransplantation, it is the major target of naturally occurring human IgM and IgG antibodies, leading to hyperacute rejection. Human and Old World monkeys make anti-Gal alpha(1,3)Gal antibodies as they lack a functional gene and do not express Gal alpha(1,3)Gal. Interestingly, the cultured fibroblasts of some other species, such as chickens, have been reported also not to express Gal alpha(1,3)Gal--if this is true for other tissues, and chickens do not express Gal alpha(1,3)Gal antigen, then they would have anti-Gal antibodies--which could have diagnostic and therapeutic value, particularly as chicken antibodies do not fix mammalian complement. Standard serological methods were used to characterize the antibodies. Several baboons received pig kidney xenografts that had been perfused with hyperimmune chicken anti-Gal antibodies. We now demonstrate that chickens do not express Gal alpha(1,3)Gal on their red cells, leukocytes, or tissues, and that their serum contains large amounts of anti-Gal alpha(1,3)Gal antibodies. In addition, chickens could be immunized to produce high-titer, high-avidity antibodies (9.5x10(9) M(-1))--an avidity considerably greater than that of the Gal alpha(1,3)Gal binding lectin IB4 (2.9x10(8) M(-1)) or Gal antibodies in human serum (2.2x10(5) M(-1)). Chicken antibodies, obtained from both normal and immunized chickens, could block the in vitro cytolysis of pig endothelial cells or lymphocytes by human or baboon antibodies. However, such antibodies tested in vivo in pig-to-baboon xenotransplantation failed to block hyperacute rejection and, indeed, may have accelerated this.

Monocytes affect cell‐cycle progression but not baseline frequency of sister chromatid exchanges of pig lymphocytes

The effect of pig monocytes (MNs) on the baseline frequency of sister chromatid exchanges (SCEs) and cell-cycle progression of pig lymphocytes was studied in plasma leukocyte (PLCs) and whole blood leukocyte cultures (WBCs). No variation in SCE frequency was observed between control WBC and PLC, nor did the addition of pig MNs to PLCs modify the baseline frequency of SCEs. Cell proliferation was slower in PLCs than in WBCs. Variations in cell-cycle progression of pig lymphocytes from PLC were observed both in the absence and presence of adherent cells in the culture. In MN-free cultures, lymphocytes proliferated faster than in parallel PLC cultures. However, when MNs were seeded into the cultures, cell-cycle progression gradually slowed as a function of the concentration of adherent cells present in the cultures. This finding shows that pig MNs modulate the in vitro cell-cycle progression of pig lymphocytes in a dose-dependent manner and that the low baseline SCE frequency of pig lymphocytes is independent of the presence or absence of MNs in the culture. Environ. Mol. Mutagen. 33:86–90, 1999 © 1999 Wiley-Liss, Inc.