Porcine Peripheral Blood Lymphocytes Research Articles

Monoclonal Antibody against Porcine LAG3 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection

Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor and the interaction between fibrinogen-like protein 1 and LAG3 can inhibit the anti-tumor effect of T cells both in vivo and in vitro, which was regarded as a new immune evasion mechanism. Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV, is an infectious disease characterized by reproductive disorders in pregnant sows and gilts and respiratory problems in pigs of all ages, seriously impacting the pig industry worldwide. In this study, monoclonal antibodies (mAbs) against porcine LAG3 (pLAG3) were developed, and one mAb (1C2) showed good reactivity with pLAG3 on PHA-activated porcine peripheral blood lymphocytes. Epitope mapping showed the epitope recognized by mAb 1C2 was located at amino acid residues 214–435 of pLAG3. LAG3 expression in the tissues of PRRSV-infected pigs was detected, using mAb 1C2 as the primary antibody, and the results revealed that PRRSV infection caused a marked increase in LAG3 expression compared to the control group. Interference of LAG3 expression on PHA-activated lymphocytes promoted PRRSV replication in the co-culture system of monocyte-derived dendritic cells and lymphocytes, whereas overexpression of LAG3 or blocking of the LAG3 signal with mAb 1C2 inhibited PRRSV replication, indicating that PRRSV infection activates the LAG3-signaling pathway, suggesting that this pathway plays an important role in PRRSV pathogenesis. The results obtained lay the foundation for subsequent research on the role of LAG3 in PRRS and other diseases with persistent infection characteristics.

Identification of Potential miRNA-mRNA Regulatory Network Associated with Regulating Immunity and Metabolism in Pigs Induced by ASFV Infection

African swine fever (ASF) is a devastating infectious disease in domestic pigs caused by African swine fever virus (ASFV) with a mortality rate of about 100%. However, the understanding of the interaction between ASFV and host is still not clear. In this study, the expression differences and functional analysis of microRNA (miRNA) in porcine peripheral blood lymphocytes of ASFV infected pigs and healthy pigs were compared based on Illumina high-throughput sequencing, then the GO and KEGG signal pathways were analyzed. The miRNA related to immunity and inflammation were screened, and the regulatory network of miRNA-mRNA was drawn. A total of 70 differentially expressed miRNAs were found (p ≤ 0.05). Of these, 45 were upregulated and 25 were downregulated in ASFV-infected pigs vs. healthy pigs. A total of 8179 mRNA genes targeted by these 70 differentially expressed miRNA were predicted, of which 1447 mRNA genes were targeted by ssc-miR-2320-5p. Five differentially expressed miRNA were validated by RT-qPCR, which were consistent with the RNA-Seq results. The GO analysis revealed that a total of 30 gene functions were significantly enriched, including 7 molecular functions (MF), 13 cellular components (CC), and 10 biological processes (BP). The KEGG enrichment analysis revealed that the differentially expressed genes were significantly enriched in pathways related to immunity, inflammation, and various metabolic processes, in which a total of two downregulated miRNAs after infection and eight upregulated miRNAs related to immunity and inflammation were screened in ASFV-infected pigs vs. healthy pigs. The network of miRNA-mRNA showed that the mRNA target genes were strongly regulated by ssc-miR-214, ssc-miR-199b-3p, and ssc-miR-199a-3p. The mRNA target genes were enriched into the MAPK signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, and IL-17 signaling pathway by using a KEGG enrichment analysis. Therefore, ASFV could regulate immunity and metabolism-related pathways in infected pigs by inducing differential expression of miRNAs. These results provided a new basis for further elucidating the interactions between ASFV and the host as well as the immunity regulation mechanisms of ASFV, which will be conducive to better controlling ASF.

Analiza fenotipa CD3+CD16+ limfocita periferne krvi svinja

The phenotype of porcine peripheral blood T cells and natural killer (NK) cells has been well-studied over the past three decades, though porcine peripheral blood lymphocytes with mixed T/NK-cell phenotype within perforin- and NKp46- positive CD3+ populations have also been identified. Despite the mixed phenotype, both populations showed in vitro NK cell-like major histocompatibility complex-unrestricted cytolysis. In this study, the peripheral blood lymphocytes of 15 crossbreed, 12-week-old pigs of both sexes, were analysed by flow cytometry for the expression of leukocyte surface antigens (cluster of differentiation, CD) that can be found on porcine T cells (CD3, TCR-γδ and CD4), NK cells (CD16) or on both cell populations (CD8α and SLA-DR). We found the presence of a minor population of CD3+CD16+ cells within peripheral blood lymphocytes (2.84%). Peripheral blood CD3+CD16+ lymphocytes consisted of all four subpopulations with respect to the expression of surface antigens CD4 and CD8α; most were CD4-CD8α+ (60.64%) and CD4-CD8α-(36.77%). While the proportion of SLA- DR+ cells within both subpopulations was similar (8.01% of CD3+CD16+CD4-CD8α+ lymphocytes and 7.41% of CD3+CD16+CD4- CD8α- lymphocytes), the proportion of TCR-γδ+ cells was noticeably higher within CD3+CD16+CD4-CD8α+ (43.48%) than CD3+CD16+CD4-CD8α- (16.55%) lymphocytes. When the expression of individual surface antigens was analysed on peripheral blood CD3+CD16+ lymphocytes, most were CD8α+ (62.44%), though some were also TCR- γδ+ (32.56%), SLA-DR+ (7.55%) or CD4+ (2.59%). Expression of CD8α on CD3+CD16+ lymphocytes was not related to co-expression of other surface antigens, though most CD3+CD16+TCR-γδ+ lymphocytes (81.04%) and most CD3+CD16+CD4+ lymphocytes (69.50%) expressed CD8α. Expression of SLA-DR was not related to the co-expression of TCR-γδ or CD8α, or to the co-expression of both antigens (TCR-γδ and CD8α) on CD3+CD16+CD4- lymphocytes. The results also showed the presence of peripheral blood lymphocytes with the combined phenotypeof T cells and NK cells in three-month old pigs. Though a functional analysis of the investigated cells was not performed in this study, future investigations should provide more insight about the functional properties of porcine peripheral blood CD3+CD16+ lymphocytes with distinct phenotypic characteristics, especially concerning antigen- specific responses and whether the results presented here are solely age-related.

Generation of anti-porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis.

T cell-mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti-porcine CD69 mAb-producing mouse hybridomas, 01-14-22-51 (IgG2b-κ) and 01-22-44-102 (IgG2a-κ), both showing fine reactivity with phorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti-interferon-γ mAb and anti-tumor necrosis factor-α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.

Changes in the Subpopulations of Porcine Peripheral Blood Lymphocytes Induced by Exposure to Low Doses of Zearalenone (ZEN) and Deoxynivalenol (DON).

Zearalenone and deoxynivalenol are secondary metabolites of fungi of the genus Fusarium. The presence of mycotoxins in cereals and the resulting contamination of feeds and foods pose health risks for animals and humans. The dangers associated with high doses of mycotoxins have been extensively researched but very little is known about NOAEL (No Observed Adverse Effect Level) doses or exposure to a combination of mycotoxins (mixed mycotoxicoses). The aim of this study was to determine the effects of six-week exposure to NOAEL doses of individual and combined mycotoxins on the subpopulations of CD4+8−, CD4−8+ and CD4+8+ lymphocytes in the peripheral blood of pigs. The experiment was performed on 72 gilts with average body weight of 25 kg, divided into three experimental groups (E1, E2 and E3, administered zearalenone (ZEN), deoxynivalenol (DON) and ZEN + DON, respectively, on a daily basis) and a control group (C) receiving placebo. Changes in lymphocyte subpopulations were evaluated by flow cytometry at weekly intervals (experimental days 7, 14, 21, 28, 35 and 42). A linear increase in the percentage of CD4+8+ lymphocytes was highly correlated with time (r = 0.682) in group C. The correlations and linear increase in the above subpopulation were disrupted in the remaining groups. In group E3, a statistically significant (p < 0.05) decrease in CD4+8+ counts was observed in week 5, which could point to a transient depletion of regulatory mechanisms of immune responses. The noted results also suggest that in mixed mycotoxicosis, ZEN and DON exerted stronger immunomodulatory effects.

Molecular cloning and characterizations of porcine SAMHD1 and its roles in replication of highly pathogenic porcine reproductive and respiratory syndrome virus

The sterile alpha motif and HD domain 1 (SAMHD1) protein is a novel innate immunity restriction factor that inhibits HIV-1 infection in myeloid cells. Here, we cloned the full-length SAMHD1 complementary DNA (cDNA) from porcine peripheral blood lymphocytes. The porcine SAMHD1 cDNA was of 3951 bp with an open reading frame of 1884 bp, encoding a polypeptide of 627 amino acids. Porcine SAMHD1 mRNA was detected in all swine tissues examined, with the higher expression in the tonsil, lung, liver, and lymph node tissues. The SAMHD1 protein was localized to the nucleus. Overexpression of SAMHD1 blocked the proliferation of HuN4, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV), in MARC-145 cells, by inhibiting the synthesis of the HuN4 complement RNA. The antiviral effects of the simian SAMHD1 protein were nearly equivalent to those of porcine SAMHD1 in the HuN4-infected MARC-145 cells. Phosphorylation analysis of SAMHD1 showed that overexpressed SAMHD1 protein was in primarily an unphosphorylated state. SAMHD1 overexpression increased the transcript abundance of IFN-stimulated genes ISG15 and ISG56. The mRNA levels of SAMHD1 and ISGs were significantly increased in porcine alveolar macrophages infected with HP-PRRSV. SAMHD1 protein level was also elevated, and the protein was not phosphorylated during infection. Collectively, our data indicate that SAMHDI inhibits HP-PRRSV proliferation through inhibiting the replication of HP-PRRSV. SAMHD1 might be the protein participating in the IFN signaling and is thus an important immunoregulatory protein in innate immunity.

Effect of cryopreservation on IL-4, IFNγ and IL-6 production of porcine peripheral blood lymphocytes

Cryopreservation of animal or human peripheral blood mononuclear cells (PBMC) is a commonly used technique. Effects of cryopreservation on functional capacity, especially the cytokine production of human PBMCs, have been extensively defined. However, certain animals, such as livestock, are a shortage of these information. Here we investigated the effects of cryopreservation on cytokine (IL-4, IFNγ and IL-6) production of porcine PBMC. The porcine PBMCs were cryopreserved at −196°C for a variety time periods for 2, 5, 25 and 50days. Viability and cytokine production of the porcine PBMCs were measured before and after cryopreservation. The results showed that about 90% cell recovery rate was obtained at each storage time, indicating that about 10% loss of PBMCs in this short-term cryopreservation was due to the freezing process rather than the duration of cryopreservation. The fresh or frozen resting porcine PBMCs produced little cytokines in the absence of stimulation. However, three cytokines were apparently increased after PMA stimulation in both fresh and frozen porcine PBMCs. The sensitivity of frozen cells to PMA simulation for IFNγ and IL-6 production was different from that of the fresh ones. IFNγ production from the frozen PBMCs was significantly higher than that from the fresh ones (P<0.01). In contrast, IL-6 level from the frozen sample was significantly lower than that from the fresh one (P<0.05). Those results indicate that cryopreservation can increase the sensitivity of porcine PBMCs stimulated by PMA for IFNγ production but not for IL-6 production. There was no significant difference of IL-4 production between fresh and frozen cells either stimulated (P>0.05) or un-stimulated (P>0.05).

Effect of the Fusarium toxin deoxynivalenol (DON) on IgA, IgM and IgG concentrations and proliferation of porcine blood lymphocytes

An important effect of the trichothecene mycotoxins is the impairment of the immune function, but immunotoxicity studies have mainly been conducted on the mouse model. In the present study, the effect of deoxynivalenol (DON) on the proliferation of ConA stimulated porcine peripheral blood lymphocytes (PBL) was assessed in vitro after adding of 70–560 ng DON per ml medium, and in vivo after chronic and acute (one single dose) dietary DON exposure (5.7 mg/kg). Immunoglobulin (IgA, IgG, IgM) concentrations were measured by ELISA in supernatants and serum of pigs. The proliferation rate was estimated with two different assays (BrdU incorporation and MTT cleavage). In vitro the ConA stimulated proliferation was inhibited to 50% (IC 50) at 200 and 309 ng DON/ml for the BrdU and MTT assay, respectively, indicating a higher sensitivity of DNA synthesis to DON. Immunoglobulin concentrations in the supernatant after in vitro proliferation of PBL with increasing DON concentrations for 72 h were significantly decreased, with IC 50 values of 120.6, 84.1 and 71.7 ng DON/ml for IgA, IgM and IgG, respectively. In vivo significant inhibition of lymphocyte proliferation was observed only in the DON acute group using the MTT assay, but values tended to be decreased in the BrdU assay and after chronic DON exposure. Immunoglobulins (IgA, IgM and IgG) in the supernatant of cultured lymphocytes were not significantly affected after dietary DON exposure. Serum IgA of pigs showed no significant differences between the groups, whereas IgM and IgG were significant increased in the DON acute group.

Evaluation of the Suitability of Monoclonal Antibodies for Flow Cytometric Intracellular Cytokine Detection in Porcine Peripheral Blood Lymphocytes

Panels of monoclonal antibodies (mAbs) specific for porcine interleukin (IL)-2, IL-4, IL-6, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were evaluated for their applicability in flow cytometric intracellular cytokine detection. Peripheral blood mononuclear cells were short-time stimulated in the presence of brefeldin-A, ionomycin and phorbol-12-myristate-13-acetate, fixed and incubated with the respective mAbs as well as phycoerythrin-conjugated second-step antibodies. Suitability of mAbs was judged by use of statistical data and by visual control of scattergrams, considering the capability of mAbs to discriminate between cytokine-positive and cytokine-negative cell populations. The number of suitable clones differed to a high degree between the investigated cytokines, but at least one mAb fitting for flow cytometric intracellular cytokine detection could be identified within each panel. Monoclonal Abs producing scattergrams with a clear demarcation between positive and negative cell populations were found within the anti-IL-2, IL-6 and IFN-gamma panels, whereas less well defined positive and negative cell populations could be generated by use of mAbs within the anti-IL-4 and TNF-alpha panels. Only one moderately fitting mAb was identified within the anti-IL-12 panel. After having evaluated the best fitting mAbs, these were used to obtain reference levels for the physiological range of porcine lymphocytic cytokine production in a second set of experiments. For that reason, 13 clinically healthy pigs aged between 6 weeks and 6 months were investigated. Data presented are given as mean +/- SD of the percentage of positive-staining lymphocytes: IL-2, 45.5 +/- 27.6; IL-4, 34.1 +/- 21.3; IL-6, 45.4 +/- 23.8; IL-12, 13.9 +/- 8.6; TNF-alpha, 43.4 +/- 11.3; IFN-gamma, 65.5 +/- 14.8.

Characterization of porcine CD5 and CD5+ B cells.

Evidence strongly supports a role for the lymphocyte transmembrane glycoprotein CD5 in intracellular signalling events, whereby antigen-dependent growth and differentiation signals are augmented. Apart from its role in activation-related signalling, CD5 has been regarded as a possible B cell lineage marker differentiating subsets, CD5+ B cells (also termed B1 cells) and conventional B cells (or B2 cells). To extend these investigations to the study of pigs, porcine B cells were examined for evidence of CD5 expression. The influence of cellular activation on CD5 expression and CD5's role in signal transduction events and lymphocyte proliferation were examined. Using an anti-porcine CD5 MoAb (b53b7), porcine B cells were shown to be heterogeneous for CD5 expression. As in other species, B lymphocyte CD5 expression is low (dull), while IgM is high (bright). Ten to 30% of pig blood B lymphocytes are CD5+, with the highest frequency in neonates. Anti-CD5 antibody treatment was sufficient to induce rapid but transient calcium ion flux in porcine peripheral blood lymphocytes (PBL). CD5 expression increased on PBL following treatment with phorbol myristate acetate (PMA), lipopolysaccharide (LPS), or immobilized anti-IgM. LPS, PMA, and concanavalin A (Con A) but not anti-CD5, anti-IgM, or combinations of these antibodies induced lymphocyte 3H-thymidine uptake. CD5+B cells are a common constituent of porcine circulating lymphocytes and resemble B1 cells of mice, man and other species in CD5 expression, frequency and lymphoid organ distribution. Porcine CD5, like CD5 in other species, mediates signal transduction, leading to changes in intracellular calcium concentration, but this signal alone is insufficient to promote cell division. A subset of porcine B cells up-regulates CD5 expression following phorbol ester activation.

Molecular cloning, characterization, and expression of porcine Fas ligand (CD95 ligand).

We isolated and sequenced cDNA that contained the coding sequence of porcine Fas ligand (FasL). Using mixed oligonucleotide primers based on the 5' and 3' nucleotide sequences conserved among human, murine, and rat FasL, we performed the reverse transcription polymerase chain reaction (RT-PCR) with total RNA prepared from porcine thymocytes stimulated with 5 microg/ml concanavalin A (ConA) to clone the cDNA of porcine FasL. The open reading frame (ORF) of porcine FasL cDNA was 849 base pairs (bp) in length and encoded 282 amino acids. The predicted amino acid sequence was 85.5%, 76.6%, and 75.5% homologous to the predicted human, murine, and rat FasL, respectively. The recombinant porcine FasL expressed by recombinant baculovirus containing the whole coding sequences of porcine FasL showed cytotoxic effect and induced apoptosis in porcine renal tubular cell line PK-15 cells sensitized by cycloheximide (CHX), which was confirmed by MTT assay, DNA fragmentation assay, and TUNEL staining, respectively. Furthermore, the mRNA expression of porcine FasL in porcine peripheral blood lymphocytes (PBL) was induced by porcine interleukin-18 (IL-18). These results indicate that porcine FasL identified in this study is biologically functional and has the ability to induce apoptosis as reported in other species.

Distribution and localization of CMP- N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes*

An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.

A COMPARISON OF FETAL AND ADULT PORCINE ISLETS WITH REGARD TO Gal?? (1,3)Gal EXPRESSION AND THE ROLE OF HUMAN IMMUNOGLOBULINS AND COMPLEMENT IN ISLET CELL CYTOTOXICITY1

It is still debated whether fetal or adult porcine islets should be the preferred choice for future clinical islet xenotransplantation. Each type of islet preparation has advantages and disadvantages compared with the other. Here we present a direct comparison between fetal and adult porcine islets with regard to Gal alpha(1,3)Gal expression, immunoglobulin and complement binding, and cytotoxicity after exposure to fresh human serum. Islet single cell suspensions were prepared from adult and fetal islets by trypsin digestion. Fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia isolectin B4 (BS-IB4) and affinity-purified chicken anti-Gal alpha(1,3)Gal antibody was used to detect Gal alpha(1,3)Gal expression. Immunoglobulin and complement binding to the islet cells and cytotoxicity for islet cells was compared after incubation with fresh and heat-inactivated human sera and with an immune serum from a diabetic patient who received a fetal porcine islet transplant. Furthermore, two pools of human AB sera were depleted of porcine endothelial cell cytotoxic human anti-Gal alpha(1,3)Gal antibodies by absorption and were used to analyze the effect of Gal alpha(1,3)Gal antibody removal on islet cell cytotoxicity. Fetal islet cells readily bound both BS-IB4 and the chicken anti-Gal alpha(1,3)Gal antibody. None of 10 adult porcine islet preparations were stained by BS-IB4. In comparison, IgY anti-Gal Ab binding was detected in two of eight adult islet isolations, whereas the other six preparations showed marginal/no binding. After incubation of fetal islet cells with fresh human serum, C3c binding was strongly positive and IgM binding variable, with occasional binding of IgG and no detectable binding of IgA. Adult islet cells were also strongly positive for C3c but did not bind detectable amounts of IgM, IgG, or IgA. Immune sera from a patient who had received fetal porcine islets showed the presence of induced antibodies that bound to fetal islet cells and to porcine peripheral blood lymphocytes, whereas binding to adult islet cells was barely detectable. Fresh human sera showed a high and similar level of complement-mediated lytic activity for both adult islet cells (78+/-22%) and fetal islet cells (75+/-16%). Cytotoxicity for fetal islet cells and peripheral blood lymphocytes was significantly reduced when the corresponding sera were depleted of anti-Gal antibodies before use (P=0.002 and P=0.003, respectively). In contrast, no difference in cytotoxicity for adult islet cells was detected when anti-Gal-depleted human sera were used. Gal alpha(1,3)Gal expression is occasionally detectable on adult porcine islet cells, but not as readily and at a lower level, compared with fetal islet cells. Thus, as porcine fetal islets mature to adult islets, the expression of the Gal alpha(1,3)Gal epitope gradually diminishes. Consequently, cytotoxic anti-Gal alpha(1,3)Gal antibodies in human serum play an important role in the lysis of fetal but not adult porcine islet cells.

Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry

We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser 65 to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.

HLA antibodies present in the sera of sensitized patients awaiting renal transplant are also reactive to swine leukocyte antigens.

To determine whether preformed HLA alloantibodies present in the sera of patients awaiting kidney transplantation will be detrimental to a potential porcine xenograft, we tested their cross-reactivity to swine leukocyte antigens (SLA). Sera obtained from patients with varying levels of HLA sensitization (high panel-reactive antibodies > 70%, n= 7; moderate panel-reactive antibodies 30-40%, n=2) were analyzed. Pooled normal human AB sera and sera from nonsensitized patients (n=3) served as negative control. IgG was purified by protein-G chromatography, and xenoreactive natural antibodies (XNA) were depleted by passing the IgG through a series of melibiose and thyroglobulin-agarose columns. The elimination of XNA from HLA IgG preparations was confirmed by GS-IB4 lectin blocking assay and by an ELISA. IgG isolated from normal AB serum and three nonsensitized patients, which was depleted of XNA (HLA-IgG), did not react to human or porcine lymphocytes (peripheral blood mononuclear cells; PBMC) either by flow cytometry or by complement-dependent microcytotoxicity assays. However, HLA-IgG isolated from nine sensitized patients were reactive to a panel of porcine peripheral blood lymphocytes (n=6) by flow cytometry (>50 mean channel shift) and in complement-dependent microcytotoxicity assays in addition to their reactivity to human PBMC. The binding of HLA-IgG to porcine PBMC was significantly reduced by preabsorption with pooled human platelet concentrate. Further, the HLA IgG showed recognition of 45-kDa affinity-purified SLA class I on Western blots. This study demonstrates that HLA antibodies present in the sera of sensitized individuals can cross-react with SLA. Thus, xenotransplantation of porcine organs into HLA-sensitized patients has the potential to be rejected by humoral mechanisms. Testing to avoid such cross-reactive antibodies should be considered.

Report on the analyses of mAb reactive with porcine CD8 for the second international swine CD workshop

Based on an analysis of their reactivity with porcine peripheral blood lymphocytes (PBL), only three of the 57 mAbs assigned to the T cell/activation marker group were grouped into cluster T9 along with the two wCD8 workshop standard mAbs 76-2-11 (CD8a) and 11/295/33 (CD8b). Their placement was verified through the use of two-color cytofluorometry which established that all three mAbs (STH101, #090; UCP1H12-2, #139; and PG164A, #051) bind exclusively to CD8 + cells. Moreover, like the CD8 standard mAbs, these three mAbs reacted with two proteins with a MW of 33 and 35 kDa from lymphocyte lysates and were, thus, given the wCD8 designation. Because the mAb STH101 inhibited the binding of mAb 76-2-11 but not of 11/295/33, it was given the wCD8a designation. The reactivity of the other two new mAbs in the T9 cluster with the various subsets of CD8 + lymphocytes were distinct from that of the other members in this cluster including the standards. Although the characteristic porcine CD8 staining pattern consisting of CD8 low and CD8 high cells was obtained with the mAb UCP1H12-2, a wider gap between the fluorescence intensity of the CD8 low and CD8 high lymphocytes was observed. In contrast, the mAb PG164A, not only exclusively reacted with CD4 −/CD8 high lymphocytes, but it also failed to recognize CD4/CD8 double positive lymphocytes. It was concluded that this mAb is specific for a previously unrecognized CD8 epitope, and was, thus, given the wCD8c designation. A very similar reactivity pattern to that of PG164A was observed for two other mAbs (STH106, #094; and SwNL554.1, #009). Although these two mAbs were not originally positioned in the T cell subgroup because of their reactivity and their ability to inhibit the binding of PG164A, they were given the wCD8c designation. Overall, five new wCD8 mAbs were identified. Although the molecular basis for the differences in PBL recognition by these mAbs is not yet understood, they will be important in defining the role of CD8 + lymphocyte subsets in health and disease.

Species‐specific detection of porcine xenografts with an antibody against a novel epitope of the lymphocyte homing receptor CD44

Abstract: We have isolated and characterized a murine monoclonal antibody, 10–14, that is reactive with an 85 kDa glycoprotein found on porcine peripheral blood lymphocytes, cultured aortic endothelial cells, fetal cardiomyocytes, and mixed neural/glial cell cultures. In contrast to the anti‐CD44 antibody Z062, which reacted with both porcine and murine CD44, the 10–14 antibody did not react with monkey, rabbit, rat, or murine cells, suggesting that 10–14 recognized a novel epitope found exclusively on the porcine CD44 molecule; 10–14 is thus a novel reagent for identification of xenotransplanted porcine cells. The reagent has been used in this study as an immunohistochemical probe to detect porcine neural cell grafts in the central nervous system of the rat and to label porcine fetal cardiomyocytes. Moreover, the antibody provides information on the expression of the CD44 molecule that is critical for lymphocyte adhesion and trafficking.

Human CD8 responder cells can be directly activated to proliferate and to produce IL‐2 following stimulation by allogeneic, but not by xenogeneic, porcine blood mononuclear cells

Abstract: In order to determine the precise nature of human T lymphocytes reactivity against porcine stimulator cells, purified CD4+ and CD8+ human peripheral T lymphocytes have been tested for their responsiveness against porcine stimulator cells. In a xenogeneic mixed lymphocyte reaction (MLR), CD4+ T cells were capable of proliferating as a result of the recognition of porcine peripheral blood lymphocytes (PBL), whereas CD8+ T cells were unresponsive. A proliferative response of CD8+ T cells could be restored by treatment with human IL‐2, but not by IL‐lα, IL‐lβ, or IL‐6. Production of IL‐2 was not detected in the xenostimulated CD8+ responder cells, nor could IL‐2 production be restored by the addition of IL‐lα, IL‐1β, or IL‐6. The presence of human CD4+ responder cells was crucial both for a xenoproliferative response and for IL‐2 synthesis. However, when the expression of the IL‐2 receptor (CD25) on the CD8+ T cells was analyzed, no difference was detected between xenostimulated and allostimulated CD8+ T cells. When the development of cytotoxic T cells in xenogeneic and allogeneic MLRs was compared, the cytotoxic activity exhibited by purified CD8+ T cells in xenogeneic MLR was significantly lower than that in the allogeneic combination. In the xenogeneic combination, exogenous IL‐2 reconstituted the cytotoxicity by purified CD8+ T cells; however, IL‐lα, IL‐lβ, or IL‐6 did not.Our results show that purified human CD4+ T cells respond directly against pig PBMCs, whereas purified CD8+ T cells do not. Furthermore, responsiveness in CD8+ T cells is completely restored by the addition of human IL‐2.

Cell membrane permeability and mitochondrial dysfunction-inducing activities in cell-free supernatants from Serpulina hyodysenteriae serotypes 1 and 2

Membrane permeability (MP) and mitochondrial dysfunction-inducing (MDI) activities were detected in cell-free supernatants (CFS) of Serpulina hyodysenteriae, using either hemoglobin release from porcine red blood cells (RBC) or cytoplasmic lactate dehydrogenase release from porcine peripheral blood lymphocytes (PBL), and reduction of the 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide dye by porcine PBL. The MP and MDI activities of CFS correlated with each other for serotype 1 and 2 isolates taken at different population densities; however, the kinetics of toxin production varied between each serotype. The loss of enteropathogenicity of two field isolates with nonpathogenic phenotypes and pathogenic isolates passaged up to 45 times in vitro was not attributable to a loss of either membrane permeability or mitochondrial dysfunction-inducing activity of cell-free supernatants. Results from this study suggested the potential for two separate toxins being involved in the pathogenesis of swine dysentery, with the MDI activity correlating with age susceptibility to clinical disease.

Translational regulation during activation of porcine peripheral blood lymphocytes: association and phosphorylation of the α and γ subunits of the initiation factor complex eIF-4F

Mature peripheral blood lymphocytes exist in a resting state both in vivo and when maintained in culture, exhibiting low translation rates consistent with their non-proliferative state. Previously we have shown that activation of these quiescent cells with either phorbol ester or concanavalin A leads to a rapid increase in the rate of protein synthesis and phosphate-labelling of initiation factor eIF-4 alpha [Morley, Rau, Kay and Pain (1993) Eur. J. Biochem. 218, 39-48]. We now show that neither the early enhanced translation rate nor the early increased phosphate-labelling of eIF-4 alpha requires the activity of the 70 kDa form of ribosomal protein S6 kinase. In addition, we demonstrate that eIF-4 gamma is phosphorylated in response to cell activation, an event which is correlated with phosphorylation of eIF-4 alpha and enhanced eIF-4F complex formation. In these studies, isoelectric focusing and immunoblot analysis of eIF-4 alpha indicate that phosphate-labelling of eIF-4 alpha following cell activation reflects a modest increase in steady-state phosphorylation, mediated by the enhanced activity of eIF-4 alpha kinase(s) and inhibition of eIF-4 alpha phosphatase activity. In the resting cell, eIF-4 alpha is associated with heat- and acid-stable insulin-responsive protein (PHAS-I; 4E-BP1); following acute stimulation with phorbol ester, there is a 40% decrease in the amount of PHAS-I associated with eIF-4 alpha. Incubation of anti-PHAS-I immunoprecipitates with extracts containing activated or immunprecipitated mitogen-activated protein kinase resulted in a small increase in phosphorylation of recovered PHAS-I and a modest release of eIF-4 alpha from the PHAS-I-eIF-4 alpha complex. These data suggest a possible role for PHAS-I in the regulation of eIF-4F complex formation and the rate of translation in primary cells.