Preliminary evaluation of selected inflammatory cytokine gene expression in lymphocytes isolated from whole human blood infected with trans-anethole-treated Staphylococcus aureus Newman strain.

In our previous study based on a whole-blood model of sepsis infected with trans-anethole (TA)-treated Staphylococcus aureus, we have found that innate immune response was more effective in comparison to non-treated cells. Due to the previous observation, in the current preliminary study, a primary adaptive immune response was analysed. This study was conducted to evaluate the expression of selected cytokine (IL1B, IL2, IL6, IL10, TNF, TGFB1, IFNG) and Toll-like receptor (TLR2) genes in lymphocytes isolated from whole human blood infected with S. aureus Newman strain treated with TA. The lymphocytes were isolated by density gradient centrifugation from blood samples infected with S. aureus, as well as from non-infected samples. Gene expression was measured using quantitative real-time PCR. The lymphocytes isolated from the blood infected with TA-treated staphylococcal cells demonstrated significantly greater IL10, IL1B, IL6, TNF and TLR2 expression. Hence, it is possible that the previously observed changes in the surface structure of TA-treated S. aureus Newman strain may significantly increase the relative expression of IL10, IL1B, IL6, TNF and TLR2 genes in lymphocytes; however, further studies are needed.

Picosecond-precision multichannel autonomous time and frequency counter.

This paper presents the design, implementation, and test results of a multichannel time interval and frequency counter developed as a desktop instrument. The counter contains four main functional modules for (1) performing precise measurements, (2) controlling and fast data processing, (3) low-noise power suppling, and (4) supplying a stable reference clock (optional rubidium standard). A fundamental for the counter, the time interval measurement is based on time stamping combined with a period counting and in-period two-stage time interpolation that allows us to achieve wide measurement range (above 1 h), high precision (even better than 4.5 ps), and high measurement speed (up to 91.2 × 106 timestamps/s). The frequency is measured up to 3.0 GHz with the use of the reciprocal method. Wide functionality of the counter includes also the evaluation of frequency stability of clocks and oscillators (Allan deviation) and phase variation (time interval error, maximum time interval error, time deviation). The 8-channel measurement module is based on a field programmable gate array device, while the control unit involves a microcontroller with a high performance ARM-Cortex core. An efficient and user-friendly control of the counter is provided either locally, through the built-in keypad or/and color touch panel, or remotely, with the aid of USB, Ethernet, RS232C, or RS485 interfaces.

Newborn baboon serum lacks natural anti-pig xenoantibody.

Discordant xenotransplantation represents an attractive alternative to allotransplantation in light of the shortage of donor organs currently available for cardiac allotransplantation. Unfortunately, discordant xenotransplantation is still limited by hyper-acute rejection, a process thought to be mediated by natural anti-xenodonor antibody. Based on data that cytotoxic natural xenoantibodies are IgM in nature, we postulated that natural xenoantibodies may be absent from newborn serum. Baboon sera were collected from infant baboons. Pooled adult baboon sera were used as controls. A whole cell ELISA was performed to determine the binding of xenoantibodies to pig aortic endothelial cells and pig lymphocytes. The cytotoxicity of both adult and newborn baboon sera to pig aortic endothelial cells was measured by a MTT (3-(4,5-dimethyl-thiazoyl-2-y) 2,5 diphenyl-tetrazolium bromide) assay. Newborn baboon sera demonstrated very low levels of binding of natural IgM xenoantibodies to pig endothelial cells and lymphocytes, whereas natural IgM xenoantibodies from adult baboon sera bound significantly to both pig aortic endothelial cells and lymphocytes. IgG natural antibodies in both adult and newborn sera bound to pig endothelial cells and pig lymphocytes. The MTT assay demonstrated high levels of cytotoxicity to pig endothelial cells from adult baboon sera and very low levels of cytotoxicity from newborn baboon sera. In this study, newborn baboon sera were demonstrated to be free of natural IgM xenoantibodies to pig endothelial cells and lymphocytes. Although natural anti-pig IgG antibodies were present in newborn sera, newborn baboon sera lack cytotoxicity to pig target cells. These findings suggest that IgM is the more important xenoantibody and that hyperacute rejection of discordant cardiac xenografts may be avoidable in the newborn.

Abrogation of baboon natural xenoantibody to pig splenocytes by DL-penicillamine.

Natural xenoantibodies are believed to be IgM in nature and are known to play a critical role in the hyperacute rejection of distantly related xenografts. The purpose of this study was to determine whether the reducing agent DL-penicillamine could inactivate baboon natural xenoantibodies to pig splenocytes. Pooled baboon serum was treated with varying concentrations of DL-penicillamine over different lengths of time and a complement-mediated cytotoxicity assay was used to determine the reactivity of baboon natural xenoantibodies to pig splenocytes. A whole-cell ELISA assay was used to assess the binding of both IgG and IgM xenoantibodies to pig splenocytes. In addition, DL-penicillamine-treated serum was dialyzed to assess its potential clinical application. These in vitro experiments indicate that both IgM and IgG baboon natural xenoantibodies bind to pig splenocytes, but only IgM xenoantibody is cytotoxic. The binding of baboon natural IgM xenoantibody can be eliminated, and the cytotoxicity of IgM xenoantibody markedly reduced by DL-penicillamine treatment despite continued binding of IgG xenoantibody to pig splenocytes. In addition, DL-penicillamine can be dialyzed, suggesting that it may be an efficacious clinical treatment, the toxicity of which can be regulated with hemodialysis.

Circulating human mononuclear cells exhibit augmented lysis of pig endothelium after activation with interleukin 2.

In immunohistochemical studies investigating the cellular infiltrates in pig xenografts undergoing delayed rejection by newborn and adult primate recipients, we observed extensive infiltration with primate macrophages and natural killer (NK) cells. To extend these studies in vitro, we investigated the functional properties of human NK cell precursors with respect to their potential interactions with pig aortic endothelial cells (PAEC). Using a short-term 51Cr release assay, human peripheral blood mononuclear cells (PBMC) demonstrated spontaneous and interleukin (IL) 2 augmented lytic activity against PAEC which increased with increasing effector to target cell ratio. Treatment of human PBMC with anti-CD2 significantly reduced this NK lytic activity by IL-2-activated PBMC. Finally, we investigated the effects of PAEC treatment with certain macrophage-derived human cytokines on adhesion of IL-2-activated human PBMC. Treatment of PAEC with IL-1 and tumor necrosis factor-alpha, in a dose-dependent manner, increased adherence of IL-2-activated human PBMC. These results demonstrate that humans contain circulating NK cells capable of lysing PAEC after activation with IL-2, that the mechanism involves interactions between CD2 and its ligand on porcine endothelium, and that these interactions may be influenced by macrophage-derived cytokines produced at the site of xenograft rejection.

Anti-GaL IgG antibodies in sera of newborn humans and baboons and its significance in pig xenotransplantation.

We have previously demonstrated that hyperacute rejection does not occur in a pig-to-newborn baboon heart transplant model, presumably because of low levels of cytotoxic antipig antibodies present in the serum of newborn baboons. Cytotoxic antipig antibodies are primarily directed to alpha-1,3-galactosyl (alpha Gal) residues on endothelial cell surface structures Twenty-one full-term humans and 5 full-term baboons were tested for complement mediated lysis (CML) of pig kidney (PK-15) cells and anti-alpha Gal activity with an ELISA using BSA-conjugated alpha Gal residues as target. To evaluate the significance of the anti-alpha Gal titers in vivo 5 newborn baboons underwent heterotopic pig cardiac xenotransplantation. Six of 21 human samples and 1 of 5 baboon samples demonstrated significant cytotoxicity to PK-15 cells. Twelve of 21 newborn humans had anti-alpha Gal IgG antibodies at titers of 1:80 or greater. None of the samples had anti-alpha Gal IgM. In newborn baboons, 1 of 5 sera had anti-alpha Gal IgG antibodies at titers greater than 1:80 and none of these samples had anti-alpha Gal IgM. Xenografts survived for an average of 3.6 days, even in the baboon with high anti-alpha Gal IgG titers. Analysis of the explanted grafts showed minimal evidence of complement-mediated hyperacute rejection (HAR), but prominent mononuclear cell infiltrates. In serum tested posttransplant there was an induced anti-alpha Gal response with cytotoxicity against PK-15 cells. These results show that anti-alpha Gal IgM is absent in newborn human and baboon sera, allowing pig grafts to avoid HAR. However, the presence of anti-alpha Gal IgG may be associated with mononuclear cell infiltration of the xenograft and its subsequent rejection.

Absence of hyperacute rejection in newborn pig-to-baboon cardiac xenografts.

The shortage of organs for transplantation is especially severe for the critically ill newborn infant, for whom donors of the appropriate size are particularly scarce. One way to overcome this problem is to use animals in lieu of humans as organ donors. The major limitation to using animals for this purpose is the susceptibility of animal organs to hyperacute rejection, a violent rejection reaction thought to be mediated by antidonor antibody and complement. To evaluate the potential application of xenotransplantation to newborns, we tested neonatal humans and neonatal baboons and found that neither population expressed significant levels of xenoreactive anti-pig antibodies. We transplanted heterotopically hearts from newborn pigs into unmanipulated newborn baboons (n = 4). There was no evidence of hyperacute rejection in any of the grafts; the animals were killed with functioning grafts at 15, 81, 82, and 82 hr. This outcome contrasts with that of newborn pig-to-mature baboon and mature pig-to-mature baboon cardiac xenografts, which were rejected within 1 hr of transplantation. The histology of pig graft biopsies from the newborn recipients was normal. Immunohistochemistry revealed only traces of IgM, C3, C4, and the membrane attack complex along graft endothelium. Fibrin deposition along endothelial surfaces was observed early after transplantation and became more extensive with time; in the absence of endothelial bound antibody or complement, this change may represent preservation injury. This study suggests that due to low levels of natural antibody, the newborn infant may permit prolonged xenograft survival.

Absence of hyperacute rejection in pig-to-primate orthotopic pulmonary xenografts.

The shortage of organ donors for transplantation is more pronounced for the lung than for any other solid organ. To address this problem, we evaluated the feasibility of pulmonary xenotransplantation. Preliminary investigations demonstrated that orthotopically placed pig lungs in cynomologous monkey recipients could be engrafted up to 9 hr after reperfusion without evidence of hyperacute rejection. In this study, the rejection reaction of pig lungs transplanted orthotopically into baboons (n = 6) was further investigated by ELISA and immunohistochemistry. Four baboon recipients were killed at 24 hr and 2 recipients were killed at 72 hr after transplantation. Pulmonary arterial flow measurements demonstrated flow to the grafts, and systemic arterial and xenograft pulmonary venous blood gas analysis suggested function of the donor lungs during the course of engraftment. Serum levels of baboon anti-pig endothelial cell xenoantibody were normal and decreased minimally over time. Immunohistochemical staining of biopsies demonstrated trace IgG and IgM along graft endothelium 2 hr after reperfusion. At 8 hr, biopsy samples showed no immunoglobulin bound to endothelial cells. Staining for complement was negative. Fibrin and platelets were detected along xenograft endothelium. Despite these findings, the lung xenografts appeared injured and clinically rejected. During the first 8 hr after reperfusion, the grafts were hyperemic and subsequently became focally ecchymotic. Chest x-rays showed progressive pulmonary congestion. These findings suggest that the lung may be relatively resistant to antibody-mediated hyperacute rejection and efforts are being directed toward identifying the mechanism of the observed xenograft lung injury.

Induction of swine major histocompatibility complex class I molecules on porcine endothelium by tumor necrosis factor-alpha reduces lysis by human natural killer cells.

BACKGROUND: Natural killer (NK) cells have been implicated in a process of delayed xenograft rejection occurring in pig-to-primate organ transplants. As tumor necrosis factor-a (TNF-a) induces expression of both adhesion receptors and major histocompatibility complex class I molecules on porcine endothelium, we investigated the effects of TNF-alpha on human NK cell adherence to and cytotoxicity of porcine aortic endothelial cell (PAEC) monolayers. METHODS: Adherence of human NK cells was measured after PAEC treatment with increasing concentrations of TNF-alpha. Monoclonal antibodies (mAbs) against adhesion molecules on NK cells and PAEC were used in inhibition studies. Resting or TNF-alpha-treated PAEC were used as targets for NK lysis. Increasing titers of anti-swine leukocyte antigen (SLA) class I antibodies or pooled human immune globulin (IVIg) were used to reverse the effects of TNF-alpha on NK lysis. RESULTS: NK cell adhesion to TNF-a-treated PAEC increased in a dose-dependent manner by a maximum of 44%, and was inhibited by mAbs against CD49d, CD11a, CD11b, CD18, and CD2, as well as porcine vascular cell adhesion molecules. In contrast, TNF-alpha treatment of PAEC reduced human NK lysis in a dose-dependent manner. Preincubation of TNF-a-treated PAEC with increasing concentrations of anti-SLA class I mAb increased NK lysis in a titer-dependent manner, and reversed the protective effect on human NK lysis by 77%. Treatment with IVIg, containing antibodies against an a-helical region of HLA class I molecules, had a similar effect. CONCLUSIONS: These results imply that SLA class I molecules can bind to inhibitory receptors on human NK cells, and that these interactions can be augmented by increasing the level of SLA class I molecule expression on porcine endothelium. Strategies that can increase porcine endothelial cell expression of either swine or human major histocompatibility complex class I molecules may reduce human NK activity against porcine xenografts.

High-level porcine endothelial cell expression of alpha(1,2)-fucosyltransferase reduces human monocyte adhesion and activation.

BACKGROUND: Monocyte binding to and activation by human endothelium requires a number of interactions, including those involving sialylated endothelial cell ligands. As porcine endothelial cell transfection with alpha(1,2)-fucosyltransferase has been shown to reduce terminal sialylation, we investigated whether high-level expression of alpha(1,2)-fucosyltransferase by porcine endothelium would reduce human monocyte adhesion and functional activation. METHOD: Purified human monocytes were labeled with 51Cr, and measured for adherence to human or porcine endothelial cell monolayers in the presence of either medium or monoclonal antibodies against monocyte lectins or sialylated endothelial cell ligands. Monocyte production of prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) was measured by enzyme-linked immunosorbent assay, using supernatants collected from cultures performed between human monocytes and human or porcine endothelial cell monolayers. Finally, monocyte adhesion and activation were measured after culture with a porcine endothelial cell line transfected with alpha(1,2)-fucosyltransferase, expressing reduced surface expression of terminal Gal alpha(1,3)-Gal and sialic acid residues. RESULTS: Human monocytes adhered by 50% higher levels to porcine endothelium than to human endothelium. This increased level of adherence was associated with augmented monocyte activation, as defined by 3.3-fold higher levels of PGE2 production and 7.3-fold higher levels of IL-1beta production. Monoclonal antibodies against CD62L (L-selectin) on monocytes or CD15s (sialylated Lewis X) on porcine endothelium reduced monocyte adhesion by 38% and 52%, respectively. Porcine endothelial cell transfection with alpha(1,2)-fucosyltransferase reduced terminal sialic acid expression by 65%, monocyte adherence by 50%, and the production of PGE2 and IL-1beta by 67% and 38%, respectively. CONCLUSIONS: Together, these results demonstrate that human monocytes use surface lectins to bind to sialylated carbohydrate structures on porcine endothelium, and indicate that reduction in porcine endothelial cell surface expression of terminally sialylated structures by high-level alpha(1,2)-fucosyltransferase activity reduces monocyte adherence and activation.

Short course single agent therapy with an LFA-3-IgG1 fusion protein prolongs primate cardiac allograft survival.

The interaction of T cell costimulatory molecules with their ligands is required for optimal T cell activation. Interference with such interactions can induce antigen unresponsiveness and delay xeno- and allograft rejection. We have previously shown that LFA3TIP, a soluble human lymphocyte function-associated antigen (LFA)-3 construct, binds CD2 and inhibits responses of human T cells in vitro. This study reports the first use of a human fusion protein, LFA3TIP, to significantly prolong primate cardiac allograft survival. Based on our observations that LFA3TIP inhibits baboon allogeneic mixed lymphocyte reactions, we gave baboon recipients of heterotopic cardiac allografts injections of LFA3TIP, 3 mg/kg i.v., for 12 consecutive days, starting 2 days before transplantation. This regimen delayed graft rejection from an average of 10.6 +/- 2.3 days for human IgG-treated controls (n = 5) to an average of 18.0 +/- 5.3 days for LFA3TIP-injected animals (n = 7; P < or = 0.01). Grafts from LFA3TIP-treated animals showed markedly diminished coronary endothelialitis as compared with control animals. LFA3TIP reached peak serum levels of approximately 100 micrograms/ml after 7-9 injections and persisted in the 10-micrograms/ml range for 1 to 2 weeks after the final injection. Despite these blood levels, circulating antibodies to LFA3TIP were not detected in the serum. No renal or hepatic toxicity was noted. The possible mechanism by which LFA3TIP acts to inhibit graft rejection is discussed; success in prolonging graft survival when LFA3TIP is used as a single-agent therapy suggests great potential for this novel therapeutic agent.

Lack of cross-species transmission of porcine endogenous retrovirus infection to nonhuman primate recipients of porcine cells, tissues, or organs.

BACKGROUND: Nonhuman primates (NHPs) have been widely used in different porcine xenograft procedures inevitably resulting in exposure to porcine endogenous retrovirus (PERV). Surveillance for PERV infection in these NHPs may provide information on the risks of cross-species transmission of PERV, particularly for recipients of vascularized organ xenografts for whom data from human clinical trials is unavailable. METHODS: We tested 21 Old World and 2 New World primates exposed to a variety of porcine xenografts for evidence of PERV infection. These NHPs included six baboon recipients of pig hearts, six bonnet macaque recipients of transgenic pig skin grafts, and nine rhesus macaque and two capuchin recipients of encapsulated pig islet cells. Serologic screening for PERV antibody was done by a validated Western blot assay, and molecular detection of PERV sequences in peripheral blood mononuclear cells (PBMCs) and plasma was performed using sensitive polymerase chain reaction and reverse transcriptase-polymerase chain reaction assays, respectively. Spleen and lymph node tissues available from six bonnet macaques and three rhesus macaques were also tested for PERV sequences. RESULTS: All plasma samples were negative for PERV RNA suggesting the absence of viremia in these xenografted animals. Similarly, PERV sequences were not detectable in any PBMC and tissue samples, arguing for the lack of latent infection of these compartments. In addition, all plasma samples were negative for PERV antibodies. CONCLUSION: These data suggest the absence of PERV infection in all 23 NHPs despite exposure to vascularized porcine organs or tissue xenografts and the use of immunosuppressive therapies in some animals. These findings suggest that PERV is not easily transmitted to these NHP species through these types of xenografts.

Role of natural killer cells, macrophages, and accessory molecule interactions in the rejection of pig-to-primate xenografts beyond the hyperacute period.

Pig-to-primate cardiac xenografts surviving beyond the period of hyperacute rejection succumb after 3-4 days to a secondary immunologic response characterized by xenograft infiltration with NK cells and macrophages. Circulating baboon mononuclear cells contain NK cell precursors which mediate lysis of porcine endothelium by two distinct mechanisms: antibody-dependent cellular cytotoxicity and lymphokine activation. IL-2 activated NK lysis of porcine endothelium was 2.4-fold stronger than lysis occurring following engagement of FcRIII by xenoreactive IgG. IL-2 augmented NK lysis involved interactions between CD2 and CD49d on baboon NK cells and their respective ligands on porcine endothelium, since NK lysis was reduced either by using Mabs against CD2, CD49d, or porcine VCAM, or by treating endothelial cells with PIPLC to cleave GPI-linked molecules. These results imply that interactions between accessory molecule receptor-ligand pairs on primate NK cells, macrophages and porcine endothelium are of critical importance in delayed xenograft rejection.

Miniature axial flow pump for ventricular assistance in children and small adults.

We investigated the efficacy of the Jarvik 2000 intraventricular assist device (Jarvik Research, Inc., New York, N.Y.) in an ovine model. The device is an axial flow pump measuring 1.8 cm in diameter by 5 cm long, has a displacement volume of 12 ml, and can deliver flow from 2 to 7 L/min. Seven devices were implanted through a left thoracotomy into the left ventricle with an outflow graft to the descending aorta. Animals were treated with warfarin sodium and aspirin to maintain prothrombin times approximately 1.5 times control. Animals were followed up for 3 to 123 days. Two animals died of operative complications at days 3 and 5. One device failed at 58 days because of thrombus formation at the inflow side of the impeller. The remaining four animals were killed at days 19, 42, 42, and 123, respectively, because of broken electric power cables. Hematocrit values rose significantly higher than preoperative levels (22.8% +/- 3.8% to 30.5% +/- 3.4%); premortem elevations of values higher than baseline values of plasma free hemoglobin (10.4 +/- 7.8 mg/dl to 17.1 +/- 7.4 mg/dl) and lactate dehydrogenase (391.5 +/- 113.7 units/L to 771.2 +/- 370.8 units/L) were statistically insignificant. Serum creatinine and bilirubin levels were normal. No end-organ dysfunction arising from long-term support was evident clinically or at postmortem examination, nor was there any evidence of embolism or damage to intracardiac structures. We found the Jarvik 2000 intraventricular assist device to be easily implantable, safe, nonhemolytic, and able to provide physiologic flow with power requirements under 10 watts.

Triple immunosuppression reduces mononuclear cell infiltration and prolongs graft life in pig-to-newborn baboon cardiac xenotransplantation.

OBJECTIVE: Pig hearts transplanted into unmedicated newborn baboons do not undergo hyperacute rejection by preformed xenoantibody and complement. These grafts are rejected at days 3 to 4 in association with the infiltration of macrophages and natural killer cells. We investigated whether an immunosuppressive regimen used widely in cardiac allotransplantation could reduce this cellular response and prolong xenograft life. METHODS: Ten newborn baboons underwent heterotopic pig cardiac xenotransplantation. Five baboons were immunosuppressed with mycophenolate mofetil (100 mg/kg), methylprednisolone acetate (0.8 mg/kg), and cyclosporine A (INN: ciclosporin; 10 mg/kg). Xenograft rejection was studied by light microscopy and immunofluorescence. The induced humoral response to porcine xenoantigens was documented by enzyme-linked immunosorbent assay using synthetic alpha-1,3-galactosyl epitopes coupled to bovine serum albumin. RESULTS: Graft life was extended from a mean of 3.6 +/- 0.5 days (n = 5) to a mean of 6.2 +/- 1.1 days (n = 5, p = 0.01). In comparison with controls, explanted grafts from medicated baboons demonstrated reduced infiltration with natural killer cells and macrophages, but increased evidence of complement-mediated rejection substantiated by increased deposition of immunoglobulin M, complement, and fibrin. In all baboons receiving transplants, levels of both immunoglobulin M and immunoglobulin G anti-galactose were significantly increased after transplantation, with immunoglobulin G levels remaining persistently elevated. CONCLUSIONS: These results indicate that cyclosporine-based triple immunosuppression marginally prolonged xenograft survival and appears to have reduced the natural killer cell and macrophage infiltrates. The immunosuppressive protocol, however, was not adequate to prevent the induced immunoglobulin M humoral response and prevent complement-mediated graft injury.

The influence of concordant xenografts on the humoral and cell-mediated immune responses to subsequent allografts in primates.

The humoral and cell-mediated immune responses to subsequent allografts were determined in primate recipients after concordant xenotransplantation as a bridge to allotransplantation. Heterotopic heart transplants (n = 4) were performed from cynomolgus monkeys into ABH type-matched olive baboons followed 2 weeks later by allotransplantation from ABH type-matched baboon donors. Allografts were explanted at 8 weeks. All recipients underwent splenectomy at the time of xenotransplantation and received immunosuppression with cyclosporine, azathioprine, and methylprednisolone. Concordant xenotransplantation in these primates did not induce humoral or cell-mediated immune responses that jeopardized subsequent allografts. The degree of xenospecific immune reactivity, as determined by specific cytotoxicity of recipient T-cell lines derived from the xenograft and extent of histologic xenograft rejection, did not predict the severity of subsequent allograft rejection. In two of the four recipients, xenotransplantation induced an alloreactive humoral response against antigens expressed by the B cells of more than 50% of members from a panel of 12 unrelated baboons. In all recipients, priming with xenogeneic splenocytes in vitro induced an accelerated proliferative T-cell response to allogeneic lymphocytes from 16% of this panel. This study affirms the role of concordant xenografts as appropriate biologic bridges to human allotransplantation. However, our results suggest that xenoreactive baboon memory CD4 T cells may recognize major histocompatibility complex class II--like structures shared between the xenogeneic and allogeneic targets. The potential allorecognition induced by a xenograft may affect the process of subsequent allograft donor selection.

Modified technique for heterotopic heart transplantation in small primates.

Cardiac transplantation in small primates represents a unique model for investigating both xenograft and allograft rejection. This report describes our technique for heterotopic transplantation of cardiac grafts into the retroperitoneal iliac vessels of newborn baboons and small primates. Small primates tolerate this position better than either cervical or abdominal placement.

Cerebral blood flow is determined by arterial pressure and not cardiopulmonary bypass flow rate.

BACKGROUND: During cardiopulmonary bypass, global hypoperfusion of the brain has been shown to result in ischemic insult and subsequent neurologic injury. Furthermore, outcome after focal cerebral ischemia depends on collateral circulation, which is determined by the parameters of global perfusion. We therefore measured cerebral blood flow during independent manipulations of arterial blood pressure and pump flow rate to determine which of these hemodynamic parameters regulates cerebral perfusion during cardiopulmonary bypass. METHODS: Seven anesthesized baboons were placed on cardiopulmonary bypass and cooled to 28 degrees C. Pump flow rate and arterial blood pressure were altered in varied sequence to each of four conditions: (1) full flow (2.23 +/- 0.06 L.min-1.m-2, mean +/- standard deviation) at high pressure (61 +/- 2 mm Hg), (2) full flow (2.23 +/- 0.06 L.min-1.m-2) at low pressure (24 +/- 3 mm Hg), (3) low flow (0.75 L.min-1.m-2) at high pressure (62 +/- 2 mm Hg), and (4) low flow (0.75 L.min-1.m-2 at low pressure (23 +/- 3 mm Hg). During each of these hemodynamic conditions cerebral blood flow was measured by washout of intracarotid xenon. RESULTS: Cerebral blood flow was greater at high blood pressure than at low pressure during cardiopulmonary bypass both at low flow (34 +/- 8.3 versus 14.1 +/- 3.7 mL.min-1 x 100 g-1) and full flow (27.6 +/- 9.9 versus 16.8 +/- 3.7 mL.min-1 x 100 g-1) (p < 0.01). At comparable mean arterial blood pressures alteration of pump flow rate produced no changes in cerebral blood flow. CONCLUSIONS: These results indicate that cerebral blood flow during moderately hypothermic cardiopulmonary bypass is regulated by arterial blood pressure and not pump flow rate.

Effects of DL-penicillamine on cytotoxic reaction between baboon performed xenoantibodies and pig endothelial cells.

The purpose of this study was to evaluate effects of DL-Penicillamine (DLP), a compound interrupting S-S bonds (IgM pentamers) on binding and cytotoxicity of adult baboon performed xenoantibodies to pig endothelial cells. Pooled baboon serum was treated with different concentrations of DLP during various periods of time. Complement-mediated cytotoxicity assay was used to determine the reactivity of baboon xenoantibodies to pig aortic endothelial cells (PAEC). To assess IgM and IgG binding to PAEC, ELISA method was applied. Serum treated with DLP revealed significant reduction of cytotoxicity in a dose dependent manner. Cytotoxicity was also reduced during time prolongation of DLP exposure to PAEC. Results indicate that baboon performed IgM and IgG xenoantibodies bind to pig endothelial cells, but only IgM is able to cause degradation of the complement. DLP significantly reduces cytotoxicity and eliminates binding of IgMs to PAEC in spite of continued binding of IgG xenoantibodies to the surface of endothelium.

Lysis of pig endothelium by IL-2 activated human natural killer cells is inhibited by swine and human major histocompatibility complex (MHC) class I gene products.

We have previously described a form of xenograft rejection, mediated by natural killer (NK) cells, occurring in pig-to-primate organ transplants beyond the period of antibody-mediated hyperacute rejection. In this study, two distinct NK activation pathways were identified as mechanisms of pig aortic endotheliual cell (PAEC) lysis by human NK cells. Using an antibody-dependent cellular cytotoxicity (ADCC) assay, a progressive increase in human NK lysis of PAEC was observed following incubation with human IgG at increasing serum titer. In the absence of IgG, a second mechanism of PAEC lysis by human NK cells was observed following activation with IL-2. IL-2 activation of human NK cells increased lysis of PAEC by over 3-fold compared with ADCC. These results indicate that IL-2 activation of human NK cells induces significantly higher levels of lytic activity than does conventional ADCC involving IgG and FcRIII. We next investigated the role of MHC class I molecules in the regulation of NK lysis following IL-2 activation. PAEC expression of SLA class I molecules was increased by up to 75% by treatment with human TNFa. Following treatment with TNFa at 1 u/ml, IL-2 activated human NK lysis of PAEC was inhibited at every effector:target (E:T) ratio tested. Maximal effect occurred at an E:T ratio of 10:1, with TNFa inhibiting specific lysis by 59% (p < 0.01). Incubation with an anti-SLA class I Mab, but not IgG isotype control, abrogated the protective effects of TNFa on NK lysis of PAEC, suggesting direct inhibitory effects of SLA class I molecules on human NK function. To investigate whether human MHC class I molecules might have similar effects on human NK lysis of PAEC, further experiments were performed using a soluble peptide derived from the alpha-helical region of HLA-B7. Incubation with the HLA-B7 derived peptide significantly reduced the IL-2 activated NK lytic activity against PAEC in a dose-dependent fashion. Maximal effect occurred at a concentration of 10 mg/ml, where an 8-fold reduction in IL-2 augmented NK lysis was observed (p < 0.01). These results suggest that IL-2 activated human NK lysis of porcine xenografts may be inhibited by strategies which increase PAEC expression of SLA class I molecules, introduce HLA class I genes into PAEC, or use soluble HLA class I peptides.