Pawel Kwiatkowski

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.

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.

A 2.9 ps equivalent resolution interpolating time counter based on multiple independent coding lines

We present the design, operation and test results of a time counter that has an equivalent resolution of 2.9 ps, a measurement uncertainty at the level of 6 ps, and a measurement range of 10 s. The time counter has been implemented in a general-purpose reprogrammable device Spartan-6 (Xilinx). To obtain both high precision and wide measurement range the counting of periods of a reference clock is combined with a two-stage interpolation within a single period of the clock signal. The interpolation involves a four-phase clock in the first interpolation stage (FIS) and an equivalent coding line (ECL) in the second interpolation stage (SIS). The ECL is created as a compound of independent discrete time coding lines (TCL). The number of TCLs used to create the virtual ECL has an effect on its resolution. We tested ECLs made from up to 16 TCLs, but the idea may be extended to a larger number of lines. In the presented time counter the coarse resolution of the counting method equal to 2 ns (period of the 500 MHz reference clock) is firstly improved fourfold in the FIS and next even more than 400 times in the SIS. The proposed solution allows us to overcome the technological limitation in achievable resolution and improve the precision of conversion of integrated interpolators based on tapped delay lines.

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.

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.

Oxygen and oxygenation in stem-cell therapy for myocardial infarction.

Myocardial infarction (MI) is caused by deprivation of oxygen and nutrients to the cardiac tissue due to blockade of coronary artery. It is a major contributor to chronic heart disease, a leading cause of mortality in the modern world. Oxygen is required to meet the constant energy demands for heart contractility, and also plays an important role in the regulation of heart function. However, reoxygenation of the ischemic myocardium upon restoration of blood flow may lead to further injury. Controlled oxygen delivery during reperfusion has been advocated to prevent this consequence. Monitoring the myocardial oxygen concentration would play a vital role in understanding the pathological changes in the ischemic heart following myocardial infarction. During the last two decades, several new techniques have become available to monitor myocardial oxygen concentration in vivo. Electron paramagnetic resonance (EPR) oximetry would appear to be the most promising and reliable of these techniques. EPR utilizes crystalline probes which yield a single sharp line, the width of which is highly sensitive to oxygen tension. Decreased oxygen tension results in a sharpening of the EPR spectrum, while an increase results in widening. In our recent studies, we have used EPR oximetry as a valuable tool to monitor myocardial oxygenation for several applications like ischemia-reperfusion injury, stem-cell therapy and hyperbaric oxygen therapy. The results obtained from these studies have demonstrated the importance of tissue oxygen in the application of stem-cell therapy to treat ischemic heart tissues. These results have been summarized in this review article.

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.

Phenylephrine increases cerebral blood flow during low-flow hypothermic cardiopulmonary bypass in baboons

Background Although low‐flow cardiopulmonary bypass (CPB) has become a preferred technique for the surgical repair of complex cardiac lesions in children, the relative hypotension and decrease in cerebral blood flow (CBF) associated with low flow may contribute to the occurrence of postoperative neurologic injury. Therefore, it was determined whether phenylephrine administered to increase arterial blood pressure during low‐flow CPB increases CBF. Methods Cardiopulmonary bypass was initiated in seven baboons during fentanyl, midazolam, and isoflurane anesthesia. Animals were cooled at a pump flow rate of 2.5 l *symbol* min‐1 *symbol* m‐2 until esophageal temperature decreased to 20 degrees C. Cardiopulmonary bypass flow was then reduced to 0.5 l *symbol* min‐1 *symbol* m‐2 (low flow). During low‐flow CPB, arterial partial pressure of carbon dioxide (PCO2) and blood pressure were varied in random sequence to three conditions: (1) PCO2 30–39 mmHg (uncorrected for temperature), control blood pressure; (2) PCO2 50–60 mmHg, control blood pressure; and (3) PCO2 30–39 mmHg, blood pressure raised to twice control by phenylephrine infusion. Thereafter, CPB flow was increased to 2.5 l *symbol* min‐1 *symbol* m‐2, and baboons were rewarmed to normal temperature. Cerebral blood flow was measured by washout of intraarterial133 Xenon before and during CPB. Results Phenylephrine administered to increase mean blood pressure from 23+/‐3 to 46+/‐3 mmHg during low‐flow CPB increased CBF from 14+/‐3 to 31+/‐9 ml *symbol* min‐1 *symbol* 100 g‐1, P < 0.05. Changes in arterial PCO2 alone during low flow bypass produced no changes in CBF. Conclusion Although low‐flow CPB resulted in a marked decrease in CBF compared with prebypass and full‐flow bypass, phenylephrine administered to double arterial pressure during low‐flow bypass produced a proportional increase in CBF.

Measurement of oxygenation at the site of stem cell therapy in a murine model of myocardial infarction.

We have developed a noninvasive EPR (electron paramagnetic resonance) oximetry, based on a new class of oxygen-sensing nano-particulate probe (LiNc-BuO), for simultaneous monitoring of stem-cell therapy and in situ oxygenation (partial pressure of oxygen, pO2) in a mouse model of acute myocardial infarction (AMI). AMI was induced by a permanent occlusion of left-anterior-descending (LAD) coronary artery. Skeletal myoblast (SM) cells were used for therapy. The oximetry probe was implanted in the mid-ventricular region using a needle. Tissue histological studies after 3 weeks of implantation of the probe revealed significant fibrosis, which was solely due to the needle track and not due to the probe particles. The feasibility of long-term monitoring of pO2 was established in control (non-infarct) group of hearts (> 3 months; pO2 = 15.0 +/- 1.2 mmHg,). A mixture of the probe with/without SM cells (1 x 10(5)) was implanted as a single injection in the infarcted region and the myocardial tissue pO2 at the site of cell therapy was measured for 4 weeks. The pO2 was significantly higher in infarcted hearts treated with SM cells (pO2 = 3.5 +/- 0.9 mmHg) compared to untreated hearts (pO2 = 1.6 +/- 0.7 mmHg). We have demonstrated, for the first time, the feasibility of monitoring pO2 in mouse hearts after stem cell therapy.

Accurate and low jitter time-interval generators based on phase shifting method.

The paper describes two time-interval generators based on the phase shifting method. The first one utilizes the digital clock manager units integrated in a field programmable gate array (FPGA) device and has jitter below 65 ps (rms) over the range of 4 ns-50 ms, while the second one utilizes a separate direct digital synthesizer and has jitter below 15 ps (rms) over the range of 10.2 ns-50 ms. The phase shifting method can be used to design new low-cost and high-precision time-interval generators using the popular FPGA technology.