Characterization of a murine monoclonal antibodv specific for swine β1 integrin

Abstract: Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160–1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM 160–1 A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human β₁ integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine α₂ integrin. Therefore, the NaM 160–1 A3 antibody was directed against porcine β₁ integrin and allowed the purification of the complex α₂β₁ also termed Very Late Antigen 2 (VLA-2). It did not recognize human β₁ integrin.     

Less Platelet Damage in the Curved Vane Centrifugal Pump: A Comparative Study with the Roller Pump in Open Heart Surgery

Abstract: The centrifugal pump with the curved vane (Lifestream Centrifugal Pump [LCP]) was applied to cardiopulmonary bypass (CPB) in 10 patients who underwent elective coronary artery bypass grafting. Serum hemoglobin levels, platelet counts, and serum β–thromboglogulin (β–TG) levels were measured during CPB. The results were compared with those for a comparative roller pump (RP) group (n = 10). There was no difference in CPB time between LCP (112 ± 22 min) and RP (121 ± 22 min) groups. Serum β–TG levels (ng/ml) were lower in the LCP group than in the RP group (34 ± 9 vs. 101 ± 80, 5 min; 81 ± 33 vs. 236 ± 112, 30 min; 120 ± 53 vs. 314 ± 100, 60 min after initiation of CPB; p < 0. 05). There were no significant differences in hemolysis and platelet depletion. The LCP showed excellent hemodynamic performance with less blood trauma in clinical application to open heart surgery.     

Intravital microscopic investigation of xenogeneic microcirculation and impact of complement depletion by cobra venom factor

Abstract: Discordant xenografts are hyperacutly rejected within minutes. Disturbances in the microcirculation are considered to be the central mechanisms of hyperacute xenogeneic rejection (HXR). In this study intravital fluorescence microscopy was applied to investigate the dynamics of microcirculatory alterations in a setting in which HXR was inhibited by complement (C) depletion. Blood flow was measured as rat livers were perfused with isogeneic rat or xenogeneic human blood to assess the pattern of either physiological isogeneic hemoperfusion or in the course of HXR. Next, the complement system of the perfusate was inactivated by cobra venom factor (CVF) in order to inhibit HXR. Liver sinusoids of the isogeneic group were homogeneously perfused (sinusoidal perfusion rate 93.6 ± 0.3%), whereas in the xenogeneic group the sinusoidal perfusion rate dropped to 67.1 ± 3%. The perfusion in the periportal zone of an acinus was significantly lower (59.0 ± 3.3%) than in the pericentral zone (76.2 ± 3.1%). Treatment with CVF improved the sinusoidal perfusion to a value of 85.6 ± 2.3%, physiological perfusion, however could not be reached. In contrast to the isogeneic group, massive white blood cell (WBC) and platelet accumulation was found in the xenogeneic group, especially in the terminal portal vessels and in the penportal zone of liver acini. WBC and platelet counts show that the adherence of these cells appears rapidly in the first 5 min after reperfusion as firm adherence. CVF was not able to inhibit WBC and platelet accumulation, indicating that WBC endothelial interactions do not require an intact complement system. Bile flow, a parameter of liver function, decreased only slightly during isogeneic perfusion. The addition of CVF to the rat blood reduced the bile flow to one half of the untreated isogeneic flow, indicating a hepatotoxic side-effect of CVF. In xenogeneic perfusion the bile flow dropped to 62.6% and with the addition of CVF to 37.5% in the first 15 min after reperfusion. The bile flow of the CVF treated groups recovered during the perfusion but could not reach isogeneic values.     

Evaluation of Platelet Damage in Two Different Centrifugal Pumps Based on Measurements of α-Granule Packing Proteins

Abstract Mechanical trauma caused by centrifugal pumps is usually evaluated in terms of hemolysis. However, platelet damage caused by centrifugal pumps has not been studied well. We evaluated platelet damage in 2 different centrifugal pumps, the Medtronic BioMedicus BP-80 and the Terumo Capiox, in vitro and compared the results in terms of hemolysis. To evaluate platelet damage, the rate of increase (RI) for β -thromboglobulin (β-TG) and platelet factor-4 (PF-4) were measured by enzyme immunoassay. RI was defined as follows: RI for β-TG is Δβ-TG/ΔN and RI for PF-4 is Δ PF-4/ΔN where Δβ-TG is the increase in β-TG, ΔPF-4 is the increase in PF-4, and ΔN is the increase of the passing number, which is defined in the following equation: N = Q_r / V ( t , time; V , priming volume; Q , flow rate). Each pump was tested in a mock circuit for 3 h under a flow rate of 5 L/min and a pressure head of 100 mm Hg using fresh human heparinized blood (n = 5). For comparison, the normalized index of hemolysis (NIH) values were calculated for both pumps. The NIH values did not indicate a significant difference between the Capiox and the BP-80 pumps (Capiox vs. BP-80, 0.0021 ± 0.0004 vs. 0.0034 ± 0.0007, NS). However, the RI values for β-TG and PF-4 in the Capiox were significantly lower than in the BP-80 (β-TG, 0.198 ± 0.047 vs. 0.376 ± 0.049; PF-4, 0.080 ± 0.014 vs. 0.268 ± 0.043, p < 0.05). In conclusion, although there was no significant difference between the 2 pumps in terms of hemolysis, the Capiox centrifugal pump induced less platelet damage than the BP-80. The results suggest that measurements of RI for β-TG and PF-4 are more sensitive parameters than NIH values for evaluating blood cell damage.     

Protein Adsorption by Artificial Membrane Materials Under Filtration Conditions

Abstract: Elevated plasma levels of numerous low molecular weight proteins (LMWP) in renal insufficiency are likely to contribute to the uremic syndrome. Dialysis-related amyloidosis, caused by the accumulation of β₂-microglobulin (β₂M), has highlighted the need for a renal replacement therapy that allows the elimination of LMWP in addition to small solutes. Synthetic membrane materials employed under hemofiltration conditions proved to be most effective in lowering elevated β₂M plasma levels. In addition to convection, protein adsorption to artificial membrane materials is an important mechanism for β₂M removal. Using an in vitro setup, 12 commercially available hemofilters representing 11 different membrane materials were perfused with human blood containing ¹²⁵I-labeled plasma proteins. Under filtration conditions, total protein adsorption ranged from 338–2,098 mg/m² of membrane surface, and the fraction of adsorbed LMWP varied between 14–70% of total protein adsorption and was negatively correlated to total protein adsorption. β₂M adsorption showed up to an 8-fold difference between membranes, and was negatively correlated with total protein adsorption and positively correlated with the adsorption of LMWPs.     

Evacuation of microscopic air bubbles from Dacron reduces complement activation and platelet aggregation

Complement activation by biomaterials may play an important role in vascular graft failure since the physiologically active polypeptides, C3a and C5a, have several relevant properties. C3a promotes platelet aggregation and release, and C5a activates neutrophils, which may stimulate platelet aggregation by liberation of platelet activating factor or by a direct neutrophil platelet interaction. Microscopic air bubbles (nuclei) are found in the surface roughness or pores of most biomaterials, and their number and size are related to the surface tension of the material. Therefore two interfaces can be postulated to exist when Dacron is exposed to blood: (1) a blood/biomaterial, and (2) a blood/air interface. These air nuclei in the surface and the biomaterial itself are capable of activating complement. The purpose of these experiments was to eliminate these surface nuclei from Dacron by a process termed denucleation and subsequently to determine the effect of this intervention on complement activation and platelet aggregation in vitro. Dacron was denucleated by pretreatment that involved serial rinsing with ethanol and degassed buffer that results in replacement of the air nuclei by buffer. Both control and denucleated pieces of Dacron (2, 4, and 6 cm2) were then incubated in human plasma. Each plasma sample was assayed for complement activation products (C3a, C5a, and C4a) by means of radioimmunoassays, and the degree of autologous platelet aggregation that resulted from the addition of a portion of each incubated plasma sample to an autologous platelet suspension was measured. There was a significant reduction in C3a and C5a in the plasma samples incubated with denucleated Dacron as compared to control Dacron (p less than 0.001, analysis of variance [ANOVA]).(ABSTRACT TRUNCATED AT 250 WORDS)     

Monitoring of the Blood Response in Blood Purification

Abstract: A principal objective of monitoring the blood response in procedures such as hemodialysis and cardiopulmonary bypass is to achieve an enhanced understanding of the relationship between blood component alterations and the biomaterials employed. The aim in a study of blood-biomaterial interactions of deriving a correlation between a characteristic of the biomaterial and a representative parameter of the blood response can be influenced in a clinical situation by antithrombotic agents, multimaterial contact, device utilization, blood condition, drug therapy, and the nature of the application. The selection of parameters representative of the blood response may require a compromise between the advantages of multiparameter assessment and the benefit of measuring a single parameter by a consistent methodology. Representative parameters are protein adsorption, platelet reactions, intrinsic coagulation and the contact activation phase, fibrinolysis, leukocyte alterations, and complement activation. Assessment during clinical application can be approached by consideration of blood response patterns.     

Raman spectroscopic study of the repair of surgical bone defects grafted or not with biphasic synthetic micro-granular HA + β-calcium triphosphate irradiated or not with λ850 nm LED light

The handling of bone losses due to different etiologic factors is difficult and many techniques are aim to improve repair, including a wide range of biomaterials and, recently, photobioengineering. This work aimed to assess, through Raman spectroscopy, the level of bone mineralization using the intensities of the Raman peaks of both inorganic (~960, ~1,070, and 1,077 cm^?1) and organic (~1,454 and ~1,666 cm^?1) contents of bone tissue. Forty rats were divided into four groups each subdivided into two subgroups according to the time of sacrifice (15 and 30 days). Surgical bone defects were made on the femur of each animal with a trephine drill. On animals of group clot, the defect was filled only by blood clot, on group LED, the defect filled with the clot was further irradiated. On animals of groups biomaterial and LED + biomaterial, the defect was filled by biomaterial and the last one was further irradiated (λ850?±?10 nm, 150 mW, Φ?~?0.5 cm², 20 J/cm²-session, 140 J/cm²-treatment) at 48-h intervals and repeated for 2 weeks. At both 15th and 30th days following sacrifice, samples were taken and analyzed by Raman spectroscopy. At the end of the experimental time, the intensity of hydroxyapatite (HA) (~960 cm^?1) were higher on group LED + biomaterial and the peaks of both organic content (~1,454 and ~1,666 cm^?1) and transitional HA (~1,070 and ~1,077 cm^?1) were lower on the same group. It is concluded that the use of LED phototherapy associated to biomaterial was effective in improving bone healing on bone defects as a result of the increasing deposition of HA measured by Raman spectroscopy.     

Plasma‐based biomaterials for the treatment of cutaneous radiation injury

Cutaneous wounds caused by an exposure to high doses of ionizing radiation remain a therapeutic challenge. While new experimental strategies for treatment are being developed, there are currently no off‐the‐shelf therapies for the treatment of cutaneous radiation injury that have been proven to promote repair of the damaged tissues. Plasma‐based biomaterials are biologically active biomaterials made from platelet enriched plasma, which can be made into both solid and semi‐solid forms, are inexpensive, and are available as off‐the‐shelf, nonrefrigerated products. In this study, the use of plasma‐based biomaterials for the mitigation of acute and late toxicity for cutaneous radiation injury was investigated using a mouse model. A 2‐cm diameter circle of the dorsal skin was irradiated with a single dose of 35 Gy followed by topical treatment with plasma‐based biomaterial or vehicle once daily for 5 weeks postirradiation. Weekly imaging demonstrated more complete wound resolution in the plasma‐based biomaterial vs. vehicle group which became statistically significant (p < 0.05) at weeks 12, 13, and 14 postmaximum wound area. Despite more complete wound healing, at 9 and 17 weeks postirradiation, there was no statistically significant difference in collagen deposition or skin thickness between the plasma‐based biomaterial and vehicle groups based on Masson trichrome staining nor was there a statistically significant difference in inflammatory or fibrosis‐related gene expression between the groups. Although significant improvement was not observed for late toxicity, plasma‐based biomaterials were effective at promoting wound closure, thus helping to mitigate acute toxicity.     

Influence of Blood Flow on Adsorption of β2-Microglobulin onto AN69 Membrane

Abstract: Adsorption onto the dialyzer membrane is a contributing factor to the elimination of β₂-microglobulin (β₂M) from the sera of uremic patients. The purpose of this prospective study was to ascertain the influence of the blood flow rate on adsorption of β₂M onto the polyacrylonitrile (AN69) hollow-fiber dialyzer membrane in 8 pa tients during regular hemodialysis (HD). Blood first passed through a low-flux polysulfone dialyzer and then through an AN69 dialyzer, which was not in contact with the dialysis fluid. During the investigation period (first hour of the HD session), the blood flow rate was 100 ml/min (first part of the study), 200 mumin (second part of the study), and 300 ml/min (third part of the study). Ultrafiltration was not performed during the investigation period. At the start of the HD sessions, the serum concentration of β₂M in the afferent blood line did not differ significantly among the 3 parts of the study. Serum β₂M was measured in samples taken from the afferent and efferent blood lines of the AN69 dialyzer at 5,10, 15, 30, 45, and 60 min. The serum β₂M concentration decreased significantly in blood that had passed through the AN69 dialyzer. This decrease, indicating membrane adsorption, was maximal during the first part and minimal during the third part of study. The decrease in the contact time between the blood and the AN69 could be the underlying cause. The calculated quantities of β₂M adsorbed onto the AN69 membrane (44.2 ± 10.2, 43.2 ± 12.1, and 42.6 ± 17.3 mg) did not differ significantly among the 3 parts of the study. These results suggest that an increase in blood flow rate from 100 to 300 ml/min did not significantly affect the quantity of β₂M adsorbed onto the AN69 membrane.     

Blocking of human anti-pig xenoantibodies by soluble GALα1–3GAL and Galα1–2GAL 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 αGal 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 Galα1–3GAL, Galα1–2Gal 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 Galα1–3Gal (155 ± 31 ml/min × 100 g^–1 kidney tissue) group compared to Galα1–2Gal (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 Galα1–3Gal and for 187 % the Galα1–2Gal. 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 × 100 g^–1 kidney tissue) compared to Galα1–3Gal (3.0) and Galα1–2Gal (3.7). Urine sodium excretion was reduced in the lactose/control groups, compared to the Galαα1- groups during the perfusions. An increase in urine potassium excretion was found in the Galαα1-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 Galαα1-saccharide groups compared to the lactose/control groups. Soluble Galαa1-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. Received: 19 August 1999 Revised: 3 May 2000 Accepted: 10 August 2000     

The Placental Transfer of Pancuronium and its Pharmacokinetics During Caesarian Section

The placental transfer of pancuronium and its pharmacokinetics were investigated in 33 pregnant women undergoing caesarean section. After a single intravenous injection of 60–100 μ g/kg , the serum pancuronium concentration was measured (using a fluorimetric method) in serial samples of maternal blood and in umbilical arterial and venous blood obtained at the time of delivery. Detectable amounts of pancuronium were found in all foetal blood samples. The cord vein to maternal concentration ratio of pancuronium averaged 0.22; it increased significantly with prolongation of the induction-delivery interval. The mean cord arterial to cord venous ratio of pancuronium was 0.66 (range 0.2–1.0), suggesting foetal drug uptake. Apgar scores were above 9 at 5 min in all cases, and there was no clinical evidence of any adverse effect in the newborns which could be related to the presence of a muscle relaxant.
The distribution and elimination of pancuronium were compared in 18 normal subjects and in 21 women undergoing caesarean section. Plasma pancuronium concentration declined biexponentially with fast (α) and slow ( β ) phases, with half-lives in the control group of 13 min and 146 min (mean values), respectively. In the women undergoing caesarean section, t 1/2 α was not changed, while t 1/2 β decreased slightly but significantly to 114 min.     

17β-Oestradiol Concentration in Spermatic Venous Blood with Increasing Age

Androstenedione, testosterone and 17β-oestradiol were measured in spermatic venous blood of 25 subjects (age range 20–70).
Blood samples were taken during surgical intervention for hernia repair. While androstenedione and testosterone decreased significantly in spermatic venous blood with advancing age, 17β-oestradiol was unchanged in the same subjects.
These results seem to demonstrate that in senescence the increase of 17β-oestradiol in systemic blood is not due to an increased secretion of this steroid by the human testis, but that it may be due to an increased peripheral conversion of the aromatizable androgens and to a reduction of the metabolic clearance rate of 17β-oestradiol.     

Intraarterial Perfusion of the Hindlimb with Pyridoxalated Hemoglobin Polyoxyethylene Conjugate Solution in Anesthetized Dogs

Abstract: To evaluate the potential clinical usefulness of a modified hemoglobin, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), the hindlimb vascular bed was perfused with PHP solution while monitoring tissue oxygen tension (Pto₂) in anesthetized dogs. The hindlimb region was perfused through the external iliac artery with a roller pump at a varying perfusion rate. Pto₂ was measured using a Po₂-monitoring probe inserted into the gra-cial muscle. After surgical preparation for perfusion, the iliac arterial flow rate was 19.9 ± 5.6 ml/min, and baseline Pto₂ was 38.4 ±1.3 mm Hg. Perfusion with autologous arterial blood with the pump increased Pto₂ and perfusion pressure (PP) in a perfusion rate-dependent manner. Perfusion with PHP solution at 20 ml/min decreased Pto₂ from the initial baseline level, but an increase in the flow rate to 40–55 ml/min restored or induced an elevation of Pto₂. Results demonstrated that PHP solution can deliver oxygen to local tissue and maintain tissue oxygen tension at the same level as autologous arterial blood at a high enough flow rate.     

Lung uptake of lidocaine during hyperoxia and hypoxia in the dog

Background: Lidocaine has been shown to accumulate in the lung following its administration. This study was undertaken to determine effects of dose of lidocaine on lung uptake during hyperoxic and hypoxic ventilation.
Methods: Using cross-circulation consisting of ventilation and constant-flow perfusion of the left lower lobe independently from all other lobes of the dog lung under nitrous oxide and halolhane anesthesia, lidocaine was infused into the inflow system, so that plasma lidocaine concentrations in the inflow blood were maintained at 5, 10, 20, 40 and 70 μg/ml respectively during ventilation with 50% O₂ or 3% O₂. During 20 μg/ml lidocaine infusion, indocyanine green (ICG), an intravascular marker, was mixed with the lidocaine solution, in such a fashion that plasma ICG concentration in the inflow blood was maintained at 20 μg/ml. Actual plasma lidocaine and ICG concentrations in blood drawn from the inflow ([Lid]pa, |ICG]pa)and the outflow ([Lid]pv, [ICG]pv) systems were measured 1, 3, 5, 7 and 10 minutes after the beginning of lidocaine infusion. Percent lung uptake of perfused lidocaine was calculated as (1-([Lid]pv/[Lid]pa)/([ICG]pv/[ICG]pa)| x100.
Results: During ventilation hyperoxia, mean percent lung uptakes of lidocaine were 41–52% 1 minute after the beginning of lidocaine infusion, and decreased in time-dependent fashion to 7–12% 10 minutes later. Curves of percent lung uptake of lidocaine over time were similar for the 5 predetermined lidocaine concentration groups (5–70 μg/ml). There were no significant differences in percent lung uptakes of lidocaine between the ventilation hyperoxia and hypoxia conditions.
Conclusions: These findings suggest that percent lung uptake of lidocaine is unaffected by hypoxic ventilation and by varying the concentration of lidocaine in the perfusion through the recipient dog lung lobe.     

Risk Factors for Infection with Extended-Spectrum and AmpC β-Lactamase-Producing Gram-Negative Rods in Renal Transplantation

Increasing prevalence of infections caused by multi-resistant gram-negative enteric bacilli due to synthesis of extended-spectrum β-lactamase (ESBL) or to desrepressed chromosomic AmpC β-lactamase (AmpC) is a major concern in the hospitalized patient population . Renal transplant recipients are especially susceptible to these infections. A cohort observational study in a 3-year period was performed. ESBL-production was determined by phenotypic analysis based on the CLSI recommendations. A multi-variate logistic regression analysis was performed to identify independent variables associated with multi-resistant gram-negative bacilli infection. The study included 417 patients (61 double kidney-pancreas recipients). The incidence of ESBL-producing and desrepressed chromosomic AmpC β-lactamase resistance was 11.8% (49 patients). The most frequent bacteria isolated was E. coli (35/60 isolations), followed by Klebsiella spp (12/60 isolations). Double kidney-pancreas transplantation (OR 3.5, CI95% 1.6–7.8), previous use of antibiotics (OR 2.1,CI95% 1.1–4.1), posttransplant dialysis requirement (OR 3.1, CI95% 1.5–6.4) and posttransplant urinary obstruction (OR 5.8, CI95% 2.2–14.9) were independent variables associated with these multi-resistant gram-negative enteric bacilli infections. The incidence of ESBL-producing and desrepressed AmpC β-lactamase gram-negative enteric bacilli infection in our population was high. These infections are associated with significant morbidity after renal transplantation.     

Bupivacaine for Intercostal Nerve Blockade in Patients on Long-term β-Receptor Blocking Therapy

Possible cardiovascular side effects of a local anaesthetic in patients on long-term β-receptor blocking therapy were studied in 26 patients given postoperative intercostal nerve blockades (ICB) with 18–28 ml of plain bupivacaine 0.5% (1.30-1.82 mg kg^-1). The patients had a history of hypertension and/or ischaemic heart disease and were scheduled for gall bladder surgery. Thirteen patients were randomized to a gradual preoperative withdrawal of the β-receptor blockers and the other 13 continued the β-receptor blockade until surgery. Cardiovascular changes were measured noninvasively and 11 patients were also monitored with pulmonary artery catheters. Blood pressure and heart rate (HR) were stable in all patients although those in whom the β-receptor blockade was withdrawn had the highest HR and most frequent arrhythmias both before and after ICB. The ICB was associated with a decrease in the overall postoperative arrhythmia incidence, but seemed most efficient ( P <0.02) concerning the ventricular arrhythmias in the β-receptor-blocked patients (even including idionodal rhythm). The bupivacaine blood levels did not modify other cardiovascular changes except in one β-receptor-blocked patient with cardiac failure in whom signs of a slight transient cardiodepression were observed. It is concluded that bupivacaine does not negatively affect cardiovascular stability in long-term β-receptor-blocked patients. In the presence of cardiac failure, however, an additive cardiodepression may be elicited.     

Inhibition of platelet GPIIbIa in an ex vivo model of hyperacute xenograft rejection does not prolong cardiac survival time

Abstract: Discordant cardiac xenografts are rapidly rejected in a process characterized by platelet activation with microvascular thrombosis, termed hyperacute rejection (HAR). The fibrinogen receptor GPIIbIIIa is crucial for the formation of platelet aggregates and potentiates platelet adhesion to subendothelial matrix. We have studied the effects of a specific GPIIbIIIa antagonist (GPI 562) in an ex vivo working heart model using discordant porcine hearts perfused with fresh, heparinized human blood. Stable plasma GPI 562 inhibitory levels were confirmed by inhibition of human platelet aggregation in vitro. Biopsies from the left ventricle of rejected hearts were analyzed by immunopathology. Control porcine hearts ( n =8) underwent HAR and ceased functioning at around 60 min. Hearts perfused with human blood containing GPI 562 at 0.5 μM ( n =5) appeared to show an initial increase in coronary blood flow relative to controls, but neither this difference nor survival times of the hearts reached significance. Mean cardiac output values were 7.3 ml/g (SEM 2.5) in the experimental group and 5 ml/g (SEM 0.6) in the control group following 5 min of working mode and were comparable at other timepoints. Platelet counts in the perfusate were maintained in the presence of GPI 562, unlike the reduction of over 50% in control samples. Immunohistochemistry suggested decreased platelet vascular plugging as determined by P-selectin staining in the GPI 562 group, with associated reduction in neutrophil adherence and fibrin deposition. The use of GPI 562 in this ex vivo model conferred no marked benefits with respect to cardiac function and explant survival despite some positive differences on histological comparison. Further studies of this agent, in association with modalities of complement inhibition, are warranted in other models of discordant xenograft rejection.     

HDAF transgenic pig livers are protected from hyperacute rejection during ex vivo perfusion with human blood

The aim of this study was to determine if human decay-accelerating factor (hDAF) protects against hyperacute rejection in an ex vivo liver perfusion system using human blood. Pig livers were perfused ex vivo via the portal vein for an average of 5–6 h using a membrane oxygenator. Three groups were studied. Group I: Wild-type pig livers were alloperfused with fresh pig blood (n=5). Group II: Wild-type pig livers were xenoperfused with fresh human blood (n=5). Group III: hDAF transgenic pig livers were xenoperfused with fresh human blood (n=5). The graft ischemic time, ratio of perfusate volume to liver weight, flow rate, and perfusate hematocrit were similar in each group. The hDAF livers perfused with human blood (Group III) had a lower ALT level, less protein and albumin losses, lower bilirubin levels in the perfusate, less weight gain, and greater bile production than the wild-type livers perfused with human blood. Histology showed classic features of hyperacute rejection in Group II, including massive hemorrhage, severe vasculitits, fibrin and C5b-9 deposition, and endothelial damage within 1 h of perfusion, whereas liver histology studies in Groups I and III were near normal. IgG and IgM deposits were seen in the xenoperfused livers. Electron microscopy (EM) and immuno-EM showed loss of endothelial cells, trapping of white blood cells and platelets, and diffuse fibrin deposits in Group II only. hDAF pig livers perfused with human blood showed superior function and histology when compared with wild-type pig livers. These data suggest that (1) hyperacute rejection may contribute to the inconsistent results using wild-type pig livers for extracorporeal liver support and (2) genetically modified pigs that express hDAF may provide a better donor source than wild-type pigs for extracorporeal liver support.     

Histochemical Demonstration of Δ5,3β-hydroxysteroid Dehydrogenase Activity in Cultured Leydig Cells under the Influence of Gonadotropic Hormones and Testosterone

The influence of gonadotropic hormones or testosterone upon activity of Δ⁵, 3β-hydroxysteroid dehydrogenase (Δ⁵,3β-OH-SDH) within cultured Leydig cells was histochemically investigated.
The following amounts of hormones were added into the culture medium: LH – 10, 100, 500, and 1000 ng/ml, FSH – 100 ng/ml, Testosterone (T) – 150 ng/ml of culture medium. Doses 10 and 100 ng LH stimulated the activity of the histochemically investigated enzyme, while 500 ng decreased enzyme activity and 1000 ng failed to support culture at all. FSH did not exert any influence on enzyme activity of cultured cells while testosterone decreased its activity beginning on day 6 in culture.