Introduction to Currently Applied Device Pathology.

Pathologic evaluation is crucial to the study of medical devices and integral to the Food and Drug Administration and other regulatory entities' assessment of device safety and efficacy. While pathologic analysis is tailored to the type of device, it generally involves at a minimum gross and microscopic evaluation of the medical device and associated tissues. Due to the complex nature of some implanted devices and specific questions posed by sponsors, pathologic evaluation inherently presents many challenges in accurately assessing medical device safety and efficacy. This laboratory's experience in numerous collaborative projects involving veterinary pathologists, biomedical engineers, physicians, and other scientists has led to a set of interrelated assessments to determine pathologic end points as a means to address these challenges and achieve study outcomes. Thorough device evaluation is often accomplished by utilizing traditional paraffin histology, plastic embedding and microground sections, and advanced imaging modalities. Combining these advanced techniques provides an integrative, comprehensive approach to medical device pathology and enhances medical device safety and efficacy assessment.

A self-cleaning, mechanically robust membrane for minimizing the foreign body reaction: towards extending the lifetime of sub-Q glucose biosensors.

Long-term, subcutaneously implanted continuous glucose biosensors have the potential to improve diabetes management and reduce associated complications. However, the innate foreign body reaction (FBR) both alters the local glucose concentrations in the surrounding tissues and compromises glucose diffusion to the biosensor due to the recruitment of high-metabolizing inflammatory cells and the formation of a dense, collagenous fibrous capsule. Minimizing the FBR has mainly focused on "passively antifouling" materials that reduce initial cellular attachment, including poly(ethylene glycol) (PEG). Instead, the membrane reported herein utilizes an "actively antifouling" or "self-cleaning" mechanism to inhibit cellular attachment through continuous, cyclic deswelling/reswelling in response to normal temperature fluctuations of the subcutaneous tissue. This thermoresponsive double network (DN) membrane is based on N-isopropylacrylamide (NIPAAm) and 2-acrylamido-2-methylpropane sulfonic acid (AMPS) (75:25 and 100:0 NIPAAm:AMPS in the 1st and 2nd networks, respectively; "DN-25%"). The extent of the FBR reaction of a subcutaneously implanted DN-25% cylindrical membrane was evaluated in rodents in parallel with a PEG-diacrylate (PEG-DA) hydrogel as an established benchmark biocompatible control. Notably, the DN-25% implants were more than 25× stronger and tougher than the PEG-DA implants while maintaining a modulus near that of subcutaneous tissue. From examining the FBR at 7, 30 and 90 days after implantation, the thermoresponsive DN-25% implants demonstrated a rapid healing response and a minimal fibrous capsule (~20-25 µm), similar to the PEG-DA implants. Thus, the dynamic self-cleaning mechanism of the DN-25% membranes represents a new approach to limit the FBR while achieving the durability necessary for long-term implantable glucose biosensors.

Cardiotoxicity of the cancer therapeutic agent imatinib mesylate.

Imatinib mesylate (Gleevec) is a small-molecule inhibitor of the fusion protein Bcr-Abl, the causal agent in chronic myelogenous leukemia. Here we report ten individuals who developed severe congestive heart failure while on imatinib and we show that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib show mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib.

Diffusible contrast-enhanced micro-CT improves visualization of stented vessels.

In this report, we showcase diffusible iodine-based contrast-enhanced computed tomography (DICE-CT) as a method for improving soft tissue visualization and reducing beam hardening artifact within a stented vessel. This technique is commonly used in our pathology lab to image soft tissue specimens with dense metal implants and to ensure reliable morphological analysis through clear delineation of tissue structures. For this report, a porcine right coronary artery with an implanted metal stent was scanned using both conventional and DICE-CT methods. Upon reconstruction, DICE-CT produced less beam hardening artifact in comparison to traditional micro-CT; furthermore, DICE-CT produced results with morphometric similarity to histology. Accordingly, these differences illustrated the clear advantage of using DICE-CT over conventional micro-CT when imaging soft tissue specimens with dense metal implants.

Image-Based Evaluation of In Vivo Degradation for Shape-Memory Polymer Polyurethane Foam.

Shape-memory polymer (SMP) polyurethane foams have been applied as embolic devices and implanted in multiple animal models. These materials are oxidatively degradable and it is critical to quantify and characterize the degradation for biocompatibility assessments. An image-based method using high-resolution and magnification scans of histology sections was used to estimate the mass loss of the peripheral and neurovascular embolization devices (PED, NED). Detailed analysis of foam microarchitecture (i.e., struts and membranes) was used to estimate total relative mass loss over time. PED foams implanted in porcine arteries showed a degradation rate of ~0.11% per day as evaluated at 30-, 60-, and 90-day explant timepoints. NED foams implanted in rabbit carotid elastase aneurysms showed a markedly faster rate of degradation at ~1.01% per day, with a clear difference in overall degradation between 30- and 90-day explants. Overall, membranes degraded faster than the struts. NEDs use more hydrophobic foam with a smaller pore size (~150-400 µm) compared to PED foams (~800-1200 µm). Previous in vitro studies indicated differences in the degradation of the two polymer systems, but not to the magnitude seen in vivo. Implant location, animal species, and local tissue health are among the hypothesized reasons for different degradation rates.

Increased artery wall stress post-stenting leads to greater intimal thickening.

Since the first human procedure in the late 1980s, vascular stent implantation has been accepted as a standard form of treatment of atherosclerosis. Despite their tremendous success, these medical devices are not without their problems, as excessive neointimal hyperplasia can result in the formation of a new blockage (restenosis). Clinical data suggest that stent design is a key factor in the development of restenosis. Additionally, computational studies indicate that the biomechanical environment is strongly dependent on the geometrical configuration of the stent, and, therefore, possibly involved in the development of restenosis. We hypothesize that stents that induce higher stresses on the artery wall lead to a more aggressive pathobiologic response, as determined by the amount of neointimal hyperplasia. The aim of this investigation was to examine the role of solid biomechanics in the development of restenosis. A combination of computational modeling techniques and in vivo analysis were employed to investigate the pathobiologic response to two stent designs that impose greater or lesser levels of stress on the artery wall. Stent designs were implanted in a porcine model (pigs) for approximately 28 days and novel integrative pathology techniques (quantitative micro-computed tomography, histomorphometry) were utilized to quantify the pathobiologic response. Concomitantly, computational methods were used to quantify the mechanical loads that the two stents place on the artery. Results reveal a strong correlation between the computed stress values induced on the artery wall and the pathobiologic response; the stent that subjected the artery to the higher stresses had significantly more neointimal thickening at stent struts (high-stress stent: 0.197±0.020 mm vs low-stress stent: 0.071±0.016 mm). Therefore, we conclude that the pathobiologic differences are a direct result of the solid biomechanical environment, confirming the hypothesis that stents that impose higher wall stresses will provoke a more aggressive pathobiological response.

Resolution of a proteinuric nephropathy associated with Babesia gibsoni infection in a dog.

A 4 yr old male castrated Labrador retriever was evaluated for a short history of inappetance, lethargy, small-bowel diarrhea, polyuria, and polydipsia. Clinicopathologic abnormalities were consistent with protein-losing nephropathy and renal azotemia. Expansive infectious disease testing implicated Babesia gibsoni via whole blood polymerase chain reaction. Renal histopathology results were consistent with membranoproliferative glomerulonephritis and immune complex deposition. The dog was treated with azithromycin, atovaquone, and one dose of corticosteroids/cyclophosphamide. Three months after therapy was completed, the dog was clinically healthy, and all clinicopathologic abnormalities (including Babesia species polymerase chain reaction) had resolved. Atypical presentations of Babesia gibsoni should be considered with proteinuric nephropathy.

Delivery of stem cells to porcine arterial wall with echogenic liposomes conjugated to antibodies against CD34 and intercellular adhesion molecule-1.

In atherosclerosis, the loss of vascular stem cells via apoptosis impairs the capacity of the vascular wall to repair or regenerate the tissue damaged by atherogenic factors. Recruitment of exogenous stem cells to the plaque tissue may repopulate vascular cells and help repair the arterial tissue. Ultrasound-enhanced liposomal targeting may provide a feasible method for stem cell delivery into atheroma. Bifunctional echogenic immunoliposomes (BF-ELIP) were generated by covalently coupling two antibodies to liposomes; the first one specific for CD34 antigens on the surface of stem cells and the second directed against the intercellular adhesion molecule-1 (ICAM-1) antigens on the inflammatory endothelium covering atheroma. CD34+ stem cells from adult bone marrow were incubated on the ICAM-1-expressing endothelium of the aorta of swine fed high cholesterol diets, which was preloaded with BF-ELIP. Significantly increased stem cell adherence and penetration were detected in particular in the aortic segments treated with 1 MHz low-amplitude continuous wave ultrasound. Fluorescence and scanning electron microscopy confirmed the presence of BF-ELIP-bound CD34+ cells in the intimal compartment of the atheromatous arterial wall. Ultrasound treatment increased the number of endothelial cell progenitors migrating into the intima. Thus, under ultrasound enhancement, BF-ELIP bound CD34+ stem cells selectively bind to the ICAM-1 expressing endothelium of atherosclerotic lesions.

Delivery of negatively charged liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits.

Liposomes have been used as imaging and therapeutic agents in various tissues but only infrequently in the cardiovascular system. We prepared a liposome to target atheromas in a Watanabe heritable hyperlipidemic (WHHL) rabbit model. Liposomes labeled with rhodamine and nanogold were injected intra-arterially into the descending thoracic aortas of WHHL rabbits. The arterial segments of interest were perfusion-fixed and evaluated with immunohistochemistry, light microscopy, and electron microscopy. Deconvolution microscopy showed that rhodamine label was concentrated in the plaque shoulder regions of advanced-stage atheromas; however, rhodamine label was not found in adjacent, non-atherosclerotic aorta. Transmission electron microscopy revealed liposome remnants and the highest concentration of nanogold label in lipid-laden areas of atheromas. Liposomes were concentrated in areas of lipoprotein-associated phospholipase A(2) expression. We conclude that modified liposomes can be delivered to the shoulder regions of advanced atheromas in WHHL rabbits and may be useful therapeutically for targeting metabolically active plaque.

Arrhythmias and elevated troponin I in a dog with steroid-responsive meningitis-arteritis.

A 10-month-old dog was presented with clinical signs of fever, lethargy, inappetence, and cardiac arrhythmias. Computed tomography scan and cerebrospinal fluid analysis supported the diagnosis of steroid-responsive meningitis-arteritis. Echocardiography, electrocardiogram, and elevated serum troponin I supported a diagnosis of myocarditis. The arrhythmias resolved during treatment of the primary neurological disease, and they were considered as secondary to the meningitis.

Microscopic Assessment of Healing and Effectiveness of a Foam-Based Peripheral Occlusion Device.

The IMPEDE Embolization Plug is a catheter-delivered vascular occlusion device that utilizes a porous shape memory polymer foam as a scaffold for thrombus formation and distal coils to anchor the device within the vessel. In this study, we investigated the biological response of porcine arteries to the IMPEDE device by assessing the extent of healing and overall effectiveness in occluding the vessel at 30, 60, and 90 days. Compared to control devices (Amplatzer Vascular Plug and Nester Embolization Coils), the host response to IMPEDE showed increased cellular infiltration (accommodated by the foam scaffold), which led to advanced healing of the initial thrombus to mature collagenous connective tissue (confirmed by transmission electron microscopy (TEM)). Over time, the host response to the IMPEDE device included degradation of the foam by multinucleated giant cells, which promoted fibrin and polymer degradation and advanced the healing response. Device effectiveness, in terms of vessel occlusion, was evaluated histologically by assessing the degree of recanalization. Although instances of recanalization were often observed at all time points for both control and test articles, the mature connective tissue within the foam scaffold of the IMPEDE devices improved percent vessel occlusion; when recanalization was observed in IMPEDE-treated vessels, channels were exclusively peri-device rather than intradevice, as often observed in the controls, and the vessels mostly remained >75% occluded. Although total vessel occlusion provides the optimal ischemic effect, in cardiovascular pathology, there is a progressive ischemic effect on the downstream vasculature as a vessel narrows. As such, we expect a sustained ischemic therapeutic effect to be observed in vessels greater than 75% occluded. Overall, the current study suggests the IMPEDE device presents advantages over controls by promoting an enhanced degree of healing within the foam scaffold, which decreases the likelihood of intradevice recanalization and ultimately may lead to a sustained ischemic therapeutic effect.

Micro-CT and histopathology methods to assess host response of aneurysms treated with shape memory polymer foam-coated coils versus bare metal coil occlusion devices.

Recent studies utilizing shape memory polymer foams to coat embolizing coils have shown potential benefits over current aneurysm treatments. In the current study utilizing a rabbit-elastase aneurysm model, the performance of test article (foam-coated coil [FCC]) and control (bare platinum coils [BPCs]) devices were compared at 30, 90, and 180 days using micro-CT and histological assessments. The host response was measured by identifying the cells regionally present within the aneurysm, and assessing the degree of residual debris and connective tissue. The 3D reconstructions of aneurysms provided context for histologic findings, and aided in the overall aneurysm assessment. At all time points, >75% of the cells categorized in each aneurysm were associated with a bioactive yet biocompatible host response (vs. the remainder of cells that were associated with acute inflammation). The extracellular matrix exhibited a transition from residual fibrin at 30 days to a greater degree of connective tissue at 90 and 180 days. Although the control BPC-treated aneurysms exhibited a greater degree of connective tissue at the earliest time point examined (30 days), by 180 days, the FCC-treated aneurysms had more connective tissue and less debris overall than the control aneurysms. When considering cell types and extracellular matrix composition, the overall host response scores were significantly better in FCC-treated aneurysms at the later time point. Based on the results of these metrics, the FCC device may lead to an advanced tissue remodeling response over BPC occlusion devices.

Foreign Body Reaction to a Subcutaneously Implanted Self-Cleaning, Thermoresponsive Hydrogel Membrane for Glucose Biosensors.

Towards achieveing a subcutaneously implanted glucose biosensor with long-term functionality, a thermoresponsive membrane previously shown to have potential to house a glucose sensing assay was evaluated herein for its ability to minimize the foriegn body reaction (FBR) and the resulting fibrous capsule. The severity of the FBR proportionally reduces diffusion of glucose to the sensor and hence sensor lifetime. However, efforts to reduce the FBR have largedly focused on anti-fouling materials that passively inhibit cellular attachment, particularly poly(ethylene glycol) (PEG). Herein, the extent of the FBR of a subcutaneously implanted "self-cleaning" cylindrical membrane was analyzed in rodents. This membrane represents an "actively anti-fouling" approach to reduce cellular adhesion. It is a thermoresponsive double network nanocomposite hydrogel (DNNC) comprised of poly(N-isopropylacrylamide) (PNIPAAm) and embedded polysiloxane nanoparticles. The membrane's cyclical deswelling/reswelling response to local body temperature fluctuations was anticipated to limit cellular accumulation. Indeed, after 30 days, the self-cleaning membrane exhibited a notably thin fibrous capsule (~30 µm) and increased microvascular density within 1 mm of the implant surface in comparison to a non-thermoresponsive, benchmark biocompatible control (PEG diacrylate, PEG-DA).

Method for preclinical pathology evaluation and analysis of cardiovascular implantable electronic device implant sites.

Cardiovascular implantable electronic devices (CIEDs) typically incorporate leads that directly contact the endocardium. Post-explant pathology evaluation of formalin-fixed CIED lead implant sites and downstream organs (i.e., lungs) can provide useful safety data to the US Food and Drug Administration; however, current regulatory guidelines do not mandate how the safety data are collected. In this paper, we outline a protocol for preclinical pathology evaluation of leads associated with CIEDs, which includes formalin fixation of the heart and lungs, gross evaluation, and qualitative and quantitative histologic evaluation. We recommend fixation of the whole heart with leads in situ alongside intratracheal formalin infusion; this enables rapid and effective preservation of target tissues and increases histologic quality to allow for accurate qualitative and quantitative pathology evaluation. Overall, we believe that our approach to pathology evaluation of leads may maximize information acquired from preclinical studies, leading to more accurate safety assessments. SUMMARY: This article introduces an established method for pathology evaluation and analysis of cardiac leads recommended for companies and researchers that seek approval from a regulatory body.

Comparison of shape memory polymer foam versus bare metal coil treatments in an in vivo porcine sidewall aneurysm model.

The endovascular delivery of platinum alloy bare metal coils has been widely adapted to treat intracranial aneurysms. Despite the widespread clinical use of this technique, numerous suboptimal outcomes are possible. These may include chronic inflammation, low volume filling, coil compaction, and recanalization, all of which can lead to aneurysm recurrence, need for retreatment, and/or potential rupture. This study evaluates a treatment alternative in which polyurethane shape memory polymer (SMP) foam is used as an embolic aneurysm filler. The performance of this treatment method was compared to that of bare metal coils in a head-to-head in vivo study utilizing a porcine vein pouch aneurysm model. After 90 and 180 days post-treatment, gross and histological observations were used to assess aneurysm healing. At 90 days, the foam-treated aneurysms were at an advanced stage of healing compared to the coil-treated aneurysms and showed no signs of chronic inflammation. At 180 days, the foam-treated aneurysms exhibited an 89-93% reduction in cross-sectional area; whereas coiled aneurysms displayed an 18-34% area reduction. The superior healing in the foam-treated aneurysms at earlier stages suggests that SMP foam may be a viable alternative to current treatment methods. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1892-1905, 2017.

A Method for Creating Artificial Thrombi In Vitro Using a Rotating Mechanical Surface.

Thromboembolism is a common concern in ventricular assist device (VAD) therapy. Precise VAD response to pass-through thromboembolism needs to be studied in a controlled in vitro setting where specific pump parameters (i.e., power consumption, flow rates, impeller RPM) can be monitored while various types of thrombi are introduced. In this article, we describe a method for creating standardized fibrin thrombi that could be introduced into a mock circulatory loop for testing VAD response to thromboembolism. Donor equine blood collected using a sodium citrate was allowed to clot by adding calcium chloride (CaCl2) while a rotating component applied shear forces to the blood. This rotating force was applied at various speeds and at various distances into the blood. Resulting clots showed similar microscopic features to thrombi taken from explanted clinical VADs. Higher RPM of the rotating component and smaller clearances between the rotating component and the blood created clots that closely resembled ante-explant clots found within VADs in vivo. This method is an effective way to create artificial fibrin clots for use in in vitro experiments to test thromboembolism in VADs.

Design and verification of a shape memory polymer peripheral occlusion device.

Shape memory polymer foams have been previously investigated for their safety and efficacy in treating a porcine aneurysm model. Their biocompatibility, rapid thrombus formation, and ability for endovascular catheter-based delivery to a variety of vascular beds makes these foams ideal candidates for use in numerous embolic applications, particularly within the peripheral vasculature. This study sought to investigate the material properties, safety, and efficacy of a shape memory polymer peripheral embolization device in vitro. The material characteristics of the device were analyzed to show tunability of the glass transition temperature (Tg) and the expansion rate of the polymer to ensure adequate time to deliver the device through a catheter prior to excessive foam expansion. Mechanical analysis and flow migration studies were performed to ensure minimal risk of vessel perforation and undesired thromboembolism upon device deployment. The efficacy of the device was verified by performing blood flow studies that established affinity for thrombus formation and blood penetration throughout the foam and by delivery of the device in an ultrasound phantom that demonstrated flow stagnation and diversion of flow to collateral pathways.

Deep Bleeder Acoustic Coagulation (DBAC)-part II: in vivo testing of a research prototype system.

BACKGROUND: Deep Bleeder Acoustic Coagulation (DBAC) is an ultrasound image-guided high-intensity focused ultrasound (HIFU) method proposed to automatically detect and localize (D&L) and treat deep, bleeding, combat wounds in the limbs of soldiers. A prototype DBAC system consisting of an applicator and control unit was developed for testing on animals. To enhance control, and thus safety, of the ultimate human DBAC autonomous product system, a thermal coagulation strategy that minimized cavitation, boiling, and non-linear behaviors was used. MATERIAL AND METHODS: The in vivo DBAC applicator design had four therapy tiles (Tx) and two 3D (volume) imaging probes (Ix) and was configured to be compatible with a porcine limb bleeder model developed in this research. The DBAC applicator was evaluated under quantitative test conditions (e.g., bleeder depths, flow rates, treatment time limits, and dose exposure time limits) in an in vivo study (final exam) comprising 12 bleeder treatments in three swine. To quantify blood flow rates, the "bleeder" targets were intact arterial branches, i.e., the superficial femoral artery (SFA) and a deep femoral artery (DFA). D&L identified, characterized, and targeted bleeders. The therapy sequence selected Tx arrays and determined the acoustic power and Tx beam steering, focus, and scan patterns. The user interface commands consisted of two buttons: "Start D&L" and "Start Therapy." Targeting accuracy was assessed by necropsy and histologic exams and efficacy (vessel coagulative occlusion) by angiography and histology. RESULTS: The D&L process (Part I article, J Ther Ultrasound, 2015 (this issue)) executed fully in all cases in under 5 min and targeting evaluation showed 11 of 12 thermal lesions centered on the correct vessel subsection, with minimal damage to adjacent structures. The automated therapy sequence also executed properly, with select manual steps. Because the dose exposure time limit (t dose ≤ 30 s) was associated with nonefficacious treatment, 60-s dosing and dual-dosing was also pursued. Thrombogenic evidence (blood clotting) and collagen denaturation (vessel shrinkage) were found in necropsy and histologically in all targeted SFAs. Acute SFA reductions in blood flow (20-30 %) were achieved in one subject, and one partial and one complete vessel occlusion were confirmed angiographically. The complete occlusion case was achieved with a dual dose (90 s total exposure) with focal intensity ≈500 W/cm(2) (spatial average, temporal average). CONCLUSIONS: While not meeting all in vivo objectives, the overall performance of the DBAC applicator was positive. In particular, D&L automation workflow was verified during each of the tests, with processing times well under specified (10 min) limits, and all bleeder branches were detected and localized. Further, gross necropsy and tissue examination confirmed that the HIFU thermal lesions were coincident with the target vessel locations in over 90 % of the multi-array dosing treatments. The SFA/DFA bleeder models selected, and the protocols used, were the most suitable practical model options for the given DBAC anatomical and bleeder requirements. The animal models were imperfect in some challenging aspects, including requiring tissue-mimicking material (TMM) standoffs to achieve deep target depths, thereby introducing device-tissue motion, with resultant imaging artifacts. The model "bleeders" involved intact vessels, which are subject to less efficient heating and coagulation cascade behaviors than true puncture injuries.

BioGlue surgical adhesive impairs aortic growth and causes anastomotic strictures.

BACKGROUND: BioGlue surgical adhesive (CryoLife, Inc, Kennesaw, GA) is currently being used to secure hemostasis at cardiovascular anastomoses in adults. Interference with vessel growth would preclude its use during congenital heart surgery. The purpose of this study was to determine if BioGlue reinforcement of aortic anastomoses impairs vessel growth and causes strictures. METHODS: Ten 4-week-old piglets (8.0 +/- 1.4 kg) underwent primary aorto-aortic anastomoses. Five piglets were randomly assigned to anastomotic reinforcement with BioGlue. After a 7-week growth period, the aortas were excised for morphometric analysis and histopathology. RESULTS: Weight gains were similar in both groups. In BioGlue animals, however, aortic circumference increased only 1.5 +/- 0.8 mm (versus 2.7 +/- 0.8 mm in controls; p = 0.054). BioGlue animals developed a 33.9% stenosis of the aortic lumen area (versus 3.7% in controls, p = 0.038). Adventitial changes reflecting tissue injury and fibrosis were present in all BioGlue animals versus none of the control animals (p = 0.008). CONCLUSIONS: BioGlue reinforcement impairs vascular growth and causes stricture when applied circumferentially around an aorto-aortic anastomosis. This adhesive should not be used on cardiovascular anastomoses in pediatric patients.

Novel anti-factor D monoclonal antibody inhibits complement and leukocyte activation in a baboon model of cardiopulmonary bypass.

BACKGROUND: Adverse outcomes after cardiopulmonary bypass (CPB) are often related to systemic inflammation triggered by complement and leukocyte activation. To determine how inhibition of the alternative complement pathway affects systemic inflammation and tissue injury, we studied a novel monoclonal antibody (Mab), anti-human factor D murine Mab 166-32, in baboons. METHODS: Fourteen baboons (mean weight, 15 kg) underwent hypothermic CPB. The treatment group (n = 7) received a single injection of anti-factor D Mab 166-32 (5 mg/kg), and the control group (n = 7) was given saline solution. After initiation of CPB, all animals were subjected to 20 minutes of core cooling (rectal temperature, 27 degrees C), followed by 60 minutes of aortic cross-clamping, 25 minutes of rewarming, and 30 minutes of normothermic CPB. Blood samples were collected before CPB, during CPB, and 1, 2, 3, 6, and 18 hours after CPB. To measure neutrophil and monocyte activation, we performed flow cytometry for CD11b expression, ELISA for complement activation (Bb, C3a, C4d, and sC5b-9) and interleukin-6 (IL-6) production, and tissue injury studies for creatine kinase MB isoenzymes (CK-MB), creatine kinase (CK), and lactic dehydrogenase (LDH) levels. RESULTS: Anti-factor D Mab almost completely inhibited plasma Bb, C3a, and sC5b-9 production during CPB (P < .001). CD11b expression on neutrophils (129 +/- 5% vs. 210 +/- 42%; P = .0006) and on monocytes (139 +/- 14% vs. 245 +/- 43%; P = .0002) was also lower in the treatment group during CPB. The treated animals had a significantly smaller increase in plasma IL-6 concentrations than did the control animals (71 +/- 27 pg/mL vs. 104 +/- 54 pg/mL; P = .0002). CK-MB levels were also lower in the treatment group 6 hours after the end of CPB (204 +/- 30 vs. 335 +/- 59 IU/L; P = .003) and 18 hours after the end of CPB (P < .05). Creatine kinase levels (6 and 18 hours after the end of CPB) and LDH levels (3 and 6 hours after the end of CPB) showed patterns similar to those of CK-MB (P < .05). CONCLUSIONS: The alternative complement pathway plays a major role in systemic inflammation during CPB. Inhibition of complement activation via the alternative pathway by anti-factor D Mab 166-32 significantly reduces leukocyte activation and tissue injury in our baboon model.