A new model of isolated perfused heart. Study of hypodermin A in hyperacute xenograft rejection]

AIMS OF THE STUDY: Isolated perfused heart (IPH) system and heart transplantation in the guinea-pig/rat combination represent a good model for the study of hyperacute xenograft rejection (HAR) in which the component plays a central role. Hypodermin A (HA), a protease cleaving the component, could be used to delay the HAR. METHODS: Creation of an original IPH working with rat serum (30 mL) and ex vivo study of HAR and I'HA. RESULTS: Study of HAR is possible with this IPH system. The mean guinea-pig heart survival after perfusion by normal rat serum was 38 +/- 7 min and was lower than survival observed after perfusion by guinea-pig serum (210 +/- 34 min) (p < 0.001), by decomplemented rat serum (177 +/- 45 min) (p < 0.001), and by rat serum with 20 micrograms/mL of HA (154 +/- 71 min) (p < 0.001). CONCLUSION: We developed an original system of isolated perfused heart allowing ex vivo study of HAR. HA delayed the occurrence of the HAR and confirmed the central role of the component in the HAR.     

Suppression of T cells results in long-term survival of mouse heart xenografts in C6-deficient rats

Abstract: The present study aimed to investigate the role of cellular immune response in the absence of membrane attack complex (MAC) formation in the concordant mouse‐to‐rat heart xenografting. Hearts from BALB/c mice were transplanted into the neck vessels of C6‐competent (C6⁺) and C6‐deficient (C6^?) PVG rats. Liposome‐encapsulated dichloro‐methylene diphosphonate (Lip‐Cl₂MDP) was administered at a dose of 10 ml/kg 2 days before transplantation and every 5 days thereafter. Cyclosporine (CsA) was administered intramuscularly (i.m.) at a dose of 15 mg/kg per day. The heart xenografts were harvested for immuno‐histological analysis at the time of rejection and the functioning grafts were removed at 70 days after transplantation. In untreated C6⁺ rats, xeno‐grafts survived for 2.3 ± 0.5 days. Treatment with CsA or Lip‐Cl₂MDP in C6⁺ rats did not significantly affect graft survival (2.5 ± 0.6 and 2.3 ± 0.4 days, respectively). In untreated C6^? rats, xenografts survived for 5.0 ± 0.6 days. However, Lip‐Cl₂MDP in C6^? rats resulted in a prolongation of graft survival to 11 ± 2.3 days (P < 0.05 vs. untreated C6^? rats), while treatment with CsA alone in these rats led to more than 70 days' survival in four out of six grafts (61 ± 16 days). In untreated C6⁺ rats, immunohistology showed a severe myocardial necrosis and thrombosis with a scarce cellular infiltrate in the rejected xenografts. By contrast, in untreated C6^? rats, xenografts were heavily infiltrated by macrophages and T cells. The number of macrophages, but not T cells, was markedly reduced in Lip‐Cl₂MDP‐treated rats. In CsA‐treated C6^? rats, the grafts harvested at 70 days after transplantation had a normal morphology, with a minimal cellular infiltrate. Our data indicate that MAC‐mediated injury plays an essential role in concordant xenograft rejection. Once this mechanism has been prevented, suppression of T cells allows for long‐term xenograft survival.     

The role of natural IgM in the hyperacute rejection of discordant heart xenografts.

The mechanism of xenograft hyperacute rejection in discordant species combinations remains controversial. The purpose of this work was to study the role of natural antibodies in the hyperacute rejection of guinea pig hearts transplanted into rats, a highly discordant combination. This study was conducted in vitro, ex vivo, and in vivo. The endothelial cells of the graft being the first targets damaged in the process of hyperacute rejection, the binding of rat natural antibodies to guinea pig endothelial cells was studied by immunofluorescence. The study was carried out in vitro on guinea pig endothelial cells in culture, and ex vivo on isolated guinea pig hearts perfused with either rat serum or immunoglobulins or immunoglobulin fragments bearing the antigen-binding site. In vitro and ex vivo, rat natural IgM were found to bind specifically to guinea pig endothelial cells, since IgM fragments bearing the antigen-binding site (Fab mu and Fab' mu) could be detected on these cells. IgM fragments were able to inhibit the fixation of native IgM molecules. In contrast, rat IgG only bound to endothelial cells through Fc portions. Thus rat natural IgM might play a role in hyperacute rejection by binding to the graft endothelial cells and triggering the complement cascade activation. In order to test the role of natural IgM in vivo, isolated guinea pig hearts were first perfused with rat Fab' mu, which inhibit the binding of IgM and are unable to activate the complement cascade. These hearts were then transplanted into Lewis rats. The rejection time of Fab' mu-perfused guinea pig hearts was prolonged compared with hearts perfused with buffer or IgG F(ab')2. Therefore, in the guinea pig to rat combination, preventing the binding of the recipient's natural IgM to the graft endothelium delays the hyperacute rejection. In addition, natural IgM are likely to play a greater role than natural IgG.     

Rat skin wound healing induced by alternagin-C, a disintegrin-like, Cys-rich protein from Bothrops alternatus venom

Alternagin‐C (ALT‐C) is a disintegrin‐like, Cys‐rich protein isolated from Bothrops alternatus snake venom, which has been shown to induce in vivo angiogenesis. Therefore, this protein could be interesting as a new approach for tissue regeneration studies. Here the effects of ALT‐C on fibroblasts and inflammatory cells, collagen type III and type I and TGF‐α expression in a rat wounded skin model were studied. Thirty‐five male Wistar rats (weight 270 ± 20 g) were divided into seven groups with five animals in each of the following groups: a control group which wounded animals received treatment with natrozol^® gel only; ALT‐C10, ALT‐C60 and ALT‐C100 groups of wounded animals that were treated with the same amount of gel containing 10, 60 and 100 ng of ALT‐C, respectively. Animals were treated once a day with 20 µl of gel associated or not with ALT‐C for 1, 3, 5 or 7 days. ALT‐C treatment increased the fibroblast density, collagen deposition and accelerated the inflammatory process, mostly in the ALT‐C60 group. These results indicate that ALT‐C improves wound repair process in rat skin. Thus, ALT‐C could be a candidate to the development of a novel therapeutic strategy for wounded skin repair.     

Hyperacute lung rejection in the pig-to-human model. 2. Synergy between soluble and membrane complement inhibition

Azimzadeh A, Zorn GL III, Blair KSA, Zhang JP, Pfeiffer S, Harrison RA, Cozzi E, White DJG, Pierson RN III. Hyperacute lung rejection in the pig‐to‐human model. 2. Synergy between soluble and membrane complement inhibition. Xenotransplantation 2003; 10: 120–131. © Blackwell Munksgaard, 2003 Background. The role of complement in hyperacute lung xenograft rejection has not been elucidated. The present study evaluates the effect of complement (C) C3/C5 convertase inhibition on hyperacute rejection of pig lung by human blood. Methods. In an established ex‐vivo model, lungs from pigs heterozygous for human decay accelerating factor (hDAF), non‐transgenic littermate control pigs, or farm‐bred pigs were perfused with fresh human blood that was either unmodified or treated with soluble complement receptor type 1 (sCR1: TP10, 100 μg/ml). Results. Non‐transgenic lungs from littermate controls had a median survival time of 35 min (range 5 to 210; P=0.25 vs. farm‐bred piglets: median 5 min, range 5 to 10). Lungs expressing hDAF survived for a median of 90 min (range 10 to 161; P=0.5 and 0.01 vs. littermate and farm‐bred controls, respectively), with sCR1, whereas hDAF (–) lungs failed by 35 min (range 6 to 307), hDAF (+) lungs survived for 330 min (range 39 to 577) [P=0.002 vs. farm‐bred; P=0.08 vs. hDAF (–); P=0.17 vs. sCR1/hDAF (–)]. The rise in pulmonary vascular resistance (PVR) at 5 min was blunted only by hDAF (+) with sCR1 (0.26 ± 0.2 vs. 0.5 to 0.7 mmHg/ml/min for other groups). Plasma C3a and sC5b‐9 and tissue deposition of C5b‐9 were dramatically diminished using sCR1, and further decreased in association with hDAF. Histamine and thromboxane were produced rapidly in all groups. Conclusion. Complement plays an important role in lung HAR. However, even potent inhibition of C3/C5 convertase, both membrane bound in lung and by a soluble‐phase inhibitor in the blood, does not prevent activation of inflammatory responses known to be particularly injurious to the lung. Our findings implicate a role for innate immune pathways resistant to efficient complement regulation. The role of anti‐species antibody, coagulation pathway dysregulation, and additional environmental or genetic influences remain to be defined.     

Human serum induces apoptosis of xenogenic cardiomyocytes in vivo and in vitro

Abstract: Discordant xenotransplantation is complicated by delayed xenograft rejection (DXR). Previous studies have demonstrated that anti‐apoptotic genes are protective against DXR. This study examines the hypothesis that apoptosis plays a role in human anti‐xenograft responses. C57BL/6 mice and NOD SCID mice were given a single intravenous injection of either a lethal dose (LD, survival < 30 min) or a sublethal dose (SLD) of human serum, and isolated pig and mouse rod‐shaped cardiomyocytes were exposed to human serum in vitro. In situ detection of apoptotic cells in mouse hearts was assessed using a terminal deoxynucleotidyl transferase‐mediated dUTP nicked‐end labeling assay. Mice transfused with human serum had approximately a 10‐fold increased percentage of apoptotic cells after SLD 18 h post‐injection compared with animals given saline, and a fourfold increase over LD. Administration of cobra venom factor (CVF) decomplemented SLD 18 h did not significantly ( P  > 0.05) alter the percentage apoptosis. The addition of 20 mM Gal‐α‐1,3‐Gal to SLD 18 h significantly ( P  < 0.05) reduced percentage apoptosis to levels comparable to saline treated control animals. In vitro using mouse and pig cardiomyocytes demonstrated parallel results as in vivo experiments.
Human serum induces apoptosis of cardiomyocytes in immunocompetent and immunoincompetent mice in vivo, as well as mouse and pig cardiomyocytes in vitro. Further, this apoptotic response can be inhibited by the addition of Gal‐α‐1,3‐Gal without affecting the capacity of the serum to cause HAR. These results demonstrate that a putative human serum factor induces a delayed apoptotic injury of xenograft tissues, and supports the hypothesis that apoptosis may be an important mediator of DXR.     

Freezing characteristics of isolated pig and human hepatocytes

The cellular response of isolated hepatocytes from pigs, humans, and human hepatoblastoma cells to freezing was characterized using cryomicroscopy and analyzed using a thermodynamic model for water transport and Intracellular Ice Formation (IIF). The value for the reference permeability, L_pg, was found to be 5.8(10)⁻¹³, 1.62(10)⁻¹³, and 2.7(10)⁻¹⁴m/Ns for pig, human, and Hep G2/C3A cells, respectively. The activation energy, E_tp, was found to be 480 kJ/mol for pig hepatocytes, 216 kJ/mol for human, and 121 kJ/mol for Hep G2/C3A cells. The average temperature at which IIF (T^avg_IIF) occurs was calculated to be −7.24 ± 2.3°C for pig hepatocytes, −8.5 ± 2.6°C for human hepatocytes, and −9.6 ± 4.5°C for Hep G2/C3A cells. These results indicate that the freezing characteristics of pig and human cells are distinct and that the specific freezing characteristics need to be understood for the development of appropriate freezing protocols.     

CCL25‐Supplemented Hyaluronic Acid Attenuates Cartilage Degeneration in a Guinea Pig Model of Knee Osteoarthritis

There is evidence that the application of mesenchymal stromal cells (MSCs) counteracts osteoarthritis (OA) progression. However, the prospect of extracting and expanding these cells might be limited. The aim of this study was to investigate whether hyaluronic acid (HA) supplemented with MSC‐recruiting chemokine C‐C motif ligand 25 (CCL25) can influence the natural course of spontaneous OA in the guinea pig. CCL25 concentration in synovial fluid (SF) was quantified with enzyme‐linked immunosorbent assay. Boyden chamber cell migration assay was used to test CCL25‐mediated migration of guinea pig MSC. Forty‐nine 11‐month‐old male guinea pigs were divided into seven groups. The main treatments consisted of five intra‐articular injections of HA in pure form and in combination with three doses of CCL25 (63, 693, and 6,993 pg) given at a weekly interval. The severity of cartilage damage was assessed by using a modified Mankin score. The measured average physiological concentration of CCL25 in SF of animals is 85 ± 39 pg/ml. MSC showed a 3.2‐fold increase in cell migration at 1,000 nM CCL25 in vitro demonstrating the biological migratory activity of CCL25 on these cells. In vivo, treatment with HA alone did not reduce OA progression. Similarly, OA scores were not found significantly reduced after treatment with 63 pg CCL25 + HA. However, when compared to pure HA, treatment with 693 pg CCL25 + HA and 6,993 pg CCL25 + HA significantly reduced the OA score from 10.1 to 7.4 (?28%) and 8.4 (?20%), respectively. These data suggest that intra‐articular injections of HA supplemented with CCL25 attenuates OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1723–1729, 2019     

Orthotopic tracheal transplantation using human bronchus: an original xenotransplant model of obliterative airway disorder

Bronchiolitis obliterans syndrome (BOS) is a main cause of allograft dysfunction and mortality after lung transplantation (LTx). A better understanding of BOS pathogenesis is needed to overcome this treatment‐refractory complication. Orthotopic tracheal transplantation using human bronchus was performed in Brown Norway (BN) and nude (RNU) rats. Allografts were recovered in both strains at Day 7 (BN₇, n = 6; RNU₇, n = 7) or Day 28 (BN₂₈, n = 6; RNU₂₈, n = 6). Immune response of the host against the bronchial graft was assessed. Human samples from BOS patients were used to compare with the histological features of the animal model. Obstruction of the allograft lumen associated with significant infiltration of CD3+ and CD68+ cells was observed in the BN group on Day 28. Immune response from type 1 T‐helper cells against the tracheal xenograft was higher in BN animals compared to nude animals on Days 7 and 28 (P < 0.001 and P = 0.035). Xenoreactive antibodies were significantly higher at Day 7 (IgM) and Day 28 (IgG) in the BN group compared to RNU (respectively, 37.6 ± 6.5 vs. 5.8 ± 0.7 mean fluorescence, P = 0.039; and 22.4 ± 3.8 vs. 6.9 ± 1.6 mean fluorescence, P = 0.011). Immunocompetent animals showed a higher infiltration of S100A4+ cells inside the bronchial wall after 28 days, associated with cartilage damage ranging from invasion to complete destruction. In vitro expression of S100A4 by human fibroblasts was higher when stimulated by mononuclear cells (MNCs) from BN rats than from RNU (2.9 ± 0.1 vs. 2.4 ± 0.1 mean fluorescence intensity, P = 0.005). Similarly, S100A4 was highly expressed in response to human MNCs compared to stimulation by T‐cell‐depleted human MNCs (4.3 ± 0.2 vs. 2.7 ± 0.1 mean fluorescence intensity, P < 0.001). Obliterative bronchiolitis has been induced in a new xenotransplant model in which chronic airway obstruction was associated with immune activation against the xenograft. Cartilage infiltration by S100A4+ cells might be stimulated by T cells.     

Influence of three different types of hypercalciuria on bone

 The relationship between bone mineral status and hypercalciuria is controversial. The effect on bone composition of different forms of hypercalciuria was studied in female rats made hypercalciuric by 7-week administration of oral furosemide (F, n=12), intraperitoneal 1,25-dihydroxy vitamin D (VD, n=11), or oral ammonium chloride (AC, n=12). Seven untreated rats served as controls (C). Hypercalciuria (mg/100 g per 24 h, mean ±SEM) of F (4.3±0.2), VD (4.1±0.4), and AC (3.9±0.3) groups was of similar intensity (C rats 1.3±0.1, P<0.01). Weight and length gains and serum CO₂, sodium, potassium, calcium, and phosphate were no different among the four groups. Bone was studied by dual-energy X-ray absorptiometry of left tibiae. AC rats had significantly less bone area (1.505±0.018 cm²) than VD and C (1.602±0.020 and 1.587±0.019 cm²). Bone mineral content was decreased in F (0.357±0.007 g) and AC (0.362±0.006 g) compared with VD (0.407±0.008 g) and C (0.389±0.009 g) groups. Bone mineral density was different between F (0.231±0.002 g/cm²) and VD and C rats (0.254±0.004 and 0.245±0.003 g/cm²), and also between AC (0.240±0.003 cm²) and VD rats. In these rat models, hypercalciuria of renal origin (F) and hypercalciuria caused by acid load (AC) adversely impaired bone mass. Received: 19 September 1997 / Revised: 28 July 1998 / Accepted: 29 July 1998     

Liposome‐Encapsulated Hemoglobin Accelerates Bronchial Healing After Pneumonectomy in the Rat With or Without Preoperative Radiotherapy

Liposome‐encapsulated hemoglobin (LEH) has been reported to accelerate wound healing in the stomach and skin in an experimental setting. LEH was tested in bronchial anastomotic healing after radiation and pneumonectomy in the rat. Sprague‐Dawley rats (n = 61) received preoperative radiation (20 Gy) to the chest and underwent left pneumonectomy with bronchial stump closure using the Sweet method 4 days later, when they were randomized to receive intravenous infusion of LEH with high O₂ affinity (P₅₀O₂ = 17 mm Hg, 10 mL/kg, n = 32) or saline (n = 29). Additional rats (n = 18) were treated in the same way without preoperative radiation. Bronchial anastomotic healing was evaluated 2 days after surgery by determining the bursting pressure and infiltration of neutrophils, monocytes, and macrophages. Bronchial bursting pressure was elevated in the rats receiving LEH both in the unirradiated group (LEH 212 ± 78 vs. saline 135 ± 63 mm Hg, P < 0.05) and in rats with preoperative radiation (LEH 162 ± 48 vs. saline 116 ± 56 mm Hg, P < 0.01). Moreover, the percentage of rats with bursting pressure <100 mm Hg tended to be smaller in the unirradiated group (LEH 1/9 [11.1%] vs. saline 4/9 [44.4%], NS) and was significantly reduced in irradiated animals (LEH 3/32 [9.4%] vs. saline 11/29 [38%], P < 0.05). There were no morphological differences except for macrophage infiltration to the anastomotic area, which was significantly prominent in the LEH‐treated rats (P < 0.05) regardless of the presence or absence of preoperative irradiation (IR). The results suggest that LEH with high O₂ affinity may improve mechanical strength and morphological findings in bronchial anastomosis in rats regardless of the presence or absence of preoperative IR. The irradiated rats later treated with LEH had equivalent or better bronchial healing than that of saline‐treated naïve animals undergoing pneumonectomy alone.     

The Protective Effect of St. Thomas Cardioplegia Enriched With Zacopride on the Isolated Rat Heart

The activation of the heart inward rectifier potassium channel (I_K1) can reduce the injury of myocardial cells by shortening the action potential duration and reducing intracellular calcium overload. Zacopride is a selective I_K1 agonist and suppresses triggered arrhythmias in rat hearts. This investigation studied the effects of St. Thomas (ST) cardioplegia enriched with Zacopride on the isolated rat heart model. Sprague‐Dawley rat hearts were harvested and perfused for 20 minutes with 37°C Krebs‐Henseleit (KH) buffer followed by 15 minute perfusion with 4°C calcium‐free KH buffer in the Control group (Con, n = 8), ST cardioplegia in the ST group (ST, n = 8) and ST cardioplegia with Zacopride in the STZ group (STZ, n = 8). After 45 minutes of arresting, all hearts were reperfused with 37°C KH buffer for 60 minutes. Hearts in the STZ group arrested faster than the Con and ST groups (9.25 ± 2.38 s vs. 72.25 ± 8.1 s, 12.75 ± 2.87 s). The recovery of the left ventricular developed pressure, ± dP/dtmax, heart rate, and coronary flow in the STZ group is significantly better than the other two groups during reperfusion. Compared with the Con and ST groups, the STZ group showed significant decreases in the maximum carciac troponin I level (P < 0.05) and the infarct size (P < 0.05). The superoxide dismutase level in the STZ group increased during the first 20 minutes of reperfusion (P < 0.05). ST cardioplegia enriched with Zacopride has beneficial effects against ischemia‐reperfusion injury in this isolated rat heart model.     

Comparative study of Nd-YAG laser versus electrocautery for liver metastatic resection in the rat

The thermal, hemostatic and lymphostatic effects of the Nd-YAG laser suggest a benefit in the treatment of multiple liver metastases. The aim of this work was to evaluate experimentally this hypothesis in a comparative study with conventional electrocautery resection of liver metastases. The original animal model was represented by syngeneic BDIX rats inoculated under the liver capsule with 1.5×10⁶ DHD/K12 tumour cells originating from a clone of a 1,2 dimethyl hydrazine induced rat colon cancer. One hundred and ten rats bearing three liver metastases were randomly treated by laser volatilization or electrocautery enucleation. The first group of 60 rats was used as a survival group: the laser treated rats survived significantly longer than rats treated by cautery (49.9 days vs 28.9 days) (mean values;p<0.015). In this group, the temperature elevation during operation at the edge of the treated lesion was found higher in the laser group than in the cautery group (56±4.6°C vs 8±3°C) (mean values±s.d.;p<0.001). Nd-YAG laser was also a faster procedure than cautery resection (21±4.2 s vs 57±6.1 s) (mean values±s.d.;p<0.001). At the time of autopsy, the infection rate with laser was found lower than in the cautery group (p<0.025) while no bile leakage was evident. A peritoneal tumour dissemination with ascites was noted in the majority of dead rats. In the second group of 50 rats, the metastatic recurrence was assessed. At day 7, no metastases were found in the laser treated rats while a mean number of 6.5 was found in the cautery group (p<0.001). At the 15th day, more metastases were present in the cautery group. There was a significant correlation between the total number of metastases and the time of death. Those findings suggest that the Nd-YAG laser destruction of experimental liver metastases by its specific effects on tumour cells delayed the recurrence of metastases when compared to the electrocautery resection, contributing to a longer survival of the laser treated rats.     

Evidence that 15-deoxyspergualin inhibits natural antibody production but fails to prevent hyperacute rejection in a discordant xenograft model.

Preventing hyperacute rejection (HAR) is a difficult and unsolved problem in xenotransplantation. This may be due, in part, to a lack of therapies that can suppress production of natural antibodies (NA), which are thought to be critical mediators of HAR. This study examined the effect of 15-deoxyspergualin (DSPG) and splenectomy (Spx) on NA production and return of NA after plasma exchange (PE) in a discordant species combination (strain 2 guinea pig to Lewis rat). A dose of 5 mg/kg/day DSPG + Spx significantly reduced Lewis rat anti-guinea pig NA titer after one week of therapy. Antibody titer was not significantly reduced in rats treated with splenectomy alone. PE alone acutely depleted NA titers; however, complete rebound was seen in 48 hr. When PE was performed in rats treated with DSPG + Spx, an additional significant NA reduction occurred; no rebound 24-48 hr after PE was seen. Except for a 20% reduction in body weight, no serious complications occurred in DSPG + Spx recipients. Despite a profound NA titer reduction, treatment with DSPG, Spx, and PE did not prolong guinea pig cardiac xenograft survival in a clinically significant fashion. Immunopathological study of rejected cardiac xenografts revealed no antibody deposition but persistent complement deposition on vascular endothelium. We conclude that DSPG + Spx effectively inhibits synthesis of rat anti-guinea pig NA, that further NA titer reduction can be achieved with the addition of PE, and that DSPG + Spx prevents post-PE antibody rebound. We also conclude that the limited prolongation in cardiac xenograft survival achieved, despite marked suppression of NA, supports a complement-mediated mechanism of HAR in our animal model.     

Elevated vitamin D receptor levels in genetic hypercalciuric stone‐forming rats are associated with downregulation of Snail

Patients with idiopathic hypercalciuria (IH) and genetic hypercalciuric stone‐forming (GHS) rats, an animal model of IH, are both characterized by normal serum Ca, hypercalciuria, Ca nephrolithiasis, reduced renal Ca reabsorption, and increased bone resorption. Serum 1,25‐dihydroxyvitamin D [1,25(OH)₂D] levels are elevated or normal in IH and are normal in GHS rats. In GHS rats, vitamin D receptor (VDR) protein levels are elevated in intestinal, kidney, and bone cells, and in IH, peripheral blood monocyte VDR levels are high. The high VDR is thought to amplify the target‐tissue actions of normal circulating 1,25(OH)₂D levels to increase Ca transport. The aim of this study was to elucidate the molecular mechanisms whereby Snail may contribute to the high VDR levels in GHS rats. In the study, Snail gene expression and protein levels were lower in GHS rat tissues and inversely correlated with VDR gene expression and protein levels in intestine and kidney cells. In human kidney and colon cell lines, ChIP assays revealed endogenous Snail binding close to specific E‐box sequences within the human VDR promoter region, whereas only one E‐box specifically bound Snail in the rat promoter. Snail binding to rat VDR promoter E‐box regions was reduced in GHS compared with normal control intestine and was accompanied by hyperacetylation of histone H₃. These results provide evidence that elevated VDR in GHS rats likely occurs because of derepression resulting from reduced Snail binding to the VDR promoter and hyperacetylation of histone H₃. © 2010 American Society for Bone and Mineral Research.     

Synthetic peptides which inhibit the interaction between C1q and immunoglobulin and prolong xenograft survival

BACKGROUND: Acute vascular xenograft rejection (AVXR), also termed delayed xenograft rejection (DXR), occurs when hyperacute rejection (HAR) is prevented by strategies directed at xenoreactive natural antibodies and/or complement activation. We have hypothesized that AVXR/DXR is initiated in part by early components of the complement cascade, notably C1q. We have developed synthetic peptides (termed CBP2 and WY) that interfere with the interaction between C1q and antibody. METHODS: CBP2 and the WY-conjugates were used as inhibitors of immunoglobulin aggregate binding to solid phase C1q. Inhibition of complement activation by the peptides of the classical system was determined using lysis assays with sensitized sheep red blood cells or porcine aortic endothelial cells as targets and of the alternate complement pathway using guinea pig red blood cells as targets. Two transplant models were used to study the effects of administering peptides to recipients: rat heart transplant to presensitized mouse, and guinea heart transplant to PVG C6-deficient rats. RESULTS: CBP2 and WY-conjugates inhibited immunoglobulin aggregate binding to C1q. The peptides also inhibited human complement-mediated lysis of sensitized sheep red blood cells and porcine aortic endothelial cells in a dose-dependent manner and the WY-conjugates prevented activation of the alternate complement pathway as shown by inhibition of guinea pig red blood cells lysis with human serum. In addition, the use of the peptides and conjugates resulted in significant prolongation of xenograft survival. CONCLUSIONS: The CBP2 and WY peptides exhibit the functional activity of inhibition of complement activation. These peptides also prolong xenograft survival and thus provide reagents for the study of the importance of C1q and other complement components in transplant rejection mechanisms.     

Adenovirus-Mediated Gene Expression of the Human c-FLIPL Gene Protects Pig Islets Against Human CD8+ Cytotoxic T Lymphocyte-Mediated Cytotoxicity

Cell-mediated immunity, especially of human CD8+ cytotoxic T lymphocytes (CTLs) is believed to have an important role in the long-term survival of pig islet xenografts. Protection against human CD8+ CTL cytotoxicity may reduce the direct damage to pig islets and enable long-term xenograft survival in pig-to-human islet xenotransplantation. We have previously reported that c-FLIP_S/L genes, which are potent inhibitors of death receptor–mediated proapoptotic signals through binding competition with caspase-8 for recruitment to the Fas-associated via death domain (FADD), markedly suppress human CD8+ CTL-mediated xenocytotoxicity. In addition, the cytoprotective effects of c-FLIP_L seem to be significantly stronger than those of c-FLIP_S. Accordingly, in the present study, expression of c-FLIP_L was induced in intact pig islets by adenoviral transduction. Consequently, the cytoprotective capacity of the transgene in pig islets was examined in in vitro and in vivo exposure to human CD8+ CTLs. Cells from untransduced islets or mock islets were sensitive to CD8+ CTL-mediated lysis (59.3% ± 15.9% and 64.0% ± 8.9% cytotoxicity, respectively). In contrast, cells from pig islets transduced with the c-FLIP_L gene were markedly protected from lysis (30.5% ± 3.5%). Furthermore, prolonged xenograft survival was elicited from pig islets transduced with this molecule as assessed using an islet transplant model using the rat kidney capsule. Thus, these data indicate that intact pig islets can be transduced to express c-FLIP_L with adenovirus. Pig islets expressing c-FLIP_L are significantly resistant to human CTL killing and further exhibit beneficial effects to prolong xenograft survival.     

Clinical Pharmacokinetics of Anti‐angiogenic Photodynamic Therapy with Benzoporphyrin Derivative Monoacid Ring‐A in Dogs Having Naturally Occurring Neoplasms

The aim of this study was to examine the pharmacokinetics of clinically applied benzoporphyrin derivative monoacid ring‐A (BPD‐MA; Verteporfin^®), a second‐generation photosensitizer, during a trial of photodynamic therapy (PDT) in nine dogs having naturally occurring neoplasms. After injecting BPD‐MA at 0.5 mg/kg intravenously, its mean half‐life (t_1/2) was found to be 8.14 ± 5.34 h, mean clearance (Cl) 35.13 ± 9.62 ml/(h kg), the mean value of the volume of distribution (V_c) 0.08 ± 0.01 l/kg and the mean steady state volume of distribution (V_ss) 0.38 ± 0.31 l/kg respectively. With the exception of a transitional increase in serum alkaline phosphatase activity, no other clinical abnormalities were observed. The t_1/2 in dogs with naturally occurring tumours was longer than that in humans, but similar to that in rats. The values of Cl and V_ss in dogs having naturally occurring neoplasms were lower than those in humans. It is suggested that the pharmacokinetics of BPD‐MA in tumour‐bearing dogs would be helpful in determining the protocol of a short drug‐light interval PDT with BPD‐MA that mainly targets the tumour vasculature.     

Silver oxide‐containing hydroxyapatite coating has in vivo antibacterial activity in the rat tibia

Bacterial infection is a serious postoperative complication of joint replacement. To prevent infections related to implantation, we have developed a novel antibacterial coating with Ag‐containing hydroxyapatite (Ag‐HA). In the present study, we examined the antibacterial activity of Ag‐HA implant coatings in the medullary cavity of rat tibiae. Forty 10‐week‐old rats received implantation of Ag‐HA‐ or HA‐coated titanium rods, then were inoculated with ～1.0 × 10² colony‐forming units of methicillin‐resistant Staphylococcus aureus. Bacterial counts were calculated for rats euthanized at 24, 48, and 72 h postoperatively. Serum levels of Ag (in the Ag‐HA group only) were calculated for rats euthanized at 24, 48, 72 h and 4 weeks. Radiographic evaluations of bone infection were also performed at 4 weeks. Tibiae from both groups showing infection were evaluated histologically. Significant differences in bacterial counts were seen at 24, 48, and 72 h. Mean concentrations of Ag in serum peaked about 48 h after implantation, then gradually decreased. Mean radiographic scores for infection were significantly lower with Ag‐HA implants than with HA implants. Histological examination showed better results for abscesses, bone resorption, and destruction of cortical bone around Ag‐HA‐coated implants. These results indicate that Ag‐HA coatings may help prevent surgical‐site infections associated with joint replacement. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1195–1200, 2013