Immune Privilege of Heart Valves.

Immune privilege is an evolutionary adaptation that protects vital tissues with limited regenerative capacity from collateral damage by the immune response. Classical examples include the anterior chamber of the eye and the brain. More recently, the placenta, testes and articular cartilage were found to have similar immune privilege. What all of these tissues have in common is their vital function for evolutionary fitness and a limited regenerative capacity. Immune privilege is clinically relevant, because corneal transplantation and meniscal transplantation do not require immunosuppression. The heart valves also serve a vital function and have limited regenerative capacity after damage. Moreover, experimental and clinical evidence from heart valve transplantation suggests that the heart valves are spared from alloimmune injury. Here we review this evidence and propose the concept of heart valves as immune privileged sites. This concept has important clinical implications for heart valve transplantation.

A review of the immune response stimulated by xenogenic tissue heart valves.

Valvular heart disease continues to afflict millions of people around the world. In many cases, the only corrective treatment for valvular heart disease is valve replacement. Valve replacement options are currently limited, and the most common construct utilized are xenogenic tissue heart valves. The main limitation with the use of this valve type is the development of valvular deterioration. Valve deterioration results in intrinsic permanent changes in the valve structure, often leading to hemodynamic compromise and clinical symptoms of valve re-stenosis. A significant amount of research has been performed regarding the incidence of valve deterioration and determination of significant risk factors for its development. As a result, many believe that the underlying driver of valve deterioration is a chronic immune-mediated rejection process of the foreign xenogenic-derived tissue. The underlying mechanisms of how this occurs are an area of ongoing research and active debate. In this review, we provide an overview of the important components of the immune system and how they respond to xenografts. A review of the proposed mechanisms of xenogenic heart valve deterioration is provided including the immune response to xenografts. Finally, we discuss the role of strategies to combat valve degeneration such as preservation protocols, epitope modification and decellularization.

Geometry influences inflammatory host cell response and remodeling in tissue-engineered heart valves in-vivo.

Regenerative tissue-engineered matrix-based heart valves (TEM-based TEHVs) may become an alternative to currently-used bioprostheses for transcatheter valve replacement. We recently identified TEM-based TEHVs-geometry as one key-factor guiding their remodeling towards successful long-term performance or failure. While our first-generation TEHVs, with a simple, non-physiological valve-geometry, failed over time due to leaflet-wall fusion phenomena, our second-generation TEHVs, with a computational modeling-inspired design, showed native-like remodeling resulting in long-term performance. However, a thorough understanding on how TEHV-geometry impacts the underlying host cell response, which in return determines tissue remodeling, is not yet fully understood. To assess that, we here present a comparative samples evaluation derived from our first- and second-generation TEHVs. We performed an in-depth qualitative and quantitative (immuno-)histological analysis focusing on key-players of the inflammatory and remodeling cascades (M1/M2 macrophages, α-SMA+- and endothelial cells). First-generation TEHVs were prone to chronic inflammation, showing a high presence of macrophages and α-SMA+-cells, hinge-area thickening, and delayed endothelialization. Second-generation TEHVs presented with negligible amounts of macrophages and α-SMA+-cells, absence of hinge-area thickening, and early endothelialization. Our results suggest that TEHV-geometry can significantly influence the host cell response by determining the infiltration and presence of macrophages and α-SMA+-cells, which play a crucial role in orchestrating TEHV remodeling.

Hydrogel hybrid porcine pericardium for the fabrication of a pre-mounted TAVI valve with improved biocompatibility.

Transcatheter aortic valve implantation (TAVI) has been developed years ago for patients who cannot undergo a surgical aortic valve replacement (SAVR). Although TAVI possesses the advantages of lower trauma and simpler manipulation compared to SAVR, the need for storage in glutaraldehyde (GLU) and a tedious intraoperative assembly process have caused great inconvenience for its further application. A pre-mounted TAVI valve assembled by mounting a dry valve frame to a delivery system is expected to address these problems. However, the currently used GLU treated leaflet cannot unfold normally after being crimped for a long-term and loses its function when the BHV is assembled to the catheter. Besides, its cytotoxicity and immune response after implantation are still problems to be solved. In the present study, a hydrogel hybrid porcine pericardium (HHPP) approach was developed to endow the BHVs with a favorable unfolding property and good biocompatibility. Three monomers with different charge characteristics (sodium acrylate, 2-methacryloyloxyethyl phosphorylcholine, and acryloyloxyethyltrimethyl ammonium chloride) were complexed with GLU treated PP (GLU-PP) to form three kinds of HHPPs (SAAH-PP, MPCH-PP, and DACH-PP). The results of the crimping simulation experiment showed that all HHPPs could quickly recover in PBS after being folded for 10 days, while the traditional BHVs (GLU-PP) could not recover under the same conditions. Bovine serum albumin adsorption and platelet adhesion test showed that SAAH-PP and MPCH-PP had good anti-adhesion abilities. A cell culture study indicated that all the three HHPPs promoted HUVEC growth and proliferation. In vivo biocompatibility studies showed that the immune response induced by MPCH-PP was reduced compared to that by GLU-PP. These studies demonstrated that the strategy of MPC hydrogel hybridization may be an effective approach to prepare a pre-mounted TAVI valve with improved biocompatibility.

Glycosphingolipids of porcine, bovine, and equine pericardia as potential immune targets in bioprosthetic heart valve grafts.

BACKGROUND: Pericardial tissue from various animal species is utilized for the production of the bioprosthetic heart valves (BHV) used clinically. Experimental data show that the eventual breakdown of BHV is partly due to immunological interactions with carbohydrate tissue antigens. To understand these processes, we have examined the glycolipid-based carbohydrate antigens in naïve porcine, bovine, and equine pericardia. EXPERIMENTAL: Total non-acid and acid glycosphingolipid fractions were isolated from porcine, bovine, and equine pericardia, and individual glycolipid compounds were characterized by thin-layer chromatography, mass spectrometry, and binding of monoclonal antibodies, lectins and bacteria in chromatogram binding assays. RESULTS: The non-acid glycolipid fractions from all species contained glycosphingolipids based on the globo- and neolacto-series, including pentaglycosylceramides with terminal Galα3 determinants. Terminal blood group A and H (O) structures based on type 2 core chains were present in porcine pericardium, while the Forssman pentaosylceramide was found in equine pericardium. All acid glycolipid fractions contained sulfatide and several gangliosides with both N-acetyl- and N-glycolyl-neuraminic acid as terminal saccharide chain determinants. CONCLUSION: Several carbohydrate antigens which are potential targets for the human immune system have been identified in the animal pericardial tissues used for the production of BHV. Which of these antigens are left in the tissues after industrial BHV production processes, as well as their potential role in eventual BHV degradation, remains to be elucidated.

Effects on human heart valve immunogenicity in vitro by high concentration cryoprotectant treatment.

It has been shown previously that cryopreservation, using an ice-free cryopreservation method with the cryoprotectant formulation VS83, beneficially modulated immune reactions in vivo and in vitro when compared with conventionally frozen tissues. In this study, we assessed the impact of a VS83 post-treatment of previously conventionally frozen human tissue on responses of human immune cells in vitro. Tissue punches of treated and non-treated (control) aortic heart valve tissue (leaflets and associated aortic root) were co-cultured for 7 days with peripheral blood mononuclear cells or enriched CD14+ monocytes. Effects on cellular activation markers, cytokine secretion and immune cell proliferation were analysed by flow cytometry. Flow cytometry studies showed that VS83 treatment of aortic root tissue promoted activation and differentiation of CD14+ monocytes, inducing both up-regulation of CD16 and down-regulation of CD14. Significantly enhanced expression levels for the C-C chemokine receptor (CCR)7 and the human leukocyte antigen (HLA)-DR on monocytes co-cultured with VS83-treated aortic root tissue were measured, while the interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 release was suppressed. However, the levels of interferon (IFN)γ and tumour necrosis factor (TNF)α remained undetectable, indicating that complete activation into pro-inflammatory macrophages did not occur. Similar, but non-significant, changes occurred with VS83-treated leaflets. Additionally, in co-cultures with T cells, proliferation and cytokine secretion responses were minimal. In conclusion, post-treatment of conventionally cryopreserved human heart valve tissue with the VS83 formulation induces changes in the activation and differentiation characteristics of human monocytes, and thereby may influence long-term performance following implantation. Copyright © 2017 John Wiley & Sons, Ltd.

Immune-Mediated Heart Disease.

The heart involvement in systemic autoimmune diseases represents a growing burden for patients and health systems. Cardiac function can be impaired as a consequence of systemic conditions and manifests with threatening clinical pictures or chronic myocardial damage. Direct injuries are mediated by the presence of inflammatory infiltrate which, even though unusual, is one of the most danger manifestations requiring prompt recognition and treatment. On the other hand, a not well-managed inflammatory status leads to accelerated atherosclerosis that precipitates ischemic disease. All cardiac structures may be damaged with different grades of intensity; moreover, lesions can appear simultaneously or more frequently at a short distance from each other leading to the onset of varied clinical pictures. The pathogenesis of heart damages in systemic autoimmune conditions is not yet completely understood for the great part of situations, even if several mechanisms have been investigated. The principal biochemical circuits refer to the damaging role of autoantibodies on cardiac tissues and the precipitation of immune complexes on endocardium. These events are finally responsible of inflammatory infiltration which leads to subsequent worsening of the previous damage. For these reasons, it appears of paramount importance a regular and deepened cardiovascular assessment to prevent a progressive evolution toward heart failure in patient affected by autoimmune diseases.

Antibody formation towards porcine tissue in patients implanted with crosslinked heart valves is directed to antigenic tissue proteins and αGal epitopes and is reduced in healthy vegetarian subjects.

BACKGROUND: Glutaraldehyde-fixed porcine heart valves (ga-pV) are one of the most frequently used substitutes for insufficient aortic and pulmonary heart valves which, however, degenerate after 10-15 years. Yet, xeno-immunogenicity of ga-pV in humans including identification of immunogens still needs to be investigated. We here determined the immunogenicity of ga-pV in patients with respect to antibody formation, identity of immunogens and potential options to reduce antibody levels. METHODS: Levels of tissue-specific and anti-αGal antibodies were determined retrospectively in patients who received ga-pV for 51 months (n=4), 25 months (n=6) or 5 months (n=4) and compared to age-matched untreated subjects (n=10) or younger subjects with or without vegetarian diet (n=12/15). Immunogenic proteins were investigated by Western blot approaches. RESULTS: Tissue-specific antibodies in patients were elevated after 5 (1.73-fold) and 25 (1.46-fold, both P<.0001) months but not after 51 months, whereas anti-Gal antibodies were induced 4.75-fold and 3.66-fold after 5 and 25 months (both P<.0001) and still were significantly elevated after 51 months (2.85-fold, P<.05). Western blots of porcine valve extracts with and without enzymatic deglycosylation revealed strong specific staining at ≈65 and ≈140 kDa by patient sera in either group which were identified by 2D Western blots and mass spectrometry as serum albumin and collagen 6A1. Vegetarian diet reduced significantly (0.63-fold, P<.01) the level of pre-formed αGal but not of tissue-specific antibodies. CONCLUSION: Immune response in patients towards ga-pV is induced by the porcine proteins albumin and collagen 6A1 as well as αGal epitopes, which seemed to be more sustained. In contrast, in healthy young subjects pre-formed anti-Gal antibodies were reduced by a meat-free nutrition.

The Immune Responses and Calcification of Bioprostheses in the α1,3-Galactosyltransferase Knockout Mouse.

BACKGROUND: The study aim was to evaluate the immune reaction, difference of degenerative calcification, and anti-calcification effect of decellularization with or without α-galactosidase in bovine pericardium and porcine heart valves, using an α1,3-galactosyltransferase (α-Gal) knockout (KO) mouse model. METHODS: In order to elucidate the anti-calcification effect of decellularization with or without α-galactosidase, bovine pericardium and porcine heart valve tissues were assigned to four groups according to the tissue preparation method: (i) glutaraldehyde (GA) fixation only; (ii) decellularization + GA fixation (Decell); (iii) α-galactosidase + GA fixation (α-galactosidase); and (iv) decellularization +α-galactosidase + GA fixation (Decell + α-galactosidase). Each prepared tissue was implanted subcutaneously into α-Gal KO mice. Anti-α-Gal immunoglobulin (Ig) G and IgM antibody titers were monitored prior to implantation and at four, eight and 12 weeks after implantation using an enzyme-linked immunosorbent assay. Calcium contents of explanted tissues were measured at 12 weeks after implantation. RESULTS: There were no significant differences in the anti-α-Gal IgG antibody titers according to the type of bioprosthetic material or tissue preparation method (p >0.05). The calcium content was significantly lower in porcine heart valves than in bovine pericardium when implanted in α-Gal-KO mice (p <0.001). Calcium contents in bovine pericardium and porcine heart valves were significantly lower in the Decell, α-galactosidase and Decell + α-galactosidase groups than in the GA group (all p <0.05). CONCLUSIONS: The porcine heart valve induced lower levels of calcium deposition than did the bovine pericardium, but the anti-α-Gal IgG antibody titers did not differ significantly between the bioprosthetic tissues. Decellularization had significant anticalcification effects in both the bovine pericardium and porcine heart valves, though there was no significant difference in the anti-α-Gal IgG antibody titers among tissue preparation methods.

Characterization of immunogenic Neu5Gc in bioprosthetic heart valves.

BACKGROUND: The two common sialic acids (Sias) in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-glycolylneuraminic acid (Neu5Gc). Unlike most mammals, humans cannot synthesize Neu5Gc that is considered foreign and recognized by circulating antibodies. Thus, Neu5Gc is a potential xenogenic carbohydrate antigen in bioprosthetic heart valves (BHV) that tend to deteriorate in time within human patients. METHODS: We investigated Neu5Gc expression in non-engineered animal-derived cardiac tissues and in clinically used commercial BHV, and evaluated Neu5Gc immunogenicity on BHV through recognition by human anti-Neu5Gc IgG. RESULTS: Neu5Gc was detected by immunohistochemistry in porcine aortic valves and in porcine and bovine pericardium. Qualitative analysis of Sia linkages revealed Siaα2-3>Siaα2-6 on porcine/bovine pericardium while the opposite in porcine aortic/pulmonary valve cusps. Similarly, six commercial BHV containing either porcine aortic valve or porcine/bovine/equine pericardium revealed Siaα2-3>Siaα2-6 expression. Quantitative analysis of Sia by HPLC showed porcine/bovine pericardium express 4-fold higher Neu5Gc levels compared to the porcine aortic/pulmonary valves, with Neu5Ac at 6-fold over Neu5Gc. Likewise, Neu5Gc was expressed on commercial BHV (186.3±16.9 pmol Sia/µg protein), with Neu5Ac at 8-fold over Neu5Gc. Affinity-purified human anti-Neu5Gc IgG showing high specificity toward Neu5Gc-glycans (with no binding to Neu5Ac-glycans) on a glycan microarray, strongly bound to all tested commercial BHV, demonstrating Neu5Gc immune recognition in cardiac xenografts. CONCLUSIONS: We conclusively demonstrated Neu5Gc expression in native cardiac tissues, as well as in six commercial BHV. These Neu5Gc xeno-antigens were recognized by human anti-Neu5Gc IgG, supporting their immunogenicity. Altogether, these findings suggest BHV-Neu5Gc/anti-Neu5Gc may play a role in valve deterioration warranting further investigation.

Human antibody recognition of xenogeneic antigens (NeuGc and Gal) on porcine heart valves: could genetically modified pig heart valves reduce structural valve deterioration?

BACKGROUND: Glutaraldehyde-fixed bioprosthetic heart valves (GBHVs) derived from wild-type (WT, genetically unmodified) pigs are widely used clinically for heart valve replacement. There is evidence that their failure is related to an immune response. The use of valves from genetically engineered pigs that do not express specific pig antigens may prolong GBHV survival. Our aims were to determine (i) expression of Gal and NeuGc on heart (aortic and pulmonary) valves and pericardium of WT, α1,3-galactosyltransferase gene knockout (GTKO) and GTKO/N-glycolylneuraminic acid gene-knockout (GTKO/NeuGcKO) pigs in comparison with three different commercially available GBHVs and (ii) to determine human antibody binding to these tissues. METHODS: Wild-type, GTKO/CD46, and GTKO/CD46/NeuGcKO pig valves and pericardium were tested (i) fresh and (ii) after fixation with glutaraldehyde (0.02%, 0.2%, 2%). Sections of GBHVs, fresh and fixed valves, and pericardium were stained for Gal and NeuGc expression, and for human IgM and IgG antibody binding. RESULTS: Gal and NeuGc expression was high on all GBHVs and WT pig valves/pericardium, but was absent after antigen-specific-knockout. There was no difference in antigen expression or antibody binding among WT aortic, pulmonary valves, and pericardium as well as GBHVs. Glutaraldehyde fixation did not alter expression of Gal or NeuGc. After incubation with human serum, human IgM and IgG bound to all GBHVs and WT pig valves/pericardium. Valves from GTKO/CD46 pigs and, particularly, GTKO/CD46/NeuGcKO pigs (with/without glutaraldehyde fixation) showed less IgM and IgG binding. CONCLUSION: Compared to WT pigs, GTKO/CD46/NeuGcKO pigs would be preferable sources of GBHVs, because the absence of Gal/NeuGc expression reduces human antibody binding.

Predicted Coverage and Immuno-Safety of a Recombinant C-Repeat Region Based Streptococcus pyogenes Vaccine Candidate.

The C-terminal region of the M-protein of Streptococcus pyogenes is a major target for vaccine development. The major feature is the C-repeat region, consisting of 35-42 amino acid repeat units that display high but not perfect identity. SV1 is a S. pyogenes vaccine candidate that incorporates five 14mer amino acid sequences (called J14i variants) from differing C-repeat units in a single recombinant construct. Here we show that the J14i variants chosen for inclusion in SV1 are the most common variants in a dataset of 176 unique M-proteins. Murine antibodies raised against SV1 were shown to bind to each of the J14i variants present in SV1, as well as variants not present in the vaccine. Antibodies raised to the individual J14i variants were also shown to bind to multiple but different combinations of J14i variants, supporting the underlying rationale for the design of SV1. A Lewis Rat Model of valvulitis was then used to assess the capacity of SV1 to induce deleterious immune response associated with rheumatic heart disease. In this model, both SV1 and the M5 positive control protein were immunogenic. Neither of these antibodies were cross-reactive with cardiac myosin or collagen. Splenic T cells from SV1/CFA and SV1/alum immunized rats did not proliferate in response to cardiac myosin or collagen. Subsequent histological examination of heart tissue showed that 4 of 5 mice from the M5/CFA group had valvulitis and inflammatory cell infiltration into valvular tissue, whereas mice immunised with SV1/CFA, SV1/alum showed no sign of valvulitis. These results suggest that SV1 is a safe vaccine candidate that will elicit antibodies that recognise the vast majority of circulating GAS M-types.

Decellularized GGTA1-KO pig heart valves do not bind preformed human xenoantibodies.

Pre-clinical and clinical data have unequivocally demonstrated the usefulness of decellularized heart valve (HV) matrices implanted for HV replacement therapy. However, human donor valves applicable for decellularization are in short supply, which prompts the search for suitable alternatives, such as porcine grafts. Since decellularization might be insufficient to remove all xenoantigens, we analysed the interaction of human preformed antibodies with decellularized porcine HV in vitro to assess potential immune reactions upon implantation. Detergent-decellularized pulmonary HV from German Landrace wild-type (wt) or α1,3-galactosyltransferase knockout (GGTA1-KO) pigs were investigated by inhibition ELISA and GSL I-B4 staining to localize and quantify matrix-bound αGal epitopes, which represent the most prominent xenoantigen. Additionally, preformed human xenoantibodies were affinity purified by perfusing porcine kidneys. Binding of purified human antibodies to decellularized HV was investigated by inhibition ELISA. Furthermore, binding of human plasma proteins to decellularized matrices was determined by western blot. Decellularized human pulmonary artery served as controls. Decellularization of wt HV led to a reduction of αGal epitopes by 70 %. Residual epitopes were associated with the subendothelial extracellular matrix. As expected, no αGal epitopes were found on decellularized GGTA1-KO matrix. The strongest binding of preformed human anti-pig antibodies was found on wt matrices, whereas GGTA1-KO matrices bound similar or even fewer xenoantibodies than human controls. These results demonstrate the suitability of GGTA1-KO pigs as donors for decellularized heart valves for human patients. Besides the presence of αGal antibodies on decellularized heart valves, no further preformed xenoantibodies against porcine matrix were detected in tested human sera.

Xeno-immunogenicity of ice-free cryopreserved porcine leaflets.

BACKGROUND: Undesirable processes of inflammation, calcification, or immune-mediated reactions are limiting factors in long-term survival of heart valves in patients. In this study, we target the modulatory effects of ice-free cryopreservation (IFC) of xenogeneic heart valve leaflet matrices, without decellularization, on the adaptive human immune responses in vitro. METHODS: We tested porcine leaflet matrices from fresh untreated, conventionally cryopreserved (CFC), and IFC pulmonary valves by culturing them with human blood mononuclear cells for 5 d in vitro. No other tissue treatment protocols to modify possible immune responses were used. Matrices alone or in addition with a low-dose second stimulus were analyzed for induction of proliferation and cytokine release by flow cytometry-based techniques. Evaluation of the α-Gal epitope expression was performed by immunohistochemistry with fluorochrome-labeled B4 isolectin. RESULTS: None of the tested leaflet treatment groups directly triggered the proliferation of immune cells. But when tested in combination with a second trigger by anti-CD3, IFC valves showed significantly reduced proliferation of T cells, especially effector memory T cells, in comparison with fresh or CFC tissue. Moreover, the cytokine levels for interferon-γ (IFNγ), tumor necrosis factor α, and interleukin-10 were reduced for the IFC-treated group being significantly different compared with the CFC group. However, no difference between treatment groups in the expression of the α-Gal antigen was observed. CONCLUSIONS: IFC of xenogeneic tissue might be an appropriate treatment method or processing step to prevent responses of the adaptive immune system.

First quantification of alpha-Gal epitope in current glutaraldehyde-fixed heart valve bioprostheses.

BACKGROUND: Glutaraldehyde fixation does not guarantee complete tissue biocompatibility in current clinical bioprosthetic heart valves (BHVs). Particularly, circulating anti-αGal human antibodies increase significantly from just 10 days after a BHV implantation. The inactivation of such epitope should be mandatory to meet the requirements for a perspectively safe clinical application; nevertheless, its quantitative assessment in commercially available BHVs has never been carried out. METHODS: In this investigation, seven different models of BHVs were tested. The number of epitopes was determined with reference to a standard αGal source by an ELISA test. The presence of xenoantigen was subsequently confirmed by immunofluorescence analysis. Porcine tissue, knockout for the αGal epitopes, was used as negative control. RESULTS: Epic™ valve was the only model among those tested, in which the αGal antigen appeared to be completely shielded. Composite Trifecta™ valve exhibited conflicting results: cusps of bovine pericardial tissue were devoid of reactive αGal epitopes, while the stent cover strip of porcine pericardium still maintained 30% of active antigens originally present in native tissue. All other tested BHVs express an αGal amount not significantly different from that exhibited by porcine Mosaic(®) valve (5.2 ± 0.6 × 10(10) each 10 mg of tissue). CONCLUSIONS: For the first time, the quantitative evaluation of the αGal epitope in heart valve bioprostheses, already in clinical practice for about 40 yrs, was finally determined. Such quantification might provide indications of biocompatibility relevant for the selection of bioprosthetic devices and an increase in the confidence of the patient. It might become a major quality control tool in the production and redirection of future investigation in the quest for αGal-free long-lasting substitutes.

Heart dysfunction induced by choline-deficiency in adult rats: the protective role of L-carnitine.

Choline is a B vitamin co-factor and its deficiency seems to impair heart function. Carnitine, a chemical analog of choline, has been used as adjunct in the management of cardiac diseases. The study investigates the effects of choline deficiency on myocardial performance in adult rats and the possible modifications after carnitine administration. Wistar Albino rats (n=24), about 3 months old, were randomized into four groups fed with: (a) standard diet (control-CA), (b) choline deficient diet (CDD), (c) standard diet and carnitine in drinking water 0.15% w/v (CARN) and (d) choline deficient diet and carnitine (CDD+CARN). After four weeks of treatment, we assessed cardiac function under isometric conditions using the Langendorff preparations [Left Ventricular Developed Pressure (LVDP-mmHg), positive and negative first derivative of LVDP were evaluated], measured serum homocysteine and brain natriuretic peptide (BNP) levels and performed histopathology analyses. In the CDD group a compromised myocardium contractility compared to control (P=0.01), as assessed by LVDP, was noted along with a significantly impaired diastolic left ventricular function, as assessed by (-) dp/dt (P=0.02) that were prevented by carnitine. Systolic force, assessed by (+) dp/dt, showed no statistical difference between groups. A significant increase in serum BNP concentration was found in the CDD group (P<0.004) which was attenuated by carnitine (P<0.05), whereas homocysteine presented contradictory results (higher in the CDD+CARN group). Heart histopathology revealed a lymphocytic infiltration of myocardium and valves in the CDD group that was reduced by carnitine. In conclusion, choline deficiency in adult rats impairs heart performance; carnitine acts against these changes.

Autoimmune myocarditis, valvulitis, and cardiomyopathy.

Myocarditis and valvulitis are inflammatory diseases affecting myocardium and valve. Myocarditis, a viral-induced disease of myocardium, may lead to dilated cardiomyopathy and loss of heart function. Valvulitis leads to deformed heart valves and altered blood flow in rheumatic heart disease. Animal models recapitulating these diseases are important in understanding the human condition. Cardiac myosin is a major autoantigen in heart, and antibodies and T cells to cardiac myosin are evident in inflammatory heart diseases. This unit is a practical guide to induction and evaluation of experimental autoimmune myocarditis (EAM) in several mouse strains and the Lewis rat. Purification protocols for cardiac myosin and protocols for induction of EAM by cardiac myosin and its myocarditis-producing peptides, and coxsackievirus CVB3, are defined. Protocols for assessment of myocarditis and valvulitis in humans and animal models provide methods to define functional autoantibodies targeting cardiac myosin, ß-adrenergic, and muscarinic receptors, and their deposition in tissues.

CXCL9/Mig mediates T cells recruitment to valvular tissue lesions of chronic rheumatic heart disease patients.

Rheumatic fever (RF) is an autoimmune disease triggered by Streptococcus pyogenes infection frequently observed in infants from developing countries. Rheumatic heart disease (RHD), the major sequel of RF, leads to chronic inflammation of the myocardium and valvular tissue. T cells are the main population infiltrating cardiac lesions; however, the chemokines that orchestrate their recruitment are not clearly defined. Here, we investigated the expression of chemokines and chemokine receptors in cardiac tissue biopsies obtained from chronic RHD patients. Our results showed that CCL3/MIP1α gene expression was upregulated in myocardium while CCL1/I-309 and CXCL9/Mig were highly expressed in valvular tissue. Auto-reactive T cells that infiltrate valvular lesions presented a memory phenotype (CD4(+)CD45RO(+)) and migrate mainly toward CXCL9/Mig gradient. Collectively, our results show that a diverse milieu of chemokines is expressed in myocardium and valvular tissue lesions and emphasize the role of CXCL9/Mig in mediating T cell recruitment to the site of inflammation in the heart.

Inflammatory response assessment of a hybrid tissue-engineered heart valve leaflet.

Despite substantial research in the past few decades, only slight progress has been made toward developing biocompatible, tissue-engineered scaffolds for heart valve leaflets that can withstand the dynamic pressure inside the heart. Recent progress on the development of hybrid scaffolds, which are composed of a thin metal mesh enclosed by multi-layered tissue, appear to be promising for heart valve engineering. This approach retains all the advantages of biological scaffolds while developing a strong extracellular matrix backbone to withstand dynamic loading. This study aims to test the inflammatory response of hybrid tissue-engineered leaflets based on characterizing the activation of macrophage cells cultured on the surfaces of the tissue construct. The results indicate that integration of biological layers around a metal mesh core-regardless of its type-may reduce the evoked inflammatory responses by THP-1 monocyte-like cells. This observation implies that masking a metal implant within a tissue construct prior to implantation can hide it from the immune system and may improve the implant's biocompatibility.