Immunologic Responses in Biological and Mechanical Valve Prostheses: Inflammation and Functionality Are Not Always Related.

BACKGROUND AND AIM OF THE STUDY: The aim of this retrospective study was to evaluate the inflammatory response in patients with aortic and/or mitral prostheses, and to correlate the level of inflammatory markers with prosthesis functionality. METHODS: A total of 48 patients with biological or mechanical prostheses was included in the study, in which levels of tumor necrosis factor-alpha (TNFα), interleukin (IL)-1, -4, and -6, interferon-gamma (IFNγ), osteopontin (OPN), intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), endothelin-1 and C-reactive protein were analyzed. Functionality of the prosthesis was evaluated using transthoracic echocardiography at three years after surgery. RESULTS: The mean period from the date of surgery was seven years. High levels of IL-1 were found in patients with mechanical prostheses compared to those with bioprostheses (p = 0.04). Patients with aortic bioprostheses and stenosis had higher levels of OPN and endothelin-1, those with aortic mechanical prostheses with stenosis had increased levels of matrix metalloproteinase (MMP)-9, OPN and ICAM, and those with aortic mechanical leakage had increased levels of MMP-1 and endothelin-1. In mitral bioprostheses with leakage of endothelin-1, ICAM and MMP-9 levels were increased, while in mechanical prostheses with leakage there were increases of ICAM and endothelin-1. Tricuspid bioprostheses with double lesions had increased levels of OPN and endothelin-1. CONCLUSIONS: Valvular dysfunction was similar across the types of prosthesis material. IL-1 was increased in subjects with mechanical prostheses independently of dysfunction, while in biological prostheses there were increases in OPN and endothelin-1, and these were related to valvular dysfunction. Given that in the analysis of durability and functionality there were no significant differences between biological and mechanical prostheses, biological prostheses may represent the first treatment option in patients with low economic resources, the elderly, and even young patients.

Circulating activated and effector memory T cells are associated with calcification and clonal expansions in bicuspid and tricuspid valves of calcific aortic stenosis.

We sought to delineate further the immunological significance of T lymphocytes infiltrating the valve leaflets in calcific aortic stenosis (CAS) and determine whether there were associated alterations in circulating T cells. Using clonotypic TCR ß-chain length and sequence analysis we confirmed that the repertoire of tricuspid CAS valves contains numerous expanded T cell clones with varying degrees of additional polyclonality, which was greatest in cases with severe calcification. We now report a similar proportion of clonal expansions in the much younger bicuspid valve CAS cases. Peripheral blood flow cytometry revealed elevations in HLA-DR(+) activated CD8 cells and in the CD8(+)CD28(null)CD57(+) memory-effector subset that were significantly greater in both bicuspid and tricuspid CAS cases with more severe valve calcification. Lesser increases of CD4(+)CD28(null) T cells were identified, principally in cases with concurrent atherosclerotic disease. Upon immunostaining the CD8 T cells in all valves were mainly CD28(null), and CD8 T cell percentages were greatest in valves with oligoclonal repertoires. T cell clones identified by their clonotypic sequence as expanded in the valve were also found expanded in the circulating blood CD28(null)CD8(+) T cells and to a lesser degree in the CD8(+)CD28(+) subset, directly supporting the relationship between immunologic events in the blood and the valve. The results suggest that an ongoing systemic adaptive immune response is occurring in cases with bicuspid and tricuspid CAS, involving circulating CD8 T cell activation, clonal expansion, and differentiation to a memory-effector phenotype, with trafficking of T cells in expanded clones between blood and the valve.

Gal-knockout bioprostheses exhibit less immune stimulation compared to standard biological heart valves.

BACKGROUND AND AIM OF THE STUDY: Current biological heart valves (BHVs) contain the major xenogeneic antigen Gal. Recipient anti-Gal antibody binding to such an implanted BHV may contribute to valve degeneration. The study aim was to compare, by implantation in non-human primates, the immune consequences of BHVs from Gal-positive wild-type (WT) pigs and those from alpha-galactosyltransferase knockout (GTKO) pigs. METHODS: Recipients were immunized prior to implant with keyhole limpet hemocyanin (KLH) conjugated to alphaGal to match the anti-Gal levels and isotypes found in humans. Stented glutaraldehyde-fixed BHVs from WT (n = 4) and GTKO (n = 3) pigs were commercially manufactured and implanted in the mitral position in non-human primates. Recipients were treated with enoxaparin (1 mg/kg b.i.d.) for five weeks which was tapered, and then discontinued. Serum antibody levels to Gal and KLH were measured using ELISA. RESULTS: Overall anti-Gal and anti-KLH antibody levels were decreased in both WT and GTKO BHV recipients after implantation. Serum anti-Gal IgG levels in GTKO BHV recipients fell rapidly within one month, matching the loss of anti-KLH reactivity. There was no significant difference in retention of anti-KLH antibody between the groups. WT BHV recipients retained significantly elevated levels of anti-Gal IgG during the first year post implant. Area under the curve analysis showed that anti-Gal IgG was significantly increased in the WT BHV group compared to GTKO BHV recipients (p < 0.01). CONCLUSION: Persistent and significantly (p < 0.01) elevated levels of anti-Gal IgG were observed in WT but not GTKO BHV non-human primate recipients, and indicated a continuing BHV-specific immune stimulation to the alphaGal antigen. These data support the hypothesis that the clinical use of Gal-positive xenogeneic bioprosthetic materials can induce an anti-Gal antibody response. Bioprosthetic devices prepared from GTKO pig tissue would eliminate immune stimulation to this major xenoreactive antigen, which may reduce the potential of immune-mediated injury and degeneration.

Upregulation of the 5-lipoxygenase pathway in human aortic valves correlates with severity of stenosis and leads to leukotriene-induced effects on valvular myofibroblasts.

BACKGROUND: The development of aortic valve stenosis is not only associated with calcification and extracellular matrix remodeling, but also with inflammation. The aim of this study was to determine the role of proinflammatory signaling through the leukotriene (LT) pathway in aortic stenosis. METHODS AND RESULTS: After macroscopic dissection of surgically removed human aortic valves, RNA was extracted from 311 preparations derived from 68 patients to differentiate normal, thickened, and calcified areas from each cusp. Subsequently, quantitative polymerase chain reaction analysis was used to correlate gene expression patterns with preoperative echocardiographic parameters. The messenger RNA levels of the LT-forming enzyme 5-lipoxygenase increased 1.6- and 2.2-fold in thickened and calcified tissue, respectively, compared with normal areas of the same valves. In thickened tissues, messenger RNA levels for 5-lipoxygenase (r= -0.35; P=0.03), its activating protein (5-lipoxygenase activating protein; r= -0.39; P=0.02), and LTA(4) hydrolase (r= -0.48; P=0.01) correlated inversely with the velocity-time integral ratio. In addition, leukotriene A(4) hydrolase transcripts correlated inversely with aortic valve area, indexed for body surface area (r= -0.52; P=0.007). Immunohistochemical stainings revealed LT receptor expression on valvular myofibroblasts. In primary cultures of human myofibroblasts derived from stenotic aortic valves, Leukotriene C(4) (LTC(4)) increased intracellular calcium, enhanced reactive oxygen species production, reduced the mitochondrial membrane potential, and led to morphological cell cytoplasm changes and calcification. CONCLUSIONS: The upregulation of the LT pathway in human aortic valve stenosis and its correlation with clinical stenosis severity, taken together with the potentially detrimental LT-induced effects on valvular myofibroblasts, suggests one possible role of inflammation in the development of aortic stenosis.

Structural Valve Deterioration Is Linked to Increased Immune Infiltrate and Chemokine Expression.

We aim to investigate whether structural valve deterioration (SVD) of bioprosthetic xenogenic tissue heart valves (XTHVs) is associated with increased immune cell infiltration and whether co-expression of several chemokines correlates with this increase in immune infiltrate. Explanted XTHVs from patients undergoing redo valve replacement for SVD were obtained. Immunohistochemical, microscopic, and gene expression analysis approaches were used. XTHVs (n = 37) were obtained from 32 patients (mean 67.7 years) after a mean time of 11.6 years post-implantation. Significantly increased immune cellular infiltration was observed in the explanted SVD valves for all immune cell types examined, including T cells, macrophages, B cells, neutrophils, and plasma cells, compared to non-SVD controls. Furthermore, a significantly increased chemokine gradient in explanted SVD valves accompanied immune cell infiltration. These data suggest the development of SVD is associated with a significantly increased burden of immune cellular infiltrate correlated to the induction of a chemokine gradient around the XHTV, representing chronic immune rejection.Graphical abstract Proposed interaction between innate and adaptive immunity leading to the development of structural valve deterioration in xenogenic tissue heart valves.

Association between antibodies against calcifying nanoparticles and mitral annular calcification.

BACKGROUND AND AIM OF THE STUDY: Mechanisms leading to vascular and tissue calcification are not yet fully understood. Previously, an association has been demonstrated between a controversial calcifying nanoparticle (CNP; also known as 'nanobacteria') and vascular calcification and kidney stone formation. The study aim was to evaluate a possible association between mitral annular calcification (MAC) and CNP infection. METHODS: A total of 93 patients with MAC, detected using echocardiography, and 94 asymptomatic subjects without valvular and coronary artery calcification, were enrolled in the study. The serum levels of anti-CNP-antibodies were monitored in all subjects. RESULTS: Patients with MAC were generally older and had a higher prevalence of systemic hypertension, diabetes mellitus, and dyslipidemia. The anti-CNP-antibody titers, which were significantly associated with MAC (p < 0.0001), were increased with older age and MAC thickness, but decreased in line with serum levels of HDL-cholesterol (p < 0.0001). In order to provide a cut-off point for anti-CNP-antibodies when detecting MAC, a receiver operating characteristic curve was created. Serum CNP-antibody levels above 0.19 units/ml showed a sensitivity of 73%, a specificity of 72%, and positive and negative predictive values of 72% and 73%, respectively. Multivariate logistic regression analysis revealed that increasing age, systemic hypertension, diabetes, HDL-cholesterol levels and high anti-CNP titers were risk factors that were independently associated with calcification in the mitral annuli. CONCLUSION: The study results suggested that CNP might play an important role in the pathogenesis of MAC.

Tissue Doppler velocities for ruling out rejection in heart transplant recipients in the context of myocardial strain imaging: a multivariate, prospective, single-center study.

To investigate the value of tissue Doppler velocities for ruling out treatment-requiring acute cellular rejection (TR-ACR), in the context of myocardial deformation analysis performed by means of speckle tracking echocardiography. We performed serial echocardiograms in 37 heart transplant recipients in their first year post-transplantation within 3 h of the routine surveillance endomyocardial biopsies (EMB). The association of the sum of lateral mitral annulus systolic (s') and early diastolic (e') velocities, in absolute values, measured by tissue Doppler echocardiography (s'+ e'), with TR-ACR (ACR grade ≥ 2R) was investigated by multivariate analysis, including classic echocardiographic parameters and myocardial deformation variables. A total of 251 pairs of EMB and echo exams were performed, 35 (14%) with rejection grade ≥ 2R (TR-ACR). s' + e' was independently associated to TR-ACR (OR 0.80, 95%CI 0.72-0.89, p < 0.0005), with a C statistic of 0.79 (95%CI 0.71-0.87, p < 0.0005) by ROC curve analysis. An s'+ e' value ≥ 23 cm/s, present in 43% of studies, had a negative predictive value of 98% for ruling out TR-ACR. Moreover, in the same patients, s'+ e' significantly decreased when TR-ACR occurred after a study without this condition (- 3.7 ± 3.3 cm/s, p = 0.003), but it was similar when rejection status was the same in the present versus the previous study. A drop in s'+ e' value < 2.7 cm/s from the previous echocardiogram, had a 99% negative predictive value for ruling out TR-ACR. Tissue Doppler velocities, a widely available echo parameter, were found to be a valuable marker for ruling out TR-ACR in this multivariate study which included myocardial deformation variables.

A rare case of enterobacter endocarditis superimposed on a mitral valve rheumatoid nodule.

We present the case of a 56-year-old man with longstanding seropositive active erosive and deforming rheumatoid arthritis with no peripheral rheumatoid nodules; he immigrated from the former Soviet Union (where he did not receive any disease-modifying antirheumatic drugs) to Israel in 1995. In February 2005, he had a buccogingival mucosal abscess on his lower lip, which was treated by surgical drainage, followed by prolonged antibiotic therapy. One and a half years later, he had 2 episodes of transient ischemic attacks characterized by speech difficulties and moderate weakness on his right side. Transesophageal echocardiogram revealed a mass on the anterior mitral valve leaflet. Repeated blood cultures were negative, and the patient was afebrile all the time. The patient underwent mitral valve replacement and the histologic findings of the mass were typical of both a rheumatoid nodule and bacterial endocarditis. The patient recovered fully after 6 weeks of antibiotic therapy. Emboli from a rheumatoid nodule should always be considered in patients with rheumatoid arthritis who present with transient ischemic attacks.

Development and Characterization of a Porcine Mitral Valve Scaffold for Tissue Engineering.

Decellularized scaffolds represent a promising alternative for mitral valve (MV) replacement. This work developed and characterized a protocol for the decellularization of whole MVs. Porcine MVs were decellularized with 0.5% (w/v) SDS and 0.5% (w/v) SD and sterilized with 0.1% (v/v) PAA. Decellularized samples were seeded with human foreskin fibroblasts and human adipose-derived stem cells to investigate cellular repopulation and infiltration, and with human colony-forming endothelial cells to investigate collagen IV formation. Histology revealed an acellular scaffold with a generally conserved histoarchitecture, but collagen IV loss. Following decellularization, no significant changes were observed in the hydroxyproline content, but there was a significant reduction in the glycosaminoglycan content. SEM/TEM analysis confirmed cellular removal and loss of some extracellular matrix components. Collagen and elastin were generally preserved. The endothelial cells produced newly formed collagen IV on the non-cytotoxic scaffold. The protocol produced acellular scaffolds with generally preserved histoarchitecture, biochemistry, and biomechanics.

Lymphocyte-rich capillary-cavernous hemangioma of the mitral valve: a case report and review of the literature.

Valvular hemangioma incidence is extremely low. In this report, we describe a 62-year-old man who presented with mild edema of the lower limbs. An echocardiogram revealed an incidental 1.3-cm diameter mass on the anterior mitral valve leaflet for which he underwent surgical resection and mitral valve replacement. Histopathological examination showed a lymphocyte-rich capillary-cavernous hemangioma. The exuberant lymphoid stroma is unusual for hemangioma and represents an undescribed pattern of cardiac hemangioma. Including the present report, only 13 cases of mitral valve hemangioma have been reported to date. Most patients are adult. Mitral hemangioma originates in the atrial aspect of the valve and involves more commonly the anterior leaflet. The average maximum diameter of the lesion is 1.7 (S.D.=0.75) cm. Pure cavernous hemangioma is the predominant type of mitral hemangioma. Most of them are described as pedunculated or polypoid. Surgical excision appears to be curative. Recurrences have not been reported. Lymphocyte-rich cardiac hemangioma represents a peculiar type of hemangioma which should be included in the differential diagnosis of other vascular lesions.

Heart infiltrating T cell clones from a rheumatic heart disease patient display a common TCR usage and a degenerate antigen recognition pattern.

CD4+ T cells are most likely the ultimate effectors of chronic heart lesions in rheumatic heart disease (RHD). We have demonstrated that infiltrating CD4+ T cell clones were able to recognize several heart tissue and streptococcal antigens by molecular mimicry. Clonality analysis of the mitral valve and myocardium infiltrating T cell lines showed several oligoclonal expansions, some of which were found in both sites of the lesions. The results presented in this study showed a degenerate pattern of reactivity of intralesional T cell clones from one RHD patient. Four mitral valve and one papillar muscle-derived T cell clones, presenting the same TCR-BV13 BJ2S7 with same sequences of the CDR3 region recognized different antigens. They expressed two alpha chains at the RNA level and the AV AJ segments were the same for mitral valve T cell clones, but not for the papillar muscle-derived T cell clone. Two other intralesional T cell clones using the same TCR-BV3 JB2S1 segments with identical CDR3 sequences also recognized different antigens. These results indicate that intralesional T cell clones with common TCR usage can recognize several epitopes that probably amplify the deleterious immune reaction. These data, allow us to hypothesize that degenerate T cell recognition may lead to intramolecular degenerate reactivity against epitopes with low homology. This can be a novel mechanism of epitope spreading, of relevance in the increase of epitopes targets that can activate cross-reactive autoimmune T cells.

Changes in the expression of Th17 cell-associated cytokines in the development of rheumatic heart disease.

BACKGROUND: Autoimmunity plays a critical role in the development of rheumatic heart disease (RHD). Recent studies have linked Th17 cells to the autoimmune mechanism associated with RHD. This study aimed to investigate changes in Th17 cell-related cytokine expression in acute and chronic RHD. METHODS: We established a Lewis rat model of experimental RHD, which was induced by inactivated Group A streptococci and complete Freund's adjuvant. After 7- and 24-week intervention treatments, we measured serum levels of interleukin-17 (IL-17) and IL-6, key cytokines associated with Th17 cells, using a Luminex liquichip method, and levels of IL-17 and IL-6 in heart tissues using immunohistochemical assays. Moreover, expression levels of IL-17, IL-21, IL-6, and IL-23 in mitral valve tissues of human RHD patients were also measured using immunohistochemistry. RESULTS: Compared with the normal control group, serum IL-17 and IL-6 concentrations were significantly increased, and the expression levels of IL-17 and IL-6 in the mitral valve were also significantly increased in 7- or 24-week RHD rats (P<.017). Compared with the control group, expression of IL-17, IL-21, IL-6, and IL-23 in mitral valve tissues was significantly increased in RHD patients (P<.05). CONCLUSIONS: Our study suggested that the increased expression of Th17 cell-associated cytokines might play an important role in the pathogenesis and development of RHD.

Doppler-derived gradients in normally functioning Monostrut Björk-Shiley prostheses.

In summary, reference values of Doppler gradients obtained in a large number of patients with normal-functioning mitral and aortic Monostrut Björk-Shiley prostheses are reported. It is shown that the value of the transprosthetic gradient increases with decreasing valve size in patients with aortic prostheses. No individual significant variations of the transprothetic Doppler gradient during a 3-year follow-up were observed.

Valvular heart disease in patients with hypocomplementemic urticarial vasculitis syndrome associated with Jaccoud's arthropathy.

BACKGROUND: Since 1973, more than 75 patients with hypocomplementemic urticarial vasculitis syndrome (HUVS) were reported, but valvular heart disease does not seem to have been noted in these patients. Since 1993, however, five patients with HUVS accompanied by Jaccoud's arthropathy (JA) were found to have serious valvular heart disease. METHODS: To characterize the cardiac valvulopathy of the third patient with HUVS/JA to have undergone valve replacement, this study included the use of routine and special tissue stains, as well as immunohistochemical staining. We compared gross and histologic findings of this patient's valve to those of two other patients with this complex syndrome who underwent valve replacement. Pathologic findings of these latter two patients were described in separate earlier reports. RESULTS: Histologic examination of the resected valves in all three patients showed an acute necrotizing endocarditis and fibrin deposition on the surface of valve leaflets. Beneath the surfaces of the leaflets, there was evidence of chronic inflammation, consisting of lymphocytes and histiocytes. A fibrocalcific degenerative change was also present in all three valves. Positive staining for IgG, IgA, IgM, and light-chain determinant-bearing proteins was detected primarily at the valve surface in special studies of the aortic valve of the patient described in the current report. CONCLUSION: Patients with HUVS and associated JA should be evaluated for the presence of valvular heart disease. The latter is probably a nonrheumatic, inflammatory, and degenerative process, mediated by immune complex, as well as cellular immune mechanisms.

Silver-coated prosthetic heart valve: a double-bladed weapon.

A St. Jude Medical Silzone was implanted in a 72-year-old female, suffering from mitral valve disease. Four months later, the patient had acute cardiac failure due to partial detachment of the prosthetic valve. The mitral annulus was ulcerated and there were multiple erosions in the myocardial tissue in contact with the prosthetic valve. Histological examination revealed chronic inflammation with hemosiderine deposits and giant cells. No allergy to silver ions was found. The silver-coated sewing cuff had caused a chronic inflammatory reaction due to a toxic reaction to silver. The Silzone valve was withdrawn from the market on January 2000.

[Cardiac lesion in psoriasis]. / Porazhenie serdtsa pri psoriaze.

The heart of 12 patients with psoriasis at the stage of exacerbation compared to that of 16 practically healthy persons who had died of trauma was studied at the necropsy medical-forensic examination. Microcirculatory vessels with the wall thickening, plasmatic impregnation, mononuclear cell proliferation, focal stromal sclerosis were observed in the myocardium. Disorganization of connective tissue with mucoid edema and diffuse mononuclear cell infiltration of mitral and aortal valves was observed. These alterations correspond to slowly progressing chronic myocarditis and diffuse valvulitis.

Analysis of the coverage capacity of the StreptInCor candidate vaccine against Streptococcus pyogenes.

Streptococcus pyogenes is responsible for infections as pharyngitis, sepsis, necrotizing fasciitis and streptococcal toxic shock syndrome. The M protein is the major bacterial antigen and consists of both polymorphic N-terminal portion and a conserved region. In the present study, we analyzed the in vitro ability of StreptInCor a C-terminal candidate vaccine against S. pyogenes to induce antibodies to neutralize/opsonize the most common S. pyogenes strains in Sao Paulo by examining the recognition by sera from StreptInCor immunized mice. We also evaluated the presence of cross-reactive antibodies against human heart valve tissue. Anti-StreptInCor antibodies were able to neutralize/opsonize at least 5 strains, showing that immunization with StreptInCor is effective against several S. pyogenes strains and can prevent infection and subsequent sequelae without causing autoimmune reactions.

Circulating cytokine concentrations in dogs with different degrees of myxomatous mitral valve disease.

Cytokines have been associated with the progression of congestive heart failure (CHF) in humans and may be implicated in the pathophysiology of myxomatous mitral valve disease (MMVD) in dogs. The aim of this study was to determine the serum concentrations of cytokines in dogs with MMVD. The study included 16 Cairn terriers with no or minimal mitral regurgitation (MR), 41 Cavalier King Charles Spaniels (CKCS) with different degrees of MR and 11 dogs of different breeds with CHF due to MMVD. Granulocyte-macrophage colony-stimulating factor, interferon-γ, interleukin (IL)-2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, keratinocyte-derived chemokine, interferon-γ-induced protein and monocyte chemoattractant protein-1 (MCP-1) were measured using a canine-specific multiplex immunoassay. CHF dogs had significantly higher MCP-1 concentrations than dogs with no or minimal MR. Among the CKCS, IL-2 and IL-7 decreased with increasing left atrial size and IL-7 also decreased with increasing MR. IL-8 decreased with increasing left ventricular end-systolic internal dimensions. MCP-1 was increased in CHF dogs compared to healthy control dogs and IL-2, IL-7 and IL-8 decreased with increasing indices of disease severity. The results suggest a role for these cytokines in canine MMVD and CHF.

PDIA3, HSPA5 and vimentin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients.

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD.