The new Ghent criteria for Marfan syndrome: what do they change?

The diagnosis of Marfan syndrome (MFS) is challenging and international criteria have been proposed. The 1996 Ghent criteria were adopted worldwide, but new diagnostic criteria for MFS were released in 2010, giving more weight to aortic root aneurysm and ectopia lentis. We aimed to compare the diagnosis reached by applying this new nosology vs the Ghent nosology in a well-known series of 1009 probands defined by the presence of an FBN1 mutation. A total of 842 patients could be classified as MFS according to the new nosology (83%) as compared to 894 (89%) according to the 1996 Ghent criteria. The remaining 17% would be classified as ectopia lentis syndrome (ELS), mitral valve prolapse syndrome or mitral valve, aorta, skeleton and skin (MASS) syndrome, or potential MFS in patients aged less than 20 years. Taking into account the median age at last follow-up (29 years), the possibility has to be considered that these patients would go on to develop classic MFS with time. Although the number of patients for a given diagnosis differed only slightly, the new nosology led to a different diagnosis in 15% of cases. Indeed, 10% of MFS patients were reclassified as ELS or MASS in the absence of aortic dilatation; conversely, 5% were reclassified as MFS in the presence of aortic dilatation. The nosology is easier to apply because the systemic score is helpful to reach the diagnosis of MFS only in a minority of patients. Diagnostic criteria should be a flexible and dynamic tool so that reclassification of patients with alternative diagnosis is possible, requiring regular clinical and aortic follow-up.

Familial dilated cardiomyopathy. Clinical and genetic characteristics.

Familial dilated cardiomyopathy (F-DCM) describes a clinically and genetically heterogeneous group of diseases, mostly inherited as autosomal dominant traits, having idiopathic left ventricular dilatation and dysfunction as a common phenotype. The age of onset, rate of progression, disease complications, as well as overall prognosis and outcome vary both amongst and within families. Clinical traits, both cardiac and extracardiac, may recur in association with the DCM phenotype. The former include conduction defects, structural abnormalities such as left ventricular noncompaction, of right ventricular involvement, and recurrence of atrial or ventricular arrhythmias; the latter commonly affect the musculoskeletal (myopathies/dystrophies, both clinically overt and subclinical), ocular, auditory, nervous, and integument systems. These traits may help guide genetic testing. In parallel to the clinical heterogeneity, F-DCM also shows genetic heterogeneity: more than 40 genes have been causally linked to F-DCM, with mutations recurring more commonly in a few known genes, and less frequently in rare, less commonly known genes. Based on the known prevalence of mutations in disease genes, more than 50% of F-DCM cases can be regarded as still genetically orphan, implying that further disease genes have to be discovered. Family screening and genetic testing are now established as the gold standard for diagnosis, care, and prevention in F-DCM. Diagnostic tests are performed using Sanger-based sequencing. Furthermore, new biotechnology tools, based on next-generation sequencing, are now being implemented in the research setting and will dramatically modify the future of the nosology of F-DCM.

Virologic and immunologic monitoring of cytomegalovirus to guide preemptive therapy in solid-organ transplantation.

Control of human cytomegalovirus (HCMV) infection during the posttransplant period was investigated in 134 solid-organ transplant recipients by monitoring in parallel virologic and immunologic parameters for at least 1 year of follow-up. Virologic monitoring was achieved by determining HCMV DNAemia with real-time PCR, using the threshold of 300 000 DNA copies/mL blood as a cutoff for starting preemptive therapy. Immunologic monitoring included measurement of HCMV-specific CD4+ and CD8+ T cells by cytokine flow cytometry, using HCMV-infected dendritic cells as a stimulus. HCMV infection was diagnosed in 110 (82%) and required treatment in 49 (36%) patients. At 12 months after transplantation 'protective' immunity (≥0.4 CD4+ and CD8+ HCMV-specific T cells/µL blood) was achieved in 115/129 (89%) patients. During the entire study period, 122 patients reconstituting HCMV-specific CD4+ and CD8+ T-cell immunity at 60 days posttransplant onward were able to control HCMV infection, except for one patient who developed HCMV disease because of a rejection episode. Patients reconstituting HCMV-specific CD8+ only did not control HCMV infection. In conclusion, the presence of both HCMV-specific CD4+ and CD8+ T cells ≥ 0.4/µL blood appears to be protective against HCMV disease. This result does not apply to patients undergoing antirejection treatment, or reconstituting HCMV-specific CD8+ T cells only.

Preemptive therapy for systemic and pulmonary human cytomegalovirus infection in lung transplant recipients.

The incidence and treatment of both systemic and pulmonary human cytomegalovirus (HCMV) infection as well as HCMV-specific T-cell immune responses were investigated in 57 consecutive lung transplant recipients (LTR) by using as cutoffs for preemptive therapy: 300 000 DNA copies/mL whole blood for systemic infections and 100 000 DNA copies/mL bronchoalveolar lavage fluid for lung infections. Results showed that out of 29/57 LTR (50.9%) needing preemptive antiviral therapy, 15 (51.7%) reached the blood cutoff, 8 (27.6%) the pulmonary cutoff and 6 (20.7%) both the blood and the lung cutoff (3 simultaneously and 3 subsequently). Recovery of HCMV-specific T-cell immune responses was achieved much earlier for CD8+ than CD4+ T cells. However, protection from HCMV reactivation was conferred by the presence of both arms of the T-cell response. In two LTR reaching the pulmonary cutoff and not preemptively treated, a full HCMV-specific CD4+ and CD8+ T-cell response was associated with resolution of lung infection. Antirejection steroid therapy suppressed T-cell immune responses, thus facilitating HCMV reactivation. In conclusion, in LTR, monitoring HCMV infection in both blood and lungs, may improve preemptive therapy efficacy. In addition, monitoring the HCMV-specific T-cell immune response appears useful for predicting control of HCMV infection in the posttransplant period.

Pathogenic FBN1 mutations in 146 adults not meeting clinical diagnostic criteria for Marfan syndrome: further delineation of type 1 fibrillinopathies and focus on patients with an isolated major criterion.

Mutations in the FBN1 gene cause Marfan syndrome (MFS) and have been associated with a wide range of milder overlapping phenotypes. A proportion of patients carrying a FBN1 mutation does not meet diagnostic criteria for MFS, and are diagnosed with "other type I fibrillinopathy." In order to better describe this entity, we analyzed a subgroup of 146 out of 689 adult propositi with incomplete "clinical" international criteria (Ghent nosology) from a large collaborative international study including 1,009 propositi with a pathogenic FBN1 mutation. We focused on patients with only one major clinical criterion, [including isolated ectopia lentis (EL; 12 patients), isolated ascending aortic dilatation (17 patients), and isolated major skeletal manifestations (1 patient)] or with no major criterion but only minor criteria in 1 or more organ systems (16 patients). At least one component of the Ghent nosology, insufficient alone to make a minor criterion, was found in the majority of patients with isolated ascending aortic dilatation and isolated EL. In patients with isolated EL, missense mutations involving a cysteine were predominant, mutations in exons 24-32 were underrepresented, and no mutations leading to a premature truncation were found. Studies of recurrent mutations and affected family members of propositi with only one major clinical criterion argue for a clinical continuum between such phenotypes and classical MFS. Using strict definitions, we conclude that patients with FBN1 mutation and only one major clinical criterion or with only minor clinical criteria of one or more organ system do exist but represent only 5% of the adult cohort.

Contribution of molecular analyses in diagnosing Marfan syndrome and type I fibrillinopathies: an international study of 1009 probands.

BACKGROUND: The diagnosis of Marfan syndrome (MFS) is usually initially based on clinical criteria according to the number of major and minor systems affected following international nosology. The number of FBN1 mutation carriers, at risk of aortic complications who would not be properly diagnosed based only on clinical grounds, is of growing importance owing to the increased availability of molecular screening. The aim of the study was to identify patients who should be considered for FBN1 mutation screening. METHODS: Our international series included 1009 probands with a known FBN1 mutation. Patients were classified as either fulfilling or not fulfilling "clinical" criteria. In patients with unfulfilled "clinical" criteria, we evaluated the percentage of additional patients who became positive for international criteria when the FBN1 mutation was considered. The aortic risk was evaluated and compared in patients fulfilling or not fulfilling the "clinical" international criteria. RESULTS: Diagnosis of MFS was possible on clinical grounds in 79% of the adults, whereas 90% fulfilled the international criteria when including the FBN1 mutation. Corresponding figures for children were 56% and 85%, respectively. Aortic dilatation occurred later in adults with unfulfilled "clinical criteria" when compared to the Marfan syndrome group (44% vs 73% at 40 years, p<0.001), but the lifelong risk for ascending aortic dissection or surgery was not significantly different in both groups. CONCLUSIONS: Because of its implications for aortic follow-up, FBN1 molecular analysis is recommended in newly suspected MFS when two systems are involved with at least one major system affected. This is of utmost importance in patients without aortic dilatation and in children.

Chemokine redundancy in BOS pathogenesis. A possible role also for the CC chemokines: MIP3-beta, MIP3-alpha, MDC and their specific receptors.

Bronchiolitis obliterans syndrome (BOS) is one of the most important factors limiting the long-term survival of lung transplant recipients (LTR), however its pathogenesis still remains unclear. We hypothesized that an increased production of certain specific proinflammatory mediators in the first post-transplant year would predispose to BOS. We retrospectively evaluated temporal kinetics of some CC chemokines that have not yet been evaluated, including CCL3/MIP1-alpha, CCL4/MIP1-beta, CCL17/TARC, CCL19/MIP3-beta, CCL20/MIP3-alpha, CCL22/MDC and CCL26/eotaxin, in broncho-alveolar lavage fluid (BAL-f) in the first post-transplant year in a cohort of 8 LTR before the development of BOS (pre-BOS LTR) and 8 LTR with long-term stable clinical conditions (stable LTR). Chemokine levels were assayed by means of a multiplex sandwich ELISA. Furthermore, for those ligands which resulted significantly predictive of BOS onset, we analyzed the expression of specific receptors (CCR) on BAL cells. The proportion of CCR-expressing BAL cells was assessed by flow cytometry. We demonstrated that MIP3-beta/CCL19, MIP3-alpha/CCL20, MDC/CCL22 levels at 6 months post-transplant significantly predicted BOS onset. In addition, the temporal behavior of these factors resulted significantly different in pre-BOS patients as compared to stable LTR. Finally the expression of CCR was documented on BAL lymphocytes and macrophages, and, in some cases, their expression was found to vary between the two groups. Within the complexity of the chemokine network, these three CCL factors could play an additive role in the pathogenesis of the inflammatory process leading to bronchiolar fibro-obliteration.

In vitro generation of human cytomegalovirus pp65 antigenemia, viremia, and leukoDNAemia.

Immunocompromised patients with disseminated human cytomegalovirus (HCMV) infection have circulating PMN carrying HCMV pp65 (antigenemia), infectious virus (viremia), and viral DNA (leukoDNAemia). Because HCMV does not fully replicate in PMN, it is generally hypothesized that virions and viral materials are taken up by phagocytosis from fully permissive HCMV-infected endothelial cells. However, no experimental evidence has ever been provided for these PMN-endothelium interactions. PMN from 11 donors were cocultured with endothelial cells infected with an endothelium-adapted HCMV strain and with human fibroblasts infected with low-passaged clinical and laboratory-adapted HCMV strains. pp65-positive PMN were detected after coculture with both HCMV-infected endothelial and fibroblast cells, provided that wild and not laboratory-adapted strains were used. In addition, cocultured PMN carried infectious virus as demonstrated by virus isolation and presence of complete virus particles by electron microscopy. Moreover, high levels of viral DNA were consistently detected by quantitative PCR in cocultured PMN. Thus, we have generated in vitro the three most important viral parameters detected in patients with disseminated HCMV infection (antigenemia, viremia, and leukoDNAemia). The failure of laboratory-adapted HCMV strain to induce this phenomenon demonstrates that important modifications have occurred in attenuated viral strains affecting basic biological functions.

Peripheral CD4+ CD25+ Treg cell expansion in lung transplant recipients is not affected by calcineurin inhibitors.

CD4+CD25+ regulatory T (Treg) cells have been shown to play a role in allograft tolerance and their peripheral counts vary according to the degree of graft acceptance in lung transplant recipients (LTR). Recent studies demonstrate that certain drugs might modulate generation, expansion and activity of Treg cells. Aim of this study was to evaluate the effect of therapeutic regimens used in our institution on peripheral CD4+CD25(high)CD69- Treg cell numbers in a group of 51 LTR with stable clinical conditions. They were treated with standard immunosuppression: calcineurin inhibitor (CNI)+azathioprine (AZA)+steroids (n=28) or with CNI+mycophenolate mofetil (MMF)+steroids (n=11) or with CNI+steroids (n=12). These stable LTR were compared with age-matched healthy controls (n=35) and with 19 LTR who developed bronchiolitis obliterans syndrome (BOS) and were treated analogously. Stable LTR showed higher peripheral Treg cell counts with respect to age-matched healthy controls (59.9+/-31.8/mul versus 42.1+/-16.9/mul, respectively; p<0.05). This increase was detectable in all patients treated with CNI either in association with AZA or MMF. During these treatments a significant expansion of Treg cell counts was detectable during acute rejection (AR) episodes (86.03+/-26.6/mul during AR versus 36.34+/-7.6 before AR; p<0,05). Moreover, the development of BOS was associated to a significant decrease of Treg cell counts irrespective to the immunosuppressive regimen used. In conclusion, therapeutic regimens based on CNI seem to allow a certain degree of peripheral Treg cell expansion in stable LTR.

Immunosuppression and cancer: A comparison of risks in recipients of organ transplants and in HIV-positive individuals.

The comparison of cancers occurring excessively among HIV-infected and transplanted individuals may help to elucidate the relationship between immune surveillance, viral infections, and cancer. A longitudinal study was conducted on 2002 HIV-infected Italian subjects, 6072 HIV-infected French individuals, and 2878 Italian recipients of solid organ transplants. Standardized incidence ratios (SIR) and 95% confidence intervals (CI) were computed to quantify the risk for cancer, compared with the French and Italian general populations. The SIRs for all cancers were 9.8 (95% CI: 9.0-10.6) for HIV-infected individuals versus 2.2 (95% CI: 1.9-2.5) for transplant recipients. In both groups, most of the excess risk was attributable to virus-related cancers, such as Kaposi's sarcoma (KS; SIR = 451 in HIV-positive individuals, 125 in transplant recipients), non-Hodgkin's lymphoma (NHL; SIR = 62.1 and 11.1, respectively), and liver cancer (SIR = 9.4 and 4.1, respectively). Significantly increased SIRs for anal cancer and Hodgkin's lymphoma were found only among HIV-positive individuals. Among women younger than 40 years of age, a more than 10-fold increase in cervical cancer risk was found in both groups. Among HIV-infected individuals treatment with highly active antiretroviral therapies drastically reduced SIRs for KS and NHL only. These results show that HIV-infected individuals and transplant recipients share a similar pattern of cancer risk, largely due to virus-related cancers.

Incidence of graft rejection in small bowel transplanted pigs after immunosuppression withdrawal.

As intestinal grafts require heavy immunosuppression, there are no reports of immunosuppression withdrawal after clinical small bowel transplantation. In this large-animal study, we investigated the occurrence of graft rejection in intestinal-transplanted pigs after withdrawal. Large-White unrelated piglets were transplanted and divided in three groups: group 1 (n = 5), intestinal transplantation (ITx) with no immunosuppression; group 2 (n = 7), Itx and 60 days of treatment with tacrolimus and mycophenolate mofetil; group 3 (n = 5), Itx and donor bone marrow infusion (DBMi) and 60 days of treatment with tacrolimus and mycophenolate mofetil. Follow-up time after withdrawal was 120 days. Group 1 pigs died of graft acute cellular rejection (ACR) after a median of 11 days. In group 2, two pigs died of ACR-related infection and another two of ACR within 90 days. The remaining three animals (43%) were sacrificed at day 180, and their grafts showed no signs of ACR. In group 3, two pigs died of ACR-related infection and one of graft versus host disease within 80 days; at day 180 the two surviving animals showed signs of chronic rejection in the allograft. This study demonstrates that total withdrawal after ITx is followed by sudden and lethal ACR (or ACR-related infection) in more than 50% of the recipients. When a tolerance-inducing strategy as DBMi is applied, lethal graft versus host disease may also occur. In group 3, the intestinal allograft, to which the recipients were partially tolerant, developed chronic rejection that was probably associated with a decline with time of donor-leukocytes chimerism, as recently demonstrated in rats.

Prevalence and characteristics of dystrophin defects in adult male patients with dilated cardiomyopathy.

OBJECTIVES: To assess the prevalence of dystrophin defects in dilated cardiomyopathy (DCM) in male patients and to formulate investigation strategies for their identification. BACKGROUND: Dystrophin defects presenting with predominant or exclusive cardiac involvement may be clinically indistinguishable from "idiopathic" DCM. Diagnosis may be missed, unless specifically investigated. METHODS: Clinical and biochemical evaluation, right ventricular endomyocardial biopsy (EMB), light and electron microscopic and immunohistochemical studies of biopsy samples, six multiplex and two single polymerase chain reactions for 38 exons and automated sequencing of exon 9 and muscle promoter-exon 1 were undertaken in 201 consecutive male patients presenting with DCM, with (n = 14) and without (n = 187) increased serum creatine phosphokinase (sCPK). RESULTS: Dystrophin defects were identified in 13 of the 201 patients (6.5%, age 16-50). Family history was positive in four patients. Serum CPK levels were increased in 11 of 13 patients. Light microscopy examination of EMB was uninformative; ultrastructural study showed multiple membrane defects. Dystrophin immunostain was abnormal. Eight patients, all older than 20, had deletions affecting midrod domain, normal or mildly increased CPK and better outcome than the five remaining cases all younger than 20, with more than five-fold increase of sCPK. Two of these latter had proximal and rod-domain deletions. Sisters of two patients were diagnosed as noncarriers with microsatellite analysis. CONCLUSIONS: Although the overall prevalence of dystrophin defects in our consecutive DCM male series is low (6.5%), immunohistochemical and molecular studies are essential to identify protein and gene defects; screening studies are justified to define prevalence, clinical profile and genotype-phenotype correlation.

Independent and additive prognostic value of right ventricular systolic function and pulmonary artery pressure in patients with chronic heart failure.

OBJECTIVES: We sought a better understanding of the coupling between right ventricular ejection fraction (RVEF) and pulmonary artery pressure (PAP), as it might improve the accuracy of the prognostic stratification of patients with heart failure. BACKGROUND: Despite the long-standing view that systolic function of the right ventricle (RV) is almost exclusively dependent on the afterload that this cardiac chamber must confront, recent studies claim that RV function is an independent prognostic factor in patients with chronic heart failure. METHODS: Right heart catheterization was performed in 377 consecutive patients with heart failure. RESULTS: During a median follow-up period of 17 +/- 9 months, 105 patients died and 35 underwent urgent heart transplantation. Pulmonary artery pressure and thermodilution-derived RVEF were inversely related (r = 0.66, p < 0.001). However, on Cox multivariate survival analysis, no interaction between such variables was found, and both turned out to be independent prognostic predictors (p < 0.001). It was found that RVEF was preserved in some patients with pulmonary hypertension, and that the prognosis of these patients was similar to that of the patients with normal PAP. In contrast, when PAP was normal, reduced RV function did not carry an additional risk. CONCLUSIONS: These observations emphasize the necessity of combining the right heart hemodynamic variables with a functional evaluation of the RV when trying to define the individual risk of patients with heart failure.

Restrictive cardiomyopathy, atrioventricular block and mild to subclinical myopathy in patients with desmin-immunoreactive material deposits.

OBJECTIVES: We present clinical data and heart and skeletal muscle biopsy findings from a series of patients with ultrastructural accumulations of granulofilamentous material identified as desmin. BACKGROUND: Desmin cardiomyopathy is a poorly understood disease characterized by abnormal desmin deposits in cardiac and skeletal muscle. METHODS: Clinical evaluation, endomyocardial and skeletal muscle biopsy, light and electron microscopy and immunohistochemistry were used to establish the presence of desmin cardiomyopathy. RESULTS: Six hundred thirty-one patients with primary cardiomyopathy underwent endomyocardial biopsy (EMB). Ultrastructural accumulations of granulofilamentous material were found in 5 of 12 biopsy samples from patients with idiopathic restrictive cardiomyopathy and demonstrated specific immunoreactivity with anti-desmin antibodies by immunoelectron microscopy. Immunohistochemical findings on light microscopy were nonspecific because of a diffuse intracellular distribution of desmin. All five patients had atrioventricular (AV) block and mild or subclinical myopathy. Granulofilamentous material was present in skeletal muscle biopsy samples in all five patients, and unlike the heart biopsy samples, light microscopic immunohistochemical analysis demonstrated characteristic subsarcolemmal desmin deposits. Two patients were first-degree relatives (mother and son); another son with first-degree AV block but without myopathy or cardiomyopathy demonstrated similar light and ultrastructural findings in skeletal muscle. Electrophoretic studies demonstrated two isoforms of desmin--one of normal and another of lower molecular weight--in cardiac and skeletal muscle of the familial cases. CONCLUSIONS: Desmin cardiomyopathy must be considered in the differential diagnosis of restrictive cardiomyopathy, especially in patients with AV block and myopathy. Diagnosis depends on ultrastructural examination of EMB samples or light microscopic immunohistochemical studies of skeletal muscle biopsy samples. Familial desminopathy may manifest as subclinical disease and may be associated with abnormal isoforms of desmin.

Detection of Epstein Barr virus in formalin-fixed paraffin tissues by fluorescent direct in situ PCR.

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. However, molecular detection is also considered the reference standard assay for diagnosis of central nervous system infections and of most cases of nasopharyngeal carcinoma (NPC). One-step or nested polymerase chain reaction (PCR) has rapidly replaced immunological assays based on virus-specific Ig antibodies for the laboratory diagnosis of Herpesvirus infections, even if serological methods are considered an additional tool for defining clinical diagnosis. In this article, we will present a rapid, sensitive and robust molecular tool for the viral detection of EBV (EBNA-1) within tissue specimens by making use of in situ PCR (IS-PCR).

Multicolor fluorescence technique to detect apoptotic cells in advanced coronary atherosclerotic plaques.

Apoptosis occurring in atherosclerotic lesions has been suggested to be involved in the evolution and the structural stability of the plaques. It is still a matter of debate whether apoptosis mainly involves vascular smooth muscle cells (vSMCs) in the fibrous tissue or inflammatory (namely foam) cells, thus preferentially affecting the cell-poor lipid core of the atherosclerotic plaques. The aim of the present investigation was to detect the presence of apoptotic cells and to estimate their percentage in a series of atherosclerotic plaques obtained either by autopsy or during surgical atherectomy. Apoptotic cells were identified on paraffin-embedded sections on the basis of cell nuclear morphology after DNA staining and/or by cytochemical reactions (TUNEL assay, immunodetection of the proteolytic poly (ADP-ribose) polymerase-1 [PARP-1] fragment); biochemical procedures (identifying DNA fragmentation or PARP-1 proteolysis) were also used. Indirect immunofluorescence techniques were performed to label specific antigens for either vSMCs or macrophages (i.e., the cells which are most likely prone to apoptosis in atherosclerotic lesions): the proper selection of fluorochrome labeling allowed the simultaneous detection of the cell phenotype and the apoptotic characteristics, by multicolor fluorescence techniques. Apoptotic cells proved to be less than 5% of the whole cell population, in atherosclerotic plaque sections: this is, in fact, a too low cell fraction to be detected by widely used biochemical methods, such as agarose gel electrophoresis of low-molecular-weight DNA or Western-blot analysis of PARP-1 degradation. Most apoptotic cells were of macrophage origin, and clustered in the tunica media, near or within the lipid-rich core; only a few TUNEL-positive cells were labeled for antigens specific for vSMCs. These results confirm that, among the cell populations in atherosclerotic plaques, macrophage foam-cells are preferentially involved in apoptosis. Their death may decrease the cell number in the lipid core and generate a possibly defective apoptotic clearance: the resulting release of matrix-degrading enzymes could contribute to weakening the fibrous cap and promote the plaque rupture with the risk of acute ischemic events, while increasing the thrombogenic pultaceous pool of the plaque core.

Phenotype commitment in vascular smooth muscle cells derived from coronary atherosclerotic plaques: differential gene expression of endothelial nitric oxide synthase.

Unstable angina and myocardial infarction are the clinical manifestations of the abrupt thrombotic occlusion of an epicardial coronary artery as a result of spontaneous atherosclerotic plaque rupture or fissuring, and the exposure of highly thrombogenic material to blood. It has been demonstrated that the proliferation of vascular smooth muscle cells (VSMCs) and impaired bioavailabilty of nitric oxide (NO) are among the most important mechanisms involved in the progression of atherosclerosis. It has also been suggested that a NO imbalance in coronary arteries may be involved in myocardial ischemia as a result of vasomotor dysfunction triggering plaque rupture and the thrombotic response. We used 5' nuclease assays (TaqMan PCRs) to study gene expression in coronary plaques collected by means of therapeutic directional coronary atherectomy from 15 patients with stable angina (SA) and 15 with acute coronary syndromes (ACS) without ST elevation. Total RNA was extracted from the 30 plaques and the cDNA was amplified in order to determine endothelial nitric oxide synthase (eNOS) gene expression. Analysis of the results showed that the expression of eNOS was significantly higher (p<0.001) in the plaques from the ACS patients. Furthermore, isolated VSMCs from ACS and SA plaques confirmed the above pattern even after 25 plating passages. In situ RT-PCR was also carried out to co-localize the eNOS messengers and the VSMC phenotype. The eNOS gene was more expressed in ACS plaques and VSMCs cultured from them, thus indicating that: a) the expression of the most important differentiation markers is retained under in vitro conditions; and b) NO may play a pivotal role in coronary artery disease. Our findings suggest a new cell system model for studying the pathophysiology of unstable angina and myocardial infarction.

The mitochondrial DNA mutation T12297C affects a highly conserved nucleotide of tRNA(Leu(CUN)) and is associated with dilated cardiomyopathy.

Mitochondrial DNA (mtDNA) mutations have been causally linked with cardiomyopathies, both dilated (DCM) and hypertrophic. We identified the T12297C mutation in the mtDNA-tRNA(Leu(CUN)) of a 36-year-old male patient diagnosed with DCM. The mutation was heteroplasmic, with high amount (88%) of mutant DNA in the myocardium, and was absent in normal (n = 120) and disease (n = 150) controls. It affects a highly conserved nucleotide (adjacent to the anticodon triplet) that allows the phospho-ribose backbone to turn and form the loop. The potential pathological role of T12297C mutation is further supported by its recent identification in another unrelated Italian family with DCM associated with endocardial fibroelastosis. In the variable loop of the same tRNA, our patient also carried the A12308G transition that is debated as pathological mutation or neutral polymorphism in progressive external ophthalmoplegia: the two defects could exert a synergistic effect on the tRNA structure and function. The endomyocardial biopsy study showed abnormal ring-like mitochondria and occasional cytochrome c oxydase negative myocytes. Overall, the heteroplasmy, the highly conserved position of the mutated nucleotide, the absence of the mutation in large series of diseased and normal controls, and the cardiac mitochondrial changes support a causative link of the mutation with the disease.