Surgical repair and replacement for native mitral valve infective endocarditis.

AIMS: The clinical benefits of mitral valve repair over replacement in the setting of mitral infective endocarditis are not clearly established. METHODS: Data of patients who underwent cardiac surgery for infective endocarditis over a 20-year period (2001-2021) at two cardiac centres were reviewed. Among them, 282 patients underwent native mitral valve surgery and were included in the study. Nearest-neighbour propensity-score matching was performed to account for differences in patients' profile between the repair and replacement subgroups. RESULTS: Mitral valve replacement was performed in 186 patients, while in 96 cases patients underwent mitral valve repair. Propensity match analysis provided 89 well matched pairs. Mean age was 60 ±â€Š15 years; 75% of the patients were male. Mitral valve replacement was more commonly performed in patients with involvement of both mitral leaflets, commissure(s) and mitral annulus. Patients with lesion(s) limited to P2 segment formed the majority of the cases undergoing mitral valve repair. There was no difference in terms of microbiological findings. In-hospital mortality was 7% with no difference between the repair and the replacement cohorts. Survival probabilities at 1, 5 and 10 years were 88%, 72% and 68%, respectively after mitral repair, and 88%, 78% and 63%, respectively after mitral replacement (log-rank P  = 0.94). CONCLUSIONS: Mitral valve repair was more commonly performed in patients with isolated single leaflet involvement and provided good early and 10-year outcomes. Patients with annular disruption, lesion(s) on both leaflets and commissure(s) were successfully served on early and mid-term course by mitral valve replacement.

Systematic coronary physiology improves level of agreement in diagnostic coronary angiography.

OBJECTIVE: The training of interventional cardiologists (ICs), non-interventional cardiologists (NICs) and cardiac surgeons (CSs) differs, and this may be reflected in their interpretation of invasive coronary angiography (ICA) and management plan. Availability of systematic coronary physiology might result in more homogeneous interpretation and management strategy compared with ICA alone. METHODS: 150 coronary angiograms from patients with stable chest pain were presented independently to three NICs, three ICs and three CSs. By consensus, each group graded (1) coronary disease severity and (2) management plan, using options: (a) optimal medical therapy alone, (b) percutaneous coronary intervention, (c) coronary artery bypass graft or (d) more investigation required. Each group was then provided with fractional flow reserve (FFR) from all major vessels and asked to repeat the analysis. RESULTS: There was only 'fair' level of agreement of management plan among ICs, NICs and CSs (kappa 0.351, 95% CI 0.295-0.408, p<0.001) based on ICA alone (complete agreement in 35% of cases), which almost doubled to 'good' level (kappa 0.635, 95% CI 0.572-0.697, p<0.001) when comprehensive FFR was available (complete agreement in 66% of cases). Overall, the consensus management plan changed in 36.7%, 52% and 37.3% of cases for ICs, NICs and CSs, respectively, when FFR data were available. CONCLUSIONS: Compared with ICA alone, the availability of systematic FFR of all major coronary arteries produced a significantly more concordant interpretation and more homogeneous management plan among IC, NIC and CS specialists. Comprehensive physiological assessment may be of value in routine care for Heart Team decision-making. TRIAL REGISTRATION NUMBER: NCT01070771.

Impact of valve size, predicted effective and indexed effective orifice area after aortic valve replacement.

INTRODUCTION: The impact of manufacturer labeled prosthesis size and predicted effective orifice area (EOA) on long-term survival after aortic valve replacement is not clear although indexed effective orifice area (iEOA) has been associated with worse survival. METHODS: Data was retrospectively collected from Jan 2000-Dec 2019 for prosthesis type, model, and size for isolated aortic valve replacements. Stratified survival was compared between groups and subgroups for labeled valve size, EOA and predicted patient prosthesis mismatch (PPM). RESULTS: A total of 3444 patients were included. Moderate and severe PPM was 15.6% and 1.6%, respectively. Cumulative lifetime hazard was worse for biological valves (mortality: biological 77.7% vs. mechanical 64.8%, p = .001). Moderate prosthetic aortic stenosis (AS), (EOA = 1-1.5 cm2 ) was 12.1% and severe prosthetic AS (EOA ≤ 1 cm2 ) was 0.8%, respectively. Survival was 10.5 ± 0.4 years with moderate to severe prosthetic AS (EOA≤1.5 cm2 ) versus 12.6 ± 0.2 years with mild to no prosthetic AS (EOA>1.5 cm2 ), p = .001. Worse survival in the presence of moderate-severe prosthetic AS was seen with biological valves (9.7 ± 0.4 years vs. 11.2 ± 0.2 years, p = .001 for EOA≤1.5, >1.5 cm2 , respectively). Moderate to severe PPM was associated with worse survival (11.1 ± 0.4 years for iEOA ≤ 0.85 cm2 /m2 vs. 12.5 ± 0.2 years with iEOA > 0.85 cm2 /m2 , p = .001). Moderate to severe PPM predicted worse long term survival (hazard ratio: 3.56; 95% confidence interval: 1.37-9.25; p = .009). CONCLUSION: Predicted prosthetic moderate to severe AS and moderate to severe PPM adversely affect long term survival. Smaller valves are associated with reduced survival.

Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state.

We have shown previously that transgene expression can be suppressed in hematopoietic cells using vectors that are responsive to microRNA (miRNA) regulation. Here we investigate the potential of this approach for more sophisticated control of transgene expression. Analysis of the relationship between miRNA expression levels and target mRNA suppression suggested that suppression depends on a threshold miRNA concentration. Using this information, we generated vectors that rapidly adjust transgene expression in response to changes in miRNA expression. These vectors sharply segregated transgene expression between closely related states of therapeutically relevant cells, including dendritic cells, hematopoietic and embryonic stem cells, and their progeny, allowing positive/negative selection according to the cells' differentiation state. Moreover, two miRNA target sites were combined to restrict transgene expression to a specific cell type in the liver. Notably, the vectors did not detectably perturb endogenous miRNA expression or regulation of natural targets. The properties of miRNA-regulated vectors should allow for safer and more effective therapeutic applications.

In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance.

Liver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFNalphabeta response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNalphabeta, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a cell-type-specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected.

Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidence indicates that miRNAs have distinct expression profiles and play crucial roles in numerous cellular processes, although the extent of miRNA regulation is not well known. By challenging mice with lentiviral vectors encoding target sequences of endogenous miRNAs, we show the efficiency of miRNAs in sharply segregating gene expression among different tissues. Transgene expression from vectors incorporating target sequences for mir-142-3p was effectively suppressed in intravascular and extravascular hematopoietic lineages, whereas expression was maintained in nonhematopoietic cells. This expression profile, which could not be attained until now, enabled stable gene transfer in immunocompetent mice, thus overcoming a major hurdle to successful gene therapy. Our results provide novel in situ evidence of miRNA regulation and demonstrate a new paradigm in vector design with applications for genetic engineering and therapeutic gene transfer.

Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction.

The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture, they display low permissivity to the vector, requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation, we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays, we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer, allowing the reach of very high levels of vector integration in their progeny in vivo. Thus, LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly, cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors, highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy.

Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two real-time calibrated PCR assays.

Sensitive and specific techniques to detect and identify Mycobacterium tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (TB). We developed two real-time PCR assays, based on the IS6110 multicopy element and on the senX3-regX3 intergenic region, which provide a rapid method for the diagnosis of mycobacterial infections. The sensitivity and specificity of both assays were established by using purified DNA from 71 clinical isolates and 121 clinical samples collected from 83 patients, 20 of whom were affected by TB. Both assays are accurate, sensitive, and specific, showing a complementary pattern of Mycobacterium recognition: broader for the IS6110-based assay and restricted to the M. tuberculosis complex for the senX3-regX3-based assay. Moreover, the addition of a synthetic DNA calibrator prior to DNA extraction allowed us to measure the efficiency of DNA recovery and to control for the presence of PCR inhibitors. The mycobacterial burden of the clinical samples, as assessed by direct microscopy, correlates with the M. tuberculosis DNA load measured by the senX3-regX3-based assay. In addition, reduced levels of M. tuberculosis DNA load are present in those patients subjected to successful therapy, suggesting a potential use of this assay for monitoring treatment efficacy. Therefore, these assays represent a fully controlled high-throughput system for the evaluation of mycobacterial burden in clinical specimens.

Detection of Pneumocystis carinii and characterization of mutations associated with sulfa resistance in bronchoalveolar lavage samples from human immunodeficiency virus-infected subjects.

One hundred ninety-four bronchoalveolar specimens were evaluated by microscopic examination and by amplification of a sequence of a Pneumocystis carinii dihidropteroate synthase gene for identification of mutations linked to sulfa resistance. PCR sensitivity and specificity were 100 and 86.7%, respectively, compared to results of microscopic examination. However, 7 out of 19 microscopy-negative, PCR-positive samples were collected from subjects with a clinically high probability of P. carinii pneumonia, suggesting that PCR may be more sensitive than microscopic examination, although the absolute performance of PCR cannot be determined. Mutations were identified in 28 out of 70 (40%) PCR-positive specimens and were significantly more common in patients exposed to sulfa drugs (21 out of 29 [72.4%]) than in those not exposed to sulfa drugs (4 out of 35 [11.4%]).

Direct detection of Helicobacter pylori mutations associated with macrolide resistance in gastric biopsy material taken from human immunodeficiency virus-infected subjects.

One hundred forty gastric biopsies were tested by microbiological methods and by amplifying a sequence of 23S rRNA and identifying mutations associated to clarithromycin resistance. Seventy-six specimens were positive for Helicobacter pylori. Mutational analysis revealed alterations in 18 (39.1%) of 46 and 2 (8.7%) of 23 samples from human immunodeficiency virus-seropositive and -seronegative persons, respectively. The results of the mutational analysis fully correlated with those of the susceptibility tests.