Improvement of the reverse transcription loop mediated isothermal amplification (RTLAMP) method for the detection of Peach latent mosaic viroid (PLMVd)

Peach latent mosaic viroid (PLMVd) is the most known peach viroid. Among the diagnostic techniques used for its detection, the most recent described being the reverse transcription loop-mediated isothermal amplification method (RT-LAMP). Several modifications were done on the basic protocol proposed by Boubourakas et al. (2009), additional experiments were preformed in order to further evaluate the method. Namely, the reaction time was further reduced and traces of leaf tissue, taken by a sterile toothpick, instead of tRNA were used as the intial material. Moreover, the AMV reverse transcriptase proved to be more effective than Thermoscript, while restriction enzyme analysis was performed on the RT-LAMP products in order to confirm that products had the respective sequences of the selected target. Finally, the extremely high efficiency and sensitivity of RT-LAMP proved to be sufficient for the detection of PLMVd in hosts other than peach. Keywords: PLMVd, RT-LAMP, peach, reverse transcriptase

Substantia Nigra Volumetry with 3-T MRI in De Novo and Advanced Parkinson Disease

Background: Magnetization transfer–prepared T1-weighted MRI can depict a hyperintense subregion of the substantia nigra involved in the degeneration process of Parkinson disease. / Purpose: To evaluate quantitative measurement of substantia nigra volume by using MRI to support clinical diagnosis and staging of Parkinson disease. / Materials and Methods: In this prospective study, a high-spatial-resolution magnetization transfer–prepared T1-weighted volumetric sequence was performed with a 3-T MRI machine between January 2014 and October 2015 for participants with de novo Parkinson disease, advanced Parkinson disease, and healthy control participants. A reproducible semiautomatic quantification analysis method that entailed mesencephalic intensity as an internal reference was used for hyperintense substantia nigra volumetry normalized to intracranial volume. A general linear model with age and sex as covariates was used to compare the three groups. / Results: Eighty participants were evaluated: 20 healthy control participants (mean age ± standard deviation, 56 years ± 11; 11 women), 29 participants with de novo Parkinson disease (64 years ± 10; 19 men), and 31 participants with advanced Parkinson disease (60 years ± 9; 16 women). Volumetric measurement of hyperintense substantia nigra from magnetization transfer–prepared T1-weighted MRI helped differentiate healthy control participants from participants with advanced Parkinson disease (mean difference for ipsilateral side, 64 mm3 ± 14, P < .001; mean difference for contralateral side, 109 mm3 ± 14, P < .001) and helped distinguish healthy control participants from participants with de novo Parkinson disease (mean difference for ipsilateral side, 45 mm3 ± 15, P < .01; mean difference for contralateral side, 66 mm3 ± 15, P < .001) and participants with de novo Parkinson disease from those with advanced Parkinson disease (mean difference for ipsilateral side, 20 mm3 ± 13, P = .40; mean difference for contralateral side, 43 mm3 ± 13, P = .004). / Conclusion: Magnetization transfer–prepared T1-weighted MRI volumetry of the substantia nigra helped differentiate the stages of Parkinson disease

Are Long Term Cryopreservation and Patency of Vein Allograft Truly Achievable?

Despite extensive experimental work, neither the effect of long term cryopreservation on vein graft architecture nor the failure of alloveins due to graft rejection have yet been investigated. Herein, we investigated ultrastructurally: a) the integrity of rabbit jugular veins following 1, 2 and 3 months of cryopreservation; b) the outcome of the three-month cryopreserved vein auto- and allografts after 1 month of implantation in the rabbit carotid artery; and c) the immunologic response to cryopreserved vein allografts with and without seeded autologous endothelium. Prior to implantation, the cryopreserved rabbit veins were well-maintained except for endothelial cell damage. Following implantation, the cryopreserved vein autografts were comparable to fresh veins with a complete endothelial lining. Conversely, only one of the allograft was still patent with features of acute rejection. After seeding with autologous endothelium , these explants failed shortly after surgery. We found absence of endothelium and necrosis of the media components with neutrophil infiltration. Although three months of cryopreservation does not affect vein graft architecture significantly, endothelial cells are damaged irrespective of the time of cryopreservation. Vein autografts promptly healed after one month of implantation at which time a viable endothelial cell lining was restored from the host artery. Conversely, vein allografts, with and without seeded autologous endothelium, failed due to graft rejection. This study highlights that current methods of cryopreservation do not reduce antigenicity of venous allografts significantly

Probable role of Brain Natriuretic Peptide (BNP) in lung hypertension secondary to scleroderma

BACKGROUND Scleroderma, when complicated with pulmonary hypertension (PHT), presents a worse prognosis; recently treatment with new drugs seems to offer good perspectives, especially in early diagnosis and treatment. The standard approach for diagnosing PHT consists in measurement of the pulmonary artery pressure (PAP) by means of echodoppler. AIM OF INVESTIGATION Aim of this work is evaluating the significance of the NT-proBNP parameter, matched to echodoppler, in diagnosing scleroderma PHT. MATERIALS AND METHODS Sixty (60) patients, who came to observation for progressive systemic sclerosis underwent echodoppler in order to measure the PAP (normal values up to 30 mmHg). NT-proBNP was determined on serum sample using ECLIA method by Modular E170 (Roche Diagnostics); manufacturer reference values for age and gender were used. Forty-three (43) patients underwent a further NT-proBNP sampling 5 days later in order to assess parameter stability. RESULTS PHT and non- PHT patients showed statistically different (p &lt; 0,001) medians (126 vs 69 pg/ml). No pathologic values of NT-proBNP were measured in the group with PAP &lt; 30 mmHg, while 27% of cases who had PAP between 30 and 40 showed pathologic concentrations. The positivity ratio increases to 57% in patients showing PAP &gt; 40 mmHg. No relevant correlation (r = 0,2) was found between PAP and NT-proBNP. Mean average between the two sampling groups was 31%. CONCLUSIONS In scleroderma patients, combination of NT-proBNP and PAP seems to improve the diagnosis of pulmonary hypertension, especially in presence of borderline pulmonary pressure values. We therefore propose the biochemical observation of NT-proBNP when PAP is &gt; 30 mmHg and in monitoring the evolution of the pathology

Low-dose oral imatinib in the treatment of systemic sclerosis interstitial lung disease unresponsive to cyclophosphamide: a phase II pilot study.

NTRODUCTION: Pulmonary involvement represents a major cause of death of systemic sclerosis (SSc) patients. Recent data suggest that tyrosine kinase inhibitors, such as imatinib, may be a therapeutic option for SSc patients. However, preliminary published clinical trials were inconclusive about imatinib efficacy and showed side effects. The purpose of this study was to verify efficacy and tolerability of low-dose imatinib on interstitial lung disease in a cohort of SSc patients unresponsive to cyclophosphamide therapy. METHODS: Thirty consecutive SSc patients with active pulmonary involvement, unresponsive to cyclophosphamide, were treated with imatinib 200 mg/day for 6 months followed by a 6-month follow-up. A "good response" was defined as an increase of forced vital capacity (FVC) by more of 15% and/or increase of diffusing capacity of carbon monoxide (DLCO) >15% and PaO2 > 90% of initial value and high-resolution computed tomography (HRCT)-scan pattern unchanged or improved. RESULTS: Twenty-six patients completed the study. Three patients died and one patient was lost to follow-up. Four patients (15.32%) had a good response, 7 worsened and 15 had a stabilized lung disease. Overall, 19 (73.07%) patients had an improved or stabilized lung disease. After a 6-month follow-up, 12 (54.5%) of the 22 patients showed an improved or stabilized lung disease. CONCLUSIONS: Lung function was stabilized in a large proportion of patients unresponsive to cyclophosphamide therapy and a beneficial outcome emerged from the analysis of HRCT lung scans. There was no significant improvement of skin involvement, and the low dose was well tolerated. These data provide useful suggestions to design future randomized clinical trials for SSc therapeutics

S.13.1 Safety and efficacy of rituximab in SSc: an analysis from the European Scleroderma Trial and Research Group

Objectives. Objective of this multicentre, observational study was to assess effects and safety of rituximab (RTX) using the European Scleroderma Trial and Research Group (EUSTAR) cohort. Methods. EUSTAR centres were asked to provide specific data about SSc patients treated with RTX. Primary endpoints were predefined for different disease manifestations and compared between baseline and follow-up. Normally distributed data, analysed by paired t-test, are shown as mean (s.d.), and non-parametric data, analysed by Wilcoxon matched paired signed-rank test, are shown as median and interquartile range. Results. Data on 72 SSc patients treated with RTX were captured from 27 EUSTAR centres (51 females/21 males, 52 diffuse/19 limited, age 51 (44-60) years, disease duration 6 (3-10) years, 47 anti-Scl-70 positive). The most frequent RTX application scheme was 1000 mg × 2 within 2 weeks (57/72 patients). Co-treatment with other immunosuppressive drugs was reported in 28 patients. The modified Rodnan skin score (mRSS) significantly decreased vs baseline at 7 (5-9) months follow-up (n = 47, 18.2 + 10.9 vs 14.5 + 9.9, P = 0.0002). This was true for both patients with later disease stages and also for patients with earlier, extended skin fibrosis (dSSc with mRSS >16 at baseline, n = 26; 26.5 + 6.8 vs 20.4 + 8.9, P < 0.0001, reduction by 29.9%). S-HAQ was unchanged, but the European SSc activity score improved after rituximab treatment [n = 10; 3.7 (2.6-6.4) vs 1.7 (0.9-2.5), P = 0.01]. RTX had no effects on lung fibrosis (FVC, DLCO, TLC, HRCT score) in n = 11 patients with evidence for SSc-ILD. In SSc-polyarthritis patients, the DAS-28 declined at 6 months follow-up without reaching statistical significance [n = 8; 4.8 (2.5-7.5) vs 3.7 (2.6-6.6); p = 0.3]. Of 8, 5patients were RF and/or anti-CCP antibody positive. Similar results were obtained for secondary outcome measures (tender and swollen joint count, VAS, CRP, ESR). Additional positive effects of RTX were seen on SSc-related myopathy (CK levels, 273 + 177 vs 184 + 139; n = 12, P = 0.03) and on digital ulcers [total number per patient 1 (1-3) vs 0 (0-1); n = 23; P = 0.0086]. During RTX treatment 14 patients had infections, 3 serum sickness, 2 allergic reactions and 1 lung fibrosis aggravation, 29 fatigue and 9 nausea. Four patients died, one possibly related to RTX treatment (pneumonia and cardiac failure 1.5 months after RTX infusion). Conclusion. This large EUSTAR cohort study points at positive effects of RTX in particular on skin fibrosis, and suggests randomized controlled trial in SSc patient

Low-dose oral imatinib in the treatment of systemic sclerosis interstitial lung disease unresponsive to cyclophosphamide: a phase II pilot study

Introduction: Pulmonary involvement represents a major cause of death of systemic sclerosis (SSc) patients. Recent data suggest that tyrosine kinase inhibitors, such as imatinib, may be a therapeutic option for SSc patients. However, preliminary published clinical trials were inconclusive about imatinib efficacy and showed side effects. The purpose of this study was to verify efficacy and tolerability of low-dose imatinib on interstitial lung disease in a cohort of SSc patients unresponsive to cyclophosphamide therapy.Methods: Thirty consecutive SSc patients with active pulmonary involvement, unresponsive to cyclophosphamide, were treated with imatinib 200 mg/day for 6 months followed by a 6-month follow-up. A "good response" was defined as an increase of forced vital capacity (FVC) by more of 15% and/or increase of diffusing capacity of carbon monoxide (DLCO) &gt; 15% and PaO2 &gt; 90% of initial value and high-resolution computed tomography (HRCT)-scan pattern unchanged or improved.Results: Twenty-six patients completed the study. Three patients died and one patient was lost to follow-up. Four patients (15.32%) had a good response, 7 worsened and 15 had a stabilized lung disease. Overall, 19 (73.07%) patients had an improved or stabilized lung disease. After a 6-month follow-up, 12 (54.5%) of the 22 patients showed an improved or stabilized lung disease.Conclusions: Lung function was stabilized in a large proportion of patients unresponsive to cyclophosphamide therapy and a beneficial outcome emerged from the analysis of HRCT lung scans. There was no significant improvement of skin involvement, and the low dose was well tolerated. These data provide useful suggestions to design future randomized clinical trials for SSc therapeutics

B lymphocytes limit senescence-driven fibrosis resolution and favor hepatocarcinogenesis in mouse liver injury

Hepatocellular carcinoma (HCC) is a frequent neoplasia and a leading cause of inflammation-related cancer mortality. Despite that most HCCs arise from persistent inflammatory conditions, pathways linking chronic inflammation to cancer development are still incompletely elucidated. We dissected the role of adaptive immunity in the Mdr2 knockout (Mdr2\u2013/\u2013) mouse, a model of inflammation-associated cancer, in which ablation of adaptive immunity has been induced genetically (Rag2\u2013/\u2013Mdr2\u2013/\u2013 and \u3bcMt-Mdr2\u2013/\u2013 mice) or with in vivo treatments using lymphocyte-specific depleting antibodies (anti-CD20 or anti-CD4/CD8). We found that activated B and T lymphocytes, secreting fibrogenic tumor necrosis factor alpha (TNF\u3b1) and other proinflammatory cytokines, infiltrated liver of the Mdr2\u2013/\u2013 mice during chronic fibrosing cholangitis. Lymphocyte ablation, in the Rag2\u2013/\u2013Mdr2\u2013/\u2013 and \u3bcMt-Mdr2\u2013/\u2013 mice, strongly suppressed hepatic stellate cell (HSC) activation and extracellular matrix deposition, enhancing HSC transition to cellular senescence. Moreover, lack of lymphocytes changed the intrahepatic metabolic/oxidative state, resulting in skewed macrophage polarization toward an anti-inflammatory M2 phenotype. Remarkably, hepatocarcinogenesis was significantly suppressed in the Rag2\u2013/\u2013Mdr2\u2013/\u2013 mice, correlating with reduced TNF\u3b1/NF-\u3baB (nuclear factor kappa B) pathway activation. Ablation of CD20+ B cells, but not of CD4+/CD8+ T cells, in Mdr2\u2013/\u2013 mice, promoted senescence-mediated fibrosis resolution and inhibited the protumorigenic TNF\u3b1/NF-\u3baB pathway. Interestingly, presence of infiltrating B cells correlated with increased tumor aggressiveness and reduced disease-free survival in human HCC. Conclusion: Adaptive immunity sustains liver fibrosis (LF) and favors HCC growth in chronic injury, by modulating innate components of inflammation and limiting the extent of HSC senescence. Therapies designed for B-cell targeting may be an effective strategy in LF. (Hepatology 2018;67:1970-1985)

Fenestrated and Branched Endografts for Post-Dissection Thoraco-Abdominal Aneurysms: Results of a National Multicentre Study and Literature Review

Objective: Fenestrated and branched endografting (F/B-EVAR) has been proposed as an endovascular solution for chronic post-dissection thoraco-abdominal aneurysms (PD-TAAAs). The aim of this study was to analyse the experience of four high volume centres nationwide and the current available literature. Methods: Data on patients undergoing F/B-EVAR in four Italian academic centres between 2008 and 2019 were collected, and those from patients with PD-TAAAs were analysed retrospectively. Peri-operative morbidity and mortality were assessed as early outcomes. Survival, freedom from re-intervention (FFR), target visceral vessel (TVV) patency, and aortic remodelling were assessed as follow up outcomes. A MEDLINE search was performed for studies published from 2008 to 2020 reporting on F/B-EVAR in PD-TAAAs. Results: Among 351 patients who underwent F/B-EVAR for TAAAs, 37 (11%) had PD-TAAAs (Crawford's extent I–III: 35% – 95%). Overall, 135 TVVs (from true lumen 120; false lumen seven; both true and false lumen eight) were accommodated by fenestrations (96% – 71%) and branches (39% – 29%). Technical success (TS) was achieved in 34 (92%) cases with three failures due to endoleaks (Ia: 1; Ic: 1; III: 1). There were no 30 day deaths. No cases of permanent spinal cord ischaemia (SCI) were recorded and six (16%) patients suffered from transient deficits. Renal function worsening (eGFR < 30% than baseline) and pulmonary complications were reported in two (5%) and four (11%) cases, respectively. From the Kaplan–Meier analysis, three year survival, FFR, and TVV patency were 81%, 66%, and 97%, respectively. Radiological imaging was available for 30 (81%) patients at 12 months with complete false lumen thrombosis in 26 (87%). Two hundred and fifty-six patients were reported in seven published papers with TS, 30 day mortality, and SCI ranging from 99% to 100%, 0 to 6%, and 0 to 16%, respectively. The mean follow up ranged from 12 to 26 months, with estimated two year survival between 81% and 90% and a re-intervention rate between 19% and 53%. Conclusion: F/B-EVAR is effective to treat PD-TAAAs. A high re-intervention rate is necessary to complete the aneurysm exclusion and promote aortic remodelling successfully

Human brain harbors single nucleotide somatic variations in functionally relevant genes possibly mediated by oxidative stress

Somatic variation in DNA can cause cells to deviate from the preordained genomic path in both disease and healthy conditions. Here, using exome sequencing of paired tissue samples, we show that the normal human brain harbors somatic single base variations measuring up to 0.48% of the total variations. Interestingly, about 64% of these somatic variations in the brain are expected to lead to non-synonymous changes, and as much as 87% of these represent G:C>T:A transversion events. Further, the transversion events in the brain were mostly found in the frontal cortex, whereas the corpus callosum from the same individuals harbors the reference genotype. We found a significantly higher amount of 8-OHdG (oxidative stress marker) in the frontal cortex compared to the corpus callosum of the same subjects (p<0.01), correlating with the higher G:C>T:A transversions in the cortex. We found significant enrichment for axon guidance and related pathways for genes harbouring somatic variations. This could represent either a directed selection of genetic variations in these pathways or increased susceptibility of some loci towards oxidative stress. This study highlights that oxidative stress possibly influence single nucleotide somatic variations in normal human brain