Completion Dissection or Observation for Sentinel-Node Metastasis in Melanoma.

Sentinel-lymph-node biopsy is associated with increased melanoma-specific survival (i.e., survival until death from melanoma) among patients with node-positive intermediate-thickness melanomas (1.2 to 3.5 mm). The value of completion lymph-node dissection for patients with sentinel-node metastases is not clear. In an international trial, we randomly assigned patients with sentinel-node metastases detected by means of standard pathological assessment or a multimarker molecular assay to immediate completion lymph-node dissection (dissection group) or nodal observation with ultrasonography (observation group). The primary end point was melanoma-specific survival. Secondary end points included disease-free survival and the cumulative rate of nonsentinel-node metastasis. Immediate completion lymph-node dissection was not associated with increased melanoma-specific survival among 1934 patients with data that could be evaluated in an intention-to-treat analysis or among 1755 patients in the per-protocol analysis. In the per-protocol analysis, the mean (±SE) 3-year rate of melanoma-specific survival was similar in the dissection group and the observation group (86±1.3% and 86±1.2%, respectively; P=0.42 by the log-rank test) at a median follow-up of 43 months. The rate of disease-free survival was slightly higher in the dissection group than in the observation group (68±1.7% and 63±1.7%, respectively; P=0.05 by the log-rank test) at 3 years, based on an increased rate of disease control in the regional nodes at 3 years (92±1.0% vs. 77±1.5%; P<0.001 by the log-rank test); these results must be interpreted with caution. Nonsentinel-node metastases, identified in 11.5% of the patients in the dissection group, were a strong, independent prognostic factor for recurrence (hazard ratio, 1.78; P=0.005). Lymphedema was observed in 24.1% of the patients in the dissection group and in 6.3% of those in the observation group. Immediate completion lymph-node dissection increased the rate of regional disease control and provided prognostic information but did not increase melanoma-specific survival among patients with melanoma and sentinel-node metastases. (Funded by the National Cancer Institute and others; MSLT-II ClinicalTrials.gov number, NCT00297895 .)

Replicating viral vector platform exploits alarmin signals for potent CD8⁺ T cell-mediated tumour immunotherapy.

Viral infections lead to alarmin release and elicit potent cytotoxic effector T lymphocyte (CTL <sup>eff</sup> ) responses. Conversely, the induction of protective tumour-specific CTL <sup>eff</sup> and their recruitment into the tumour remain challenging tasks. Here we show that lymphocytic choriomeningitis virus (LCMV) can be engineered to serve as a replication competent, stably-attenuated immunotherapy vector (artLCMV). artLCMV delivers tumour-associated antigens to dendritic cells for efficient CTL priming. Unlike replication-deficient vectors, artLCMV targets also lymphoid tissue stroma cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, artLCMV elicits CTL <sup>eff</sup> responses of higher magnitude and functionality than those induced by replication-deficient vectors. Superior anti-tumour efficacy of artLCMV immunotherapy depends on interleukin-33 signalling, and a massive CTL <sup>eff</sup> influx triggers an inflammatory conversion of the tumour microenvironment. Our observations suggest that replicating viral delivery systems can release alarmins for improved anti-tumour efficacy. These mechanistic insights may outweigh safety concerns around replicating viral vectors in cancer immunotherapy

Age-related endothelial dysfunction in human skeletal muscle feed arteries: the role of free radicals derived from mitochondria in the vasculature

Aim This study sought to determine the role of free radicals derived from mitochondria in the vasculature in the recognized age-related endothelial dysfunction of human skeletal muscle feed arteries (SMFAs). Methods A total of 44 SMFAs were studied with and without acute exposure to the mitochondria-targeted antioxidant MitoQ and nitric oxide synthase (NOS) blockade. The relative abundance of proteins from the electron transport chain, phosphorylated (p-) to endothelial (e) NOS ratio, manganese superoxide dismutase (MnSOD) and the mitochondria-derived superoxide () levels were assessed in SMFA. Endothelium-dependent and endothelium-independent SMFA vasodilation was assessed in response to flow-induced shear stress, acetylcholine (ACh) and sodium nitroprusside (SNP). Results MitoQ restored endothelium-dependent vasodilation in the old to that of the young when stimulated by both flow (young: 68 ± 5; old: 25 ± 7; old + MitoQ 65 ± 9%) and ACh (young: 97 ± 4; old: 59 ± 10; old + MitoQ: 98 ± 5%), but did not alter the initially uncompromised, endothelium-independent vasodilation (SNP). Compared to the young, MitoQ in the old diminished the initially elevated mitochondria-derived levels and appeared to attenuate the breakdown of MnSOD. Furthermore, MitoQ increased the ratio of p-eNOS to NOS and the restoration of endothelium-dependent vasodilation in the old by MitoQ was ablated by NOS blockade. Conclusion This study demonstrated that MitoQ reverses age-related vascular dysfunction by what appears to be an NO-dependent mechanism in human SMFAs. These findings suggest that mitochondria-targeted antioxidants may have utility in terms of counteracting the attenuated blood flow and vascular dysfunction associated with advancing age

Human skeletal muscle feed arteries: evidence of regulatory potential

Aim Recently, it has been recognized that human skeletal muscle feed arteries can be harvested during exploratory surgery for melanoma. This approach provides vessels for in vitro study from a wide spectrum of relatively healthy humans. Although, the regulatory role of skeletal muscle feed arteries in rodent models has been documented, whether such vessels in humans possess this functionality is unknown. Methods Therefore, skeletal muscle feed arteries (~950 μm OD) from 10 humans (48 ± 4, 27–64 years) were studied using pressure myography. Vessel function was assessed using potassium chloride (KCl), phenylephrine (PE), acetylcholine (ACh) and sodium nitroprusside (SNP) concentration–response curves (CRCs) to characterize non-receptor and receptor-mediated vasoconstriction as well as endothelium-dependent and independent vasodilation respectively. To understand the physiological relevance of the diameter changes as a result of pharmacological stimulation, the estimated conductance ratio (CR) was calculated. Results Vessel function protocols revealed significant vasoconstriction in response to PE and KCl (35 ± 6; 43 ± 9%vasoconstriction, respectively) and significant vasodilation with ACh and SNP (85 ± 7; 121 ± 17% vasodilation, respectively). Both PE and KCl significantly reduced the CR (0.26 ± 0.05 and 0.23 ± 0.07, respectively), whereas ACh and SNP increased the CR (2.56 ± 0.10 and 5.32 ± 1.3, respectively). Conclusion These novel findings provide evidence that human skeletal muscle feed arteries are capable of generating significant diameter changes that would translate into significant changes in vascular conductance. Thus, human skeletal muscle feed arteries likely play a significant role in regulating vascular conductance and subsequently blood flow in vivo

α1-Adrenergic responsiveness in human skeletal muscle feed arteries: the impact of reducing extracellular pH

What is the central question of this study? In human arteries involved in the regulation of muscle blood flow, there is a lack of data about whether acidosis alters vascular sensitivity to vasoactive agents, as well as altering endothelium dependent vasorelaxation. Little is known about the interaction of metabolites and vascular function in human skeletal muscle feed arteries. • What is the main finding and its importance? Increasing acidosis attenuated the response and sensitivity of the arteries to phenylephrine; this effect was selective to the receptor over smooth muscle. Acidosis did not alter endothelium dependent vasorelaxation. Impaired vasoconstriction coupled with intact vasorelaxation, promotes decreased vascular tone with exposure to acidosis, and may contribute to sympatholysis during exercise

TRPV1 channels in human skeletal muscle feed arteries: implications for vascular function

New Findings What is the central question of this study? We sought to determine whether human skeletal muscle feed arteries (SFMAs) express TRPV1 channels and what role they play in modulating vascular function. What is the main finding and its importance? Human SMFAs do express functional TRPV1 channels that modulate vascular function, specifically opposing α-adrenergic receptor-mediated vasocontraction and potentiating vasorelaxation, in an endothelium-dependent manner, as evidenced by the α1-receptor-mediated responses. Thus, the vasodilatory role of TRPV1 channels, and their ligand capsaicin, could be a potential therapeutic target for improving vascular function. Additionally, given the ‘sympatholytic’ effect of TRPV1 activation and known endogenous activators (anandamide, reactive oxygen species, H+, etc.), TRPV1 channels might contribute to functional sympatholysis during exercise. To examine the role of the transient receptor potential vanilloid type 1 (TRPV1) ion channel in the vascular function of human skeletal muscle feed arteries (SMFAs) and whether activation of this heat-sensitive receptor could be involved in modulating vascular function, SMFAs from 16 humans (63 ± 5 years old, range 41–89 years) were studied using wire myography with capsaicin (TRPV1 agonist) and without (control). Specifically, phenylephrine (α1-adrenergic receptor agonist), dexmedetomidine (α2-adrenergic receptor agonist), ACh and sodium nitroprusside concentration–response curves were established to assess the role of TRPV1 channels in α-receptor-mediated vasocontraction as well as endothelium-dependent and -independent vasorelaxation, respectively. Compared with control conditions, capsaicin significantly attenuated maximal vasocontraction in response to phenylephrine [control, 52 ± 8% length–tensionmax (LTmax) and capsaicin, 21 ± 5%LTmax] and dexmedetomidine (control, 29 ± 12%LTmax and capsaicin, 2 ± 3%LTmax), while robustly enhancing maximal vasorelaxation with ACh (control, 78 ± 8% vasorelaxation and capsaicin, 108 ± 13% vasorelaxation) and less clearly enhancing the sodium nitroprusside response. Denudation of the endothelium greatly attenuated the maximal ACh-induced vasorelaxation equally in the control and capsaicin conditions (∼17% vasorelaxation) and abolished the attenuating effect of capsaicin on the maximal phenylephrine response (denuded + capsaicin, 61 ± 20%LTmax). Immunohistochemistry identified a relatively high density of TRPV1 channels in the endothelium compared with the smooth muscle of the SMFAs, but because of the far greater volume of smooth muscle, total TRPV1 protein content was not significantly attenuated by denudation. Thus, SMFAs ubiquitously express functional TRPV1 channels, which alter vascular function, in terms of α1-receptors, in a predominantly endothelium-dependent manner, conceivably contributing to the functional sympatholysis and unveiling a therapeutic target

Vasodilatory and vascular mitochondrial respiratory function with advancing age: evidence of a free radically mediated link in the human vasculature

Recognizing the age-related decline in skeletal muscle feed artery (SMFA) vasodilatory function, this study examined the link between vasodilatory and mitochondrial respiratory function in the human vasculature. Twenty-four SMFAs were harvested from young (35 ± 6 yr, n = 9) and old (71 ± 9 yr, n = 15) subjects. Vasodilation in SMFAs was assessed, by pressure myography, in response to flow-induced shear stress, acetylcholine (ACh), and sodium nitroprusside (SNP) while mitochondrial respiration was measured, by respirometry, in permeabilized SMFAs. Endothelium-dependent vasodilation was significantly attenuated in the old, induced by both flow (young: 92 ± 3, old: 45 ± 4%) and ACh (young: 92 ± 3, old: 54 ± 5%), with no significant difference in endothelium-independent vasodilation. Complex I and I + II state 3 respiration was significantly lower in the old (CI young: 10.1 ± 0.8, old: 7.0 ± 0.4 pmol·s−1·mg−1; CI + II young: 12.3 ± 0.6, old: 7.6 ± 0.4 pmol·s−1·mg−1). The respiratory control ratio (RCR) was also significantly attenuated in the old (young: 2.2 ± 0.1, old: 1.1 ± 0.1). Furthermore, state 3 (CI + II) and 4 respiration, as well as RCR, were significantly correlated (r = 0.49–0.86) with endothelium-dependent, but not endothelium-independent, function. Finally, the direct intervention with mitochondrial-targeted antioxidant (MitoQ) significantly improved endothelium-dependent vasodilation in the old but not in the young. Thus, the age-related decline in vasodilatory function is linked to attenuated vascular mitochondrial respiratory function, likely by augmented free radicals

Vascular mitochondrial respiratory function: the impact of advancing age

Little is known about vascular mitochondrial respiratory function and the impact of age. Therefore, skeletal muscle feed arteries were harvested from young (33 ± 7 yr, n = 10), middle-aged (54 ± 5 yr, n = 10), and old (70 ± 7 yr, n = 10) subjects, and mitochondrial respiration as well as citrate synthase (CS) activity were assessed. Complex I (CI) and complex I + II (CI+II) state 3 respiration were greater in young (CI: 10.4 ± 0.8 pmol·s−1·mg−1 and CI+II: 12.4 ± 0.8 pmol·s−1·mg−1, P \u3c 0.05) than middle-aged (CI: 7 ± 0.6 pmol·s−1·mg−1 and CI+II: 8.3 ± 0.5 pmol·s−1·mg−1) and old (CI: 7.2 ± 0.4 pmol·s−1·mg−1 and CI+II: 7.6 ± 0.5 pmol·s−1·mg−1) subjects and, as in the case of complex II (CII) state 3 respiration, were inversely correlated with age [r = −0.56 (CI), r = −0.7 (CI+II), and r = 0.4 (CII), P \u3c 0.05]. In contrast, state 4 respiration and mitochondria-specific superoxide levels were not different across groups. The respiratory control ratio was greater in young (2.2 ± 0.2, P \u3c 0.05) than middle-aged and old (1.4 ± 0.1 and 1.1 ± 0.1, respectively) subjects and inversely correlated with age (r = −0.71, P \u3c 0.05). As CS activity was inversely correlated with age (r = −0.54, P \u3c 0.05), when normalized for mitochondrial content, the age-related differences and relationships with state 3 respiration were ablated. In contrast, mitochondrion-specific state 4 respiration was now lower in young (15 ± 1.4 pmol·s−1·mg−1·U CS−1, P \u3c 0.05) than middle-aged and old (23.4 ± 3.6 and 27.9 ± 3.4 pmol·s−1·mg−1·U CS−1, respectively) subjects and correlated with age (r = 0.46, P \u3c 0.05). Similarly, superoxide/CS levels were lower in young (0.07 ± 0.01) than old (0.19 ± 0.41) subjects and correlated with age (r = 0.44, P \u3c 0.05). Therefore, with aging, vascular mitochondrial respiratory function declines, predominantly as a consequence of falling mitochondrial content. However, per mitochondrion, aging likely results in greater mitochondrion-derived oxidative stress, which may contribute to age-related vascular dysfunction

oHSV Genome Editing by Means of galK Recombineering

open8noThis work was supported by European Research Council (ERC) Advanced Grant number 340060, VII framework program to G. C.-F., by RFO (University of Bologna) to L.M. and T.G, and by Fondi Pallotti to T.G.Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.embargoed_20201017Laura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-FiumeLaura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-Fium