Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia

Livers from donors after circulatory death (DCD) are a promising option to increase the donor pool, but their use is associated with higher complication rate and inferior graft survival. Normothermic machine perfusion (NMP) keeps the graft at 37°C, providing nutrients and oxygen supply. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) are able to reduce liver injury and promote regeneration. We investigated the efficacy of a reconditioning strategy with HLSC-EVs in an experimental model of NMP. Following total hepatectomy, rat livers were divided into 4 groups: (i) healthy livers, (ii) warm ischemic livers (60 min of warm ischemia), (iii) warm ischemic livers treated with 5 × 10⁸ HLSC-EVs/g-liver, and (iv) warm ischemic livers treated with a 25 × 10⁸ HLSC-EVs/g-liver. NMP lasted 6 h and HLSC-EVs (Unicyte AG, Germany) were administered within the first 15 min. Compared to controls, HLSC-EV treatment significantly reduced transaminases release. Moreover, HLSC-EVs enhanced liver metabolism by promoting phosphate utilization and pH self-regulation. As compared to controls, the higher dose of HLSC-EV was associated with significantly higher bile production and lower intrahepatic resistance. Histologically, this group showed reduced necrosis and enhanced proliferation. In conclusion, HLSC-EV treatment during NMP was feasible and effective in reducing injury in a DCD model with prolonged warm ischemia.     

Clonal composition and persistence of antigen-specific circulating T follicular helper cells

T follicular helper (T_FH) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4⁺ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC T_FH cells. However, the antigen specificity and relationship of these circulating T_FH (cT_FH) cells with other memory CD4⁺ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vβ sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cT_FH and non-cT_FH subsets, although with different frequencies and effector functions. Interestingly, cT_FH and non-cT_FH cells specific for C.alb or TT had a largely overlapping TCR Vβ repertoire while the repertoire of Flu-specific cT_FH and non-cT_FH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1⁺ cT_FH cells had a “GC T_FH-like” phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cT_FH and non-cT_FH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cT_FH with non-cT_FH cells and on the heterogeneity and persistence of antigen-specific human cT_FH cells.     

p15INK4b, p14ARF, and p16INK4a inactivation in sporadic and neurofibromatosis type 1-related malignant peripheral nerve sheath tumors.

PURPOSE: Malignant peripheral nerve sheath tumor (MPNST) can arise sporadically or in association with neurofibromatosis type 1. Deletions at the 9p21 locus have been reported in these tumors. To additionally characterize the status of this chromosomal region, in this study we performed a comprehensive, mostly PCR-based molecular analysis of the three tumor suppressor genes p15(INK4b), p14(ARF) and p16(INK4a) located at the 9p21 locus in 26 cryopreserved MPNSTs. EXPERIMENTAL DESIGN: Fourteen neurofibromatosis type 1-related and 12 sporadic cases were investigated for homozygous deletion coupled with fluorescent in situ hybridization, promoter methylation, and mutational analysis, as well as m-RNA expression. RESULTS: The results showed that an inactivation of one or more genes occurred in 77% of MPNSTs and was mainly achieved through homozygous deletion (46%), which, in turn, encompassed all of the three tandemly linked genes in 83% of the deleted cases. Promoter methylation was at a less extent involved in gene silencing (18%), and no mutations were found. Loss of function at DNA level strongly correlated with loss of mRNA expression accounting for 80% of the cases. Because of the close relationship between p14(ARF) and TP53 and between p15(INK4b)/p16(INK4a) and Rb, these results support a model of a coinactivation of TP53 and Rb pathways in 75% of MPNSTs, with functional consequences on cell growth control and apoptosis. CONCLUSIONS: The inactivation of the 9p21 locus is a frequent and peculiar hallmark of MPNST genetic profile leading also to an impaired apoptosis that could be taken into account in treatment planning of these tumors.     

Significance of low birthweight for gestational age among very preterm infants

Objective To estimate the risk of specific adverse neonatal events resulting from the combined effects of prematurity and low birthweight in very preterm infants (delivered at 24–31 weeks of gestation)
Design A cohort study of specific adverse neonatal events in preterm infants born at between 24 and 31 weeks of gestation.
Setting Pavia, Italy.
Population Two hundred and thirty singleton infants with sonographically confirmed gestational age, delivered at 24 to 31 weeks of gestation.
Methods To evaluate the impact of a lower than expected birthweight on selected neonatal events independently of gestational age, we calculated birthweight standard deviation scores (differences between actual birthweight and fitted birthweight divided by fitted standard deviation) for each week of gestation.
Results After adjustment for gestational age and other confounders, there was a significant linear trend relating a decreasing birthweight SDS to an increased likelihood of neonatal death, intraventricular haemorrhage, severe respiratory distress syndrome, and acidosis. Compared with infants with SDS 0 ( 50th centile of birthweight), infants with birthweight SDS < −1 (< 16th centile) had increased odds for neonatal death [odds ratio (OR) 3.7, 95% confidence interval (CI) 1.42–9.6], grade III-IV intraventricular haemorrhage (OR 17.5, 95% CI 4.04–75.9), and neonatal acidosis (OR 3.22, 95% CI 1.41–7.4). The significance of birthweight SDS as a predictor of neonatal outcome, however, was lower than that of gestational age.
Conclusions A lower than expected birthweight affects the likelihood of several adverse neonatal events in very preterm infants. However, a decreasing birthweight SDS affects neonatal outcome less than decreasing gestation does.     

男性与女性糖尿病患者致死性冠心病的额外危险：37个前瞻性队列研究的汇总分析

目的：估计男性和女性糖尿病患者致死性冠心病的相对危险。设计：前瞻性队列研究的汇总分析。数据来源：自1966年至2005年3月间Embase和Medline确认并发表的研究，加上源自亚太队列研究协作组的研究，采用正文词组与MeSH主题词相结合的策略进行检索。综述方法：对入选研究的要求为报告有或无糖尿病的男性与女性致死性冠心病的相对危险比较的估计值。如果对估计值仅做年龄调整（而其它因素来经调整——泽者注），则将该研究排除在外。结果：总共纳入37项2型糖尿病和致死性冠心病的研究，涉及447064例患者。糖尿病患者的致死性冠心病发生率显著高于非糖尿病患者（5．4％比1．6％）。与无糖尿病者相比，糖尿病患者发生致死性冠心病的总的相对危险，女性为3．50，95％可信区间为2．70～4．53，显著高于男性患者（2．06，95％可信区间1．81～2．34）。在除外8项仅根据年龄调整的研究后，两性之间危险的差异大大减小，但仍然具有极显著统计学意义。汇集29项经多因素调整估计值的研究，总的相对危险比（女性比男性）为1．46（1．14～1．88）。结论：糖尿病相关的致死性冠心病相对危险，女性比男性高出50％。这种额外的冠心病危险可以通过女性糖尿病患者具有更多不利的心血管危险因素特点以及倾向于对男性患者更注重治疗的可能偏差来解释。     

The wide clinical spectrum of nocturnal frontal lobe epilepsy

Nocturnal frontal lobe epilepsy (NFLE) has become clinically relevant in recent years. NFLE represents a spectrum of clinical manifestations, ranging from brief, stereotyped, sudden arousals, often recurring several times per night, sometimes with a quasi-periodic pattern, to more complex dystonic-dyskinetic seizures and to prolonged "somnambulic" behaviour. Episodes of increasing intensity have been labelled as paroxysmal arousal (PA), nocturnal paroxysmal dystonia (NPD) and episodic nocturnal wandering (ENW). NFLE affects both sexes with a higher prevalence for men, is frequently cryptogenetic and displays a strong familial trait for parasomnias and epilepsy (NFLE). Seizures appear more frequently between 14 and 20 years of age, but can affect any age and tend to increase in frequency during life. Interictal and ictal scalp electroencephalography (EEG) are often normal, the use of sphenoidal leads may be helpful. Carbamazepine taken at night is often effective at low doses, but a third of the patients are resistant to anti-epileptic drugs (AED) treatment. A familial form, characterized by an autosomal dominant transmission, has also been described. Autosomal dominant nocturnal frontal lobe epilepsy is a genetic variant of NFLE, in itself both clinically and biologically heterogeneous. NFLE should be suspected in the presence of frequent stereotyped paroxysmal nocturnal motor events arising or persisting into adulthood. Videopolysomnography is mandatory to confirm the diagnosis.     

In vitro development of engineered muscle using a scaffold based on the pressure‐activated microsyringe (PAM) technique

The development of new human skeletal muscle tissue is an alternative approach to the replacement of tissue after severe damage, for example in the case of traumatic injury, where surgical reconstruction is often needed following major loss of natural tissue. Treatment to date has involved the transfer of muscle tissue from other sites, resulting in a functional loss and volume deficiency of donor sites. Approaches that seek to eliminate these problems include the relatively new solution of skeletal muscle engineering. Here there are two main components to consider: (a) the cells with their regenerative potential; and (b) the polymeric structure onto which cells are seeded and where they must perform their activities. In this paper we describe well‐defined two‐ and three‐dimensional polymeric structures able to drive the myoblast process of adhesion, proliferation and differentiation. We examine a series of polymers and protein adhesions with which to functionalize the structures, and cell‐seeding methods, with a view to defining the optimal protocol for engineering skeletal muscle tissue. All polymer samples were tested for their mechanical and biological properties, to support the validity of our results in the real context of muscle tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.