Automatic generation of alloreactivity-reduced donor lymphocytes and hematopoietic stem cells from the same mobilized apheresis product.

INTRODUCTION: In vitro or in vivo depletion of alloreactive T cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). Very satisfactory transplant outcomes were thus reported for TCRαß/CD19-depleted hematopoietic stem/progenitor cell (HSPC) grafts. The current semi-automatic manufacturing process on the CliniMACS Plus, although robust, still requires a significant amount of manual labor to be completed. Towards advancing and further facilitating large scale cell processing, a new TCRαß/CD19 depletion module combined with the previously described CD45RA depletion module (to serve as allo-reactivity attenuated donor lymphocyte infusion) was established on the CliniMACS Prodigy. METHODS: We evaluated six apheresis products from G-CSF-mobilized volunteer donors which were split automatically by the Prodigy, one portion each depleted of CD45RA+ or of TCRαß+ and CD19+ cells. We investigated critical quality attributes for both products. Products were assessed for recovery of HSPCs and mature subsets, as well as depletion efficiency of targeted cells using flow cytometry. Effects of apheresis and product age post 48 h storage at 2-6 °C as well as freeze-thawing on product viability and recovery of WBC and HPSCs were assessed by flow cytometry. RESULTS: Ten sequential automatic processes were completed with minimal hands-on time beyond tubing set installation. Depletion efficiency of CD45RA+ resp. TCRαß+ and CD19+ cells was equivalent to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγδ+ and NK cells. 48 h storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions remained efficient. Depleted products were stable until at least 72 h after apheresis with stem cell viabilities > 90%. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45+ and HSPCs was > 70% and in line with expectation. CONCLUSION: The closed, GMP-compatible process generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαß/CD19-depleted products included HSPCs, TCRγδ+ and NK cells. Both products are predicted to be effectively depleted of GVH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.

Comparison between rice husk ash grown in different regions for stabilizing fly ash from a solid waste incinerator.

The Stabilization of heavy metals from municipal solid waste incineration (MSWI) fly ash by rice husk ash (RHA) is under intense study as an effective strategy to recover and reuse industrial and agricultural waste together. We compare the metal entrapment performances of RHA from different Asian rice sources ­ namely from Japonica rice grown in Italy and Indica rice grown in India ­ Physicochemical and morphological characterization of the final stabilized material show that the same thermal treatment may result in marked structural differences in the silica contained in the two RHA. Remarkably, one of them displays a crystalline silica content, although obtained by a thermal treatment below 800 °C. We also find that the presence of an alkali metal ion (potassium) in the rice husk plays a crucial role in the attainment of the final silica phase. These physicochemical differences are mirrored by different stabilization yields by the two RHA.

Effects of a 25-km trial on psychological, physiological and stroke characteristics of short- and mid-distance swimmers.

AIM: The aim of this paper was to evaluate the effects of a very long distance swimming task on psychological, biomechanical and physiological responses. Eight swimmers (age 21.0 ± 1.2 years; stature 1.80 ± 0.07 m; body mass 76.7 ± 11.0 kg; means ± SD) participated in a 25-km trial in a swimming pool. METHODS: Before and immediately after the trial, swimmers underwent a 50-m sprint test, during which we assessed velocity, stroke rate (SR), stroke length (SL) and psychological condition (rate of perceived exertion [RPE] and profile of mood state [POMS] questionnaire). During the 25-km trial we determined also elbow angle, heart rate (HR) and lactate concentration ([La]). RESULTS: Velocity, SR and SL during the sprint test after the trial decreased compared to before from 1.91 ± 0.01 m·s⁻¹, 0.94 ± 0.01 cycles·s⁻¹ and 1.99 ± 0.02 m·cycle⁻¹ to 1.45 ± 0.01 m·s⁻¹, 0.78 ± 0.01 cycles·s⁻¹ and 1.84 ± 0.03 m·cycle⁻¹, respectively (P<0.05). During the 25-km trial, velocity and SL decreased significantly, while SR and elbow angle did not change. Velocity and SR during the sprint test after the trial were significantly higher than those during the trial. RPE and fatigue (POMS subscale) increased significantly, while the other negative POMS subscales and vigor decreased significantly. HR decreased significantly at 20 km, then increasing significantly at 25 km, while [La] did not change. CONCLUSION: These results suggest that, despite the occurrence of fatigue, as evidenced by the drop in velocity and changes in psychological profile, swimmers were able to complete the 25-km trial by adopting a conservative pacing, unveiling also a reserve in maximum performance.

Metronomic Temozolomide in Heavily Pretreated Patients With Recurrent Isocitrate Dehydrogenase Wild-type Glioblastoma: A Large Real-Life Mono-Institutional Study.

AIMS: Glioblastoma (GBM) is the most common primary malignant brain tumour in adults and frequently relapses. The aim of this study was to assess the efficacy and safety of metronomic temozolomide (TMZ) in the recurrent GBM population. MATERIALS AND METHODS: All patients treated at our centre between September 2013 and March 2021 were retrospectively reviewed. The main inclusion criteria were first-line therapy with the Stupp protocol, relapse after the first or subsequent line of therapy, treatment with a metronomic TMZ schedule (50 mg/m2 continuously) and histological diagnosis of isocitrate dehydrogenase wild-type GBM according to World Health Organization 2016 classification. RESULTS: In total, 120 patients were enrolled. The median follow-up was 15.6 months, the median age was 59 years, Eastern Cooperative Oncology Group performance status (ECOG-PS) was 0-2 in 107 patients (89%). O6-methylguanine-DNA-methyltransferase (MGMT) was methylated in 66 of 105 (62%) evaluable patients. The median number of prior lines of treatment was 2 (range 1-7). Three (2%) patients showed a partial response; 48 (40%) had stable disease; 69 (57%) had progressive disease. The median overall survival from the start of metronomic TMZ was 5.4 months (95% confidence interval 4.3-6.4), whereas the median progression-free survival (PFS) was 2.6 months (95% confidence interval 2.3-2.8). At univariate analysis, MGMT methylated and unmethylated patients had a median PFS of 2.9 and 2.1 months (P = 0.001) and a median overall survival of 5.6 and 4.4 months (P = 0.03), respectively. At multivariate analysis, the absence of MGMT methylation (hazard ratio = 2.3, 95% confidence interval 1.3-3.9, P = 0.004) and ECOG-PS ≤ 2 (hazard ratio = 0.5, 95% confidence interval 0.3-0.9, P = 0.017) remained significantly associated with PFS, whereas ECOG-PS ≤ 2 (hazard ratio = 0.4, 95% confidence interval 0.3-07, P = 0.001) was the only factor associated with overall survival. The most common grade 3-4 toxicities were haematological (lymphopenia 10%, thrombocytopenia 3%). CONCLUSIONS: Rechallenge with metronomic TMZ is a well-tolerated option for recurrent GBM, even in pretreated patients. Patients with methylated MGMT disease and good ECOG-PS seem to benefit the most from this treatment.

Physiological and anthropometric characteristics of junior cyclists of different specialties and performance levels.

This study analyzes the anthropometric and physiological characteristics of junior cyclists within different cycling specialties and different performance levels. One hundred and thirty-two junior riders (16.8 ± 0.6 years, 177 ± 6 cm, 66.3 ± 6.7 kg) were tested for anthropometric, aerobic and anaerobic parameters. Cyclists were classified within specialties [uphill (UH) flat terrain (FT) all terrain (AT) and sprint (SP)] and performance levels, based on a seasonal ranking [low level (LL) medium level (ML) and high level (HL)]. The results of the two-way analysis of variance showed that FT and SP have greater body dimensions than UH and AT (P<0.001). Concerning the relative aerobic parameters, AT and UH have higher values (P<0.001) than FT and SP [maximal oxygen uptake (VO(2max) ): 69.4 ± 3.6, 67.5 ± 5.0, 62.8 ± 4.5 and 61.9 ± 4.1 mL/kg/min, respectively] while absolute parameters resulted higher for FT and AT (P≤0.008). The relative power produced in the 5 s test was higher (P<0.001) for AT and SP than FT and UH (16.7 ± 1.1, 16.6 ± 0.6, 14.9 ± 1.7 and 14.4 ± 1.7 W/kg, respectively). Concerning the performance level, only the age and the aerobic parameters resulted differently within levels (VO(2max) : HL=67.3 ± 4.9, ML=65.5 ± 5.1 and LL=63.3 ± 5.2 mL/kg/min), with the highest values for HL (P≤0.007). In conclusion, juniors are specialized in the same way as professional cyclists and the aerobic characteristics are confirmed as significant in the performance level assessment.

Accuracy of a spontaneous breathing trial for extubation of neonates.

BACKGROUND: Prevalence of extubation failure in neonates may be up to 80%, but evidence to determine if a neonate is ready for extubation remains unclear. We aim to evaluate a spontaneous breathing trial accuracy with minimum pressure support to predict success in neonates' extubation and identify variables related to failures. METHODS: This is a diagnostic accuracy study based on a cohort study in an intensive care unit with all eligible newborn infants subjected to invasive mechanical ventilation for at least 24 hours submitted to the trial for 10 minutes before extubations. The outcome was failures of extubations, considered if reintubation was needed until 72 hours. RESULTS: The incidence of failure was 14.7%among 170 extubations. There were 145 successful extubations; of these, 140 also passed the trial with a sensitivity of 96.5%(95%CI: 92.1-98.9). Of the 25 extubations that eventually failed, 16 failed the test with a specificity of 64.0%(95%CI: 42.5-82.0). The negative predictive value was 76.2%, and the positive predictive value was 94%. In stratifying by weight, the accuracy was >98.7%for neonates weighting >2500 g, but 72.5%for those weighing <1250 g. Extubation failures occurred more frequently in smaller (p = 0.01), preterm infants (p = 0.17), with longer ventilation time (p = 0.05), and having a hemodynamically significant persistent arterial duct (p = 0.01), compared with infants whose extubation was successful. CONCLUSION: The spontaneous breathing trial with minimum pressure support ventilation seems to predict extubation success with great accuracy in full-term and larger neonates.

Management of 49 antebrachial and crural fractures in dogs using circular external fixators.

OBJECTIVES: To evaluate frame constructs, postoperative fracture reduction, postoperative care, complications, treatment duration and outcome of circular external fixation for management of 49 antebrachial and crural fractures in dogs. METHODS: Medical records of dogs that underwent surgery with circular external fixation were examined for fracture location, frame configuration, postoperative fracture reduction, treatment duration, complications and outcome. RESULTS: Forty-nine fractures in 48 dogs were stabilised using circular external fixation. Frame removal occurred on average 61 days after surgery (median+/-sd, 52+/-30 days). Minor complications were observed in 35 cases (71 per cent) and major complications in seven (14 per cent). Radiographic outcome at frame removal was judged as excellent in 18 cases (37 per cent), as good in 26 (53 per cent), as fair in four (8 per cent) and poor in one (2 per cent). After frame removal, one fracture was stabilised with a plate, and eight patients were lost to follow-up. Functional and cosmetic outcome at follow-up was judged as excellent in 32 cases (80 per cent), good in seven (18 per cent) and fair in one (2 per cent). CLINICAL SIGNIFICANCE: Circular external fixation can effectively treat antebrachial and crural fractures in dogs, even geometrically complex fractures. Minor complications are frequent but easily managed in most instances. Healing time is comparable to that of other external fixation methods.

Fertilizing a Patient Engagement Ecosystem to Innovate Healthcare: Toward the First Italian Consensus Conference on Patient Engagement.

Currently we observe a gap between theory and practices of patient engagement. If both scholars and health practitioners do agree on the urgency to realize patient engagement, no shared guidelines exist so far to orient clinical practice. Despite a supportive policy context, progress to achieve greater patient engagement is patchy and slow and often concentrated at the level of policy regulation without dialoguing with practitioners from the clinical field as well as patients and families. Though individual clinicians, care teams and health organizations may be interested and deeply committed to engage patients and family members in the medical course, they may lack clarity about how to achieve this goal. This contributes to a wide "system" inertia-really difficult to be overcome-and put at risk any form of innovation in this filed. As a result, patient engagement risk today to be a buzz words, rather than a real guidance for practice. To make the field clearer, we promoted an Italian Consensus Conference on Patient Engagement (ICCPE) in order to set the ground for drafting recommendations for the provision of effective patient engagement interventions. The ICCPE will conclude in June 2017. This document reports on the preliminary phases of this process. In the paper, we advise the importance of "fertilizing a patient engagement ecosystem": an oversimplifying approach to patient engagement promotion appears the result of a common illusion. Patient "disengagement" is a symptom that needs a more holistic and complex approach to solve its underlined causes. Preliminary principles to promote a patient engagement ecosystem are provided in the paper.

Myelin glycolipids and their functions.

During myelination, oligodendrocytes in the CNS and Schwann cells in the PNS synthesise myelin-specific proteins and lipids for the assembly of the axon myelin sheath. A dominant class of lipids in the myelin bilayer are the glycolipids, which include galactocerebroside (GalC), galactosulfatide (sGalC) and galactodiglyceride (GalDG). A promising approach for unravelling the roles played by various lipids in the myelin membrane involves knocking out the genes encoding important enzymes in lipid biosynthesis. The recent ablation of the ceramide galactosyltransferase ( cgt) gene in mice is the first example. The cgt gene encodes a key enzyme in glycolipid biosynthesis. Its absence causes glycolipid deficiency in the lipid bilayer, breakdown of axon insulation and loss of saltatory conduction. Additional knock-out studies should provide important insights into the various functions of glycolipids in myelinogenesis and myelin structure.

'Engage me in taking care of my heart': a grounded theory study on patient-cardiologist relationship in the hospital management of heart failure.

OBJECTIVE: In approaching the study and practice of heart failure (HF) management, authors recognise that the patient-doctor relationship has a central role in engaging patients in their care. This study aims at identifying the features and the levers of HF patient engagement and suggestions for orienting clinical encounters. DESIGN: Using a grounded theory approach, we conducted 22 in-depth interviews (13 patients with HF, 5 physicians and 4 caregivers). Data were collected and analysed using open, axial and selective coding procedures according to the grounded theory principles. SETTINGS: All interviews were conducted in an office in a university hospital located in a metropolitan area of Milan, Italy. PARTICIPANTS: The data comprised a total of 22 patient, hospital cardiologist and caregiver interviews. Patients aged ≥18 years with New York Heart Association (NYHA) Functional Class of II or III were eligible to take part. Patients were recruited primarily through their referral cardiologist. RESULTS: The HF patient engagement process develops in four main phases that are characterised by different patients' emotional, cognitive and behavioural dynamics that contribute to shape the process of a patient's meaning making towards health and illness regarding their care. The emerging model illustrates that HF patient engagement entails a meaning-making process enacted by the patient after the critical event. This implies patients' ability to give sense to their care experience and to their disease, symptomatology and treatments, and their changes along their illness course. Doctors are recognised as crucial in fostering patients' engagement along all the phases of the process as they contribute to providing patients with self-continuity and give new meaning to their illness experience. CONCLUSIONS: This study identifies the core experiential domains and the main levers involved in driving patients with HF to effectively engage in their disease management. The model emerging from this study may help clinicians think in a fresh way about encounters with patients and their role in fostering their patients' health engagement.

Reliability of the assessment of peripheral muscle fatigue induced by high-intensity intermittent exercise.

AIM: Reliability of the assessment of peripheral fatigue induced in knee extensors by high-intensity intermittent running exercise (HIE) was established in ten amateur soccer players (age: 18±1, height: 178±5 cm, weight: 68±8 kg). METHODS: Transcutaneous electrical stimulations before and after HIE determined muscle contractile properties on three separate occasions (T1, T2 and T3), each separated by 7 days. RESULTS: No significant differences were noted for any of the parameters measured (P=0.101). The ICC values for peak torque (PT) varied from moderate to high, with the exception of PT at 10-Hz calculated comparing T2 vs. T1 (range PRE=0.78-0.92; POST=0.76-0.97). The ICC derived from PT percentage decrements data were all low, with the exception of PT decrements at 1 Hz calculated comparing T3 vs. T2 (ICC=0.85, moderate). The TE for all contractile parameters obtained using 1-Hz and 100-Hz electrical stimulations were below 10%, including some that demonstrated a TE lower than 5%. Muscle contractile properties determined using 10-Hz stimulations showed a higher level of TE (range: 3.2-15.1%). Similar results were obtained for maximal rate of torque development and torque relaxation. CONCLUSION: From the results of this study it can be concluded that muscle contractile properties express a good level of reliability in baseline and postexercise measures following familiarization.

Action of ethanol and salsolinol on opiate receptor function.

Ethanol may act at the enkephalinergic receptor level through condensation products such as salsolinol. This fact has been demonstrated by studying the 'in vitro' and 'in vivo' salsolinol interaction on enkephalinergic receptor sites labeled by [3H-Met] enkephalin. The modification induced by chronic ethanol and salsolinol on this neuronal system is a reduction of the affinity of the receptor for its ligand. These data suggest that a down regulation process due to the continuous opiate receptor stimulation occurs after ethanol administration.

Effect of zinc deficiency on the mRNA expression pattern in liver and jejunum of adult rats: monitoring gene expression using cDNA microarrays combined with real-time RT-PCR.

In the study presented here, the effect of zinc deficiency on mRNA expression levels in liver and jejunum of adult rats was analyzed. Feed intake was restricted to 8 g/day. The semi-synthetic diet was fortified with pure phytate and contained either 2 microg Zn/g (Zn deficiency, n = 6) or 58 microg Zn/g (control, n = 7). After 29 days of Zn depletion feeding, entire jejunum and liver were retrieved and total RNA was extracted. Tissue specific expression pattern were screened and quantified by microarray analysis and verified individually via real-time RT-PCR. A relative quantification was performed with the newly developed Relative Expression Software Tool Copyright on numerous candidate genes which showed a differential expression. This study provides the first comparative view of gene expression regulation and fully quantitative expression analysis of 35 candidate genes in a non-growing Zn deficient adult rat model. The expression results indicate the existence of individual expression pattern in liver and jejunum and their tissue specific regulation under Zn deficiency. In addition, in jejunum a number of B-cell related genes could be demonstrated to be suppressed at Zn deficiency. In liver, metallothionein subtype 1 and 2 (MT-1 and MT-2) genes could be shown to be dramatically repressed and therefore represent putative markers for Zn deficiency. Expression results imply that some genes are expressed constitutively, whereas others are highly regulated in tissues responsible for Zn homeostasis.

Evaluation of endorphin content in the CSF of patients with trigeminal neuralgia before and after Gasserian ganglion thermocoagulation.

The beta-endorphin content in cerebrospinal fluid (CSF) was evaluated in 10 patients with idiopathic trigeminal neuralgia during medical treatment (with or without carbamazepine) and after selective thermocoagulation of the Gasserian ganglion. These values were compared with those obtained in a control group of seven patients without pain problems. No statistically significant difference was found between patients suffering from trigeminal neuralgia and those without pain. Furthermore, neither pharmacological treatment nor surgery changed CSF endorphin values. It is concluded that there is no pathogenetic relationship between trigeminal neuralgia and endorphins.

Neuronal mechanisms regulating ethanol effects on the dopaminergic system.

Chronic ethanol consumption induces an increase in striatal 3H-Spiroperidol and 3H(-)Sulpiride specific binding by enhancing the affinity between the different dopaminergic recognition sites and the labelled ligands. Dopamine (DA) receptor supersensitivity is also suggested by the enhanced effect of neuroleptics in inducing hypomotility in rats treated with ethanol. The results, obtained by means of the administration of neuroleptics in comparison to ethanol treated rats, indicate a lack of cross tolerance between ethanol and other drugs acting on the dopaminergic recognition sites. These data suggest that ethanol effects on the dopaminergic system are mediated by events involving other neurotransmitter systems.

Immunoreactive met-enkephalin plasma concentrations in chronic alcoholics and in children born from alcoholic mothers.

Several experimental and clinical observations indicate that ethanol ingestion induces specific neurochemical modifications in the Central Nervous System. In particular, an involvement of endogenous opiates has been suggested in the case of alcohol addiction. In this light, the plasma concentrations of met-enkephalin immunoreactive peptides (ME-IR) have been measured in selected groups of chronic alcoholics and in children whose mothers were ethanol addicts. Both groups revealed a marked reduction of ME-IR plasma concentrations when compared with sex and age matched controls.

Pharmacokinetics of cyclophosphamide in patients with systemic necrotizing angiitis.

Cyclophosphamide pharmacokinetics were investigated following administration to patients with systemic necrotizing angiitis. Ten patients (eight women and two men) received cyclophosphamide as a 1-h-rate-constant intravenous infusion at doses ranging from 600 to 1200 mg. All patients received concomitant oral prednisone (1 mg/kg/d). Blood samples were collected at the end of drug infusion and 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 h later. Serum cyclophosphamide concentrations were assayed by high pressure liquid chromatography. The peak serum cyclophosphamide levels ranged from 15.7 to 29.4 mg/L. The mean cyclophosphamide elimination half-life was 6.2 +/- 1.3 h (mean +/- SD). The mean apparent volume of distribution and mean total plasma clearance were, respectively, 0.75 +/- 0.22 L/kg (mean +/- SD) and 83 +/- 22 mL/min (mean +/- SD). These results obtained in systemic vasculitic diseases were consistent with those observed in other studies with cancer patients receiving comparable doses of cyclophosphamide.

Central toxic effects of chronic ethanol treatment: actions on GABA and benzodiazepine recognition sites.

Prolonged ethanol treatment modifies various neurotransmitter systems. GABAergic neuronal function was particularly affected. On the other hand, clinical reports have indicated an interaction between ethyl alcohol and benzodiazepine receptors. These observations suggest a possible site of action of ethanol at the level of the GABA-benzodiazepine receptor complex. Our results showed that ethanol treatment differentially affected GABA receptor function and benzodiazepine binding sites. When [3H]GABA binding in the cerebellum, striatum and hippocampus was increased, [3H]diazepam binding remained unchanged in the same areas. The possibility of modulation of ethanol effects on GABAergic neurons through benzodiazepine receptors is discussed.

Induction of hepatic drug metabolizing enzymes and interaction with carbon tetrachloride in rats after a single oral exposure to atrazine.

A single oral dose (430 mg/kg) of atrazine, a widely employed s-triazine herbicide, was administered to young male rats. There was a significant increase of the in vivo elimination of hexobarbital and a significant induction of the activity of 7-pentoxyresorufin-O-dealkylase, while cytochrome P-450 content and other mixed function oxidase activities remained unaltered. The administration of carbon tetrachloride (CCl4) to atrazine pretreated rats did not substantially augment the impairment of drug metabolizing enzymes brought about by CCl4 alone. Results suggest that atrazine behaves like a relatively weak inducer of phenobarbital-inducible families of cytochrome P-450.

Exit-site infection prevention and treatment protocol.

Infections are universally known as one of the main causes of drop-out in peritoneal dialysis. Exit-site infections are often a problem, as they are difficult to treat, tend to become chronic and may lead to the development of continuity peritonitis, resulting in the need for removal of the peritoneal catheter. The purpose of this paper is to describe the application of an exit-site infection prevention and treatment protocol.