Surfing on the waves of the human γδ T cell ontogenic sea.

While γδ T cells are present virtually in all vertebrates, there is a remarkable lack of conservation of the TRG and TRD loci underlying the generation of the γδ T cell receptor (TCR), which is associated with the generation of species-specific γδ T cells. A prominent example is the human phosphoantigen-reactive Vγ9Vδ2 T cell subset that is absent in mice. Murine γδ thymocyte cells were among the first immune cells identified to follow a wave-based layered development during embryonic and early life, and since this initial observation, in-depth insight has been obtained in their thymic ontogeny. By contrast, less is known about the development of human γδ T cells, especially regarding the generation of γδ thymocyte waves. Here, after providing an overview of thymic γδ wave generation in several vertebrate classes, we review the evidence for γδ waves in the human fetal thymus, where single-cell technologies have allowed the breakdown of human γδ thymocytes into functional waves with important TCR associations. Finally, we discuss the possible mechanisms contributing to the generation of waves of γδ thymocytes and their possible significance in the periphery.

Long-lived central memory γδ T cells confer protection against murine cytomegalovirus reinfection.

The involvement of γδ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. γδ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αß T cell deficient mice in order to analyze the memory potential of γδ T cells. As for CMV-specific αß T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ γδ TEM that principally localized in infected organ vasculature. Typifying T cell memory, γδ T cell expansion in organs and blood was higher after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection was prevented when masking γδ T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire composed of a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, long-term-primed γδ TCM cells, but not γδ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. This better survival potential of TCM cells was confirmed by a detailed scRNASeq analysis of the two γδ T cell memory subsets which also revealed their similarity to classically adaptive αß CD8 T cells. Overall, our study uncovered memory properties of long-lived TCM γδ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches.

Sepsis shapes the human γδ TCR repertoire in an age- and pathogen-dependent manner.

Sepsis affects 25 million children per year globally, leading to 2.9 million deaths and substantial disability in survivors. Extensive characterization of interactions between the host and bacteria in children is required to design novel preventive and therapeutic strategies tailored to this age group. Vγ9Vδ2 T cells are the first T cells generated in humans. These cells are defined by the expression of Vγ9Vδ2 T-cell receptors (TCRs, using the TRGV9 and TRDV2 gene segments), which react strongly against the prototypical bacterial phosphoantigen HMBPP. We investigated this reactivity by analyzing the TCR δ (TRD) repertoire in the blood of 76 children (0-16 years) with blood culture-proven bacterial sepsis caused by HMBPP-positive Escherichia coli or by HMBPP-negative Staphylococcus aureus or by HMBPP-negative Streptococcus pneumoniae. Strikingly, we found that S. aureus, and to a lesser extent E. coli but not S. pneumoniae, shaped the TRDV2 repertoire in young children (<2 years) but not in older children or adults. This dichotomy was due to the selective expansion of a fetal TRDV2 repertoire. Thus, young children possess fetal-derived Vγ9Vδ2 T cells that are highly responsive toward specific bacterial pathogens.

Management of seizures in patients with primary mitochondrial diseases: consensus statement from the InterERNs Mitochondrial Working Group.

BACKGROUND AND PURPOSE: Primary mitochondrial diseases (PMDs) are common inborn errors of energy metabolism, with an estimated prevalence of one in 4300. These disorders typically affect tissues with high energy requirements, including heart, muscle and brain. Epilepsy may be the presenting feature of PMD, can be difficult to treat and often represents a poor prognostic feature. The aim of this study was to develop guidelines and consensus recommendations on safe medication use and seizure management in mitochondrial epilepsy. METHODS: A panel of 24 experts in mitochondrial medicine, pharmacology and epilepsy management of adults and/or children and two patient representatives from seven countries was established. Experts were members of five different European Reference Networks, known as the Mito InterERN Working Group. A Delphi technique was used to allow the panellists to consider draft recommendations on safe medication use and seizure management in mitochondrial epilepsy, using two rounds with predetermined levels of agreement. RESULTS: A high level of consensus was reached regarding the safety of 14 out of all 25 drugs reviewed, resulting in endorsement of National Institute for Health and Care Excellence guidelines for seizure management, with some modifications. Exceptions including valproic acid in POLG disease, vigabatrin in patients with γ-aminobutyric acid transaminase deficiency and topiramate in patients at risk for renal tubular acidosis were highlighted. CONCLUSIONS: These consensus recommendations describe our intent to improve seizure control and reduce the risk of drug-related adverse events in individuals living with PMD-related epilepsy.

Branched-chain amino acids as adjunctive-alternative treatment in patients with autism: a pilot study.

The branched-chain amino acid (BCAA) is a group of essential amino acids that are involved in maintaining the energy balance of a human being as well as the homoeostasis of GABAergic, glutamatergic, serotonergic and dopaminergic systems. Disruption of these systems has been associated with the pathophysiology of autism while low levels of these amino acids have been discovered in patients with autism. A pilot open-label, prospective, follow-up study of the use of BCAA in children with autistic behaviour was carried out. Fifty-five children between the ages of 6 and 18 participated in the study from May 2015 to May 2018. We used a carbohydrate-free BCAA-powdered mixture containing 45·5 g of leucine, 30 g of isoleucine and 24·5 g of valine in a daily dose of 0·4 g/kg of body weight which was administered every morning. Following the initiation of BCAA administration, children were submitted to a monthly psychological examination. Beyond the 4-week mark, BCAA were given to thirty-two people (58·18 %). Six of them (10·9 %) discontinued after 4-10 weeks owing to lack of improvement. The remaining twenty-six children (47·27 %) who took BCAA for longer than 10 weeks displayed improved social behaviour and interactions, as well as improvements in their speech, cooperation, stereotypy and, principally, their hyperactivity. There were no adverse reactions reported during the course of the treatment. Although these data are preliminary, there is some evidence that BCAA could be used as adjunctive treatment to conventional therapeutic methods for the management of autism.

Innate and adaptive γδ T cells: How, when, and why.

γδ T cells comprise the third cell lineage of lymphocytes that use, like αß T cells and B cells, V(D)J gene rearrangement with the potential to generate a highly diverse T cell receptor (TCR) repertoire. There is no obvious conservation of γδ T cell subsets (based on TCR repertoire and/or function) between mice and human, leading to the notion that human and mouse γδ T cells are highly different. In this review, we focus on human γδ T cells, building on recent studies using high-throughput sequencing to analyze the TCR repertoire in various settings. We make then the comparison with mouse γδ T cell subsets highlighting the similarities and differences and describe the remarkable changes during lifespan of innate and adaptive γδ T cells. Finally, we propose mechanisms contributing to the generation of innate versus adaptive γδ T cells. We conclude that key elements related to the generation of the γδ TCR repertoire and γδ T cell activation/development are conserved between human and mice, highlighting the similarities between these two species.

Fetal public Vγ9Vδ2 T cells expand and gain potent cytotoxic functions early after birth.

Vγ9Vδ2 T cells are a major human blood γδ T cell population that respond in a T cell receptor (TCR)-dependent manner to phosphoantigens which are generated by a variety of microorganisms. It is not clear how Vγ9Vδ2 T cells react toward the sudden microbial exposure early after birth. We found that human Vγ9Vδ2 T cells with a public/shared fetal-derived TCR repertoire expanded within 10 wk postpartum. Such an expansion was not observed in non-Vγ9Vδ2 γδ T cells, which possessed a private TCR repertoire. Furthermore, only the Vγ9Vδ2 T cells differentiated into potent cytotoxic effector cells by 10 wk of age, despite their fetal origin. Both the expansion of public fetal Vγ9Vδ2 T cells and their functional differentiation were not affected by newborn vaccination with the phosphoantigen-containing bacillus Calmette-Guérin (BCG) vaccine. These findings suggest a strong and early priming of the public fetal-derived Vγ9Vδ2 T cells promptly after birth, likely upon environmental phosphoantigen exposure.

Neurotrophin Analog ENT-A044 Activates the p75 Neurotrophin Receptor, Regulating Neuronal Survival in a Cell Context-Dependent Manner.

Neuronal cell fate is predominantly controlled based on the effects of growth factors, such as neurotrophins, and the activation of a variety of signaling pathways acting through neurotrophin receptors, namely Trk and p75 (p75NTR). Despite their beneficial effects on brain function, their therapeutic use is compromised due to their polypeptidic nature and blood-brain-barrier impermeability. To overcome these limitations, our previous studies have proven that DHEA-derived synthetic analogs can act like neurotrophins, as they lack endocrine side effects. The present study focuses on the biological characterization of a newly synthesized analog, ENT-A044, and its role in inducing cell-specific functions of p75NTR. We show that ENT-A044 can induce cell death and phosphorylation of JNK protein by activating p75NTR. Additionally, ENT-A044 can induce the phosphorylation of TrkB receptor, indicating that our molecule can activate both neurotrophin receptors, enabling the protection of neuronal populations that express both receptors. Furthermore, the present study demonstrates, for the first time, the expression of p75NTR in human-induced Pluripotent Stem Cells-derived Neural Progenitor Cells (hiPSC-derived NPCs) and receptor-dependent cell death induced via ENT-A044 treatment. In conclusion, ENT-A044 is proposed as a lead molecule for the development of novel pharmacological agents, providing new therapeutic approaches and research tools, by controlling p75NTR actions.

Identifying barriers to the educational role of midwives in Cyprus and defining determinants in behaviour terms using the Behaviour Change Wheel: a mixed-method formative study.

BACKGROUND: Τhe Baby Buddy Cyprus webapp was co-created with parents and health professionals within a Participatory Action Research framework. While using Baby Buddy in routine consultations can support the educational role of mother-child healthcare providers (HP), antenatal education (AE) may be currently perceived as a formal activity within the physical space of the antenatal class. We aimed to gain an understanding of influences on midwives engaging in an educational role during routine appointments and identify potential interventions using the Behaviour Change Wheel (BCW) framework. METHODS: This is a formative mixed-methods research study, with a convergent parallel design, guided by the COM-B model and related Theoretical Domains Framework (TDF). Complimentary methods were used to collect information from in-training and registered midwives: focus group (N = 11), questionnaire survey (N = 24) and Nominal Group Technique during workshops (N = 40). Deductive content analysis of qualitative data and quantitative survey analysis shaped the behaviour diagnosis along the 6 COM-B and 14 TDF domains, and informed the selection of relevant intervention functions and related Behaviour Change Techniques from the BCW taxonomy. RESULTS: AE is viewed as a core function of the professional role, yet neither supported nor prioritized by current practices. Problematic areas relate to organizational context, such as weak interprofessional collaboration and lack of policy, protocols and resources. In addition, medicalization of birth and related socio-cultural norms, pertaining to users and providers, are sustaining alienation of the midwife and conditions of power dynamics. AE was perceived as a means to enhance the autonomy of the profession but there might be issues with procedural knowledge and the need for skill development was identified. Several intervention functions were identified as promising, however cognitive re-framing through strategic communication and modelling may also be needed both in terms of providing "credible models" for the role itself as well as re-framing AE through the concept of "making every contact count". CONCLUSIONS: AE is currently perceived to be a 'bad fit' with routine practice. The study identified several barriers to the educational role of midwives, influencing Capacity, Opportunity and Motivation. While digital tools, such as Baby Buddy, can facilitate aspects of the process, a much wider behaviour and system change intervention is needed to enhance midwives' educational role and professional identity. In addition to proposing a theory-driven research-informed intervention, the process functioned as a participatory learning experience through collective reflection.

Behavioral Data Analysis of Robot-Assisted Autism Spectrum Disorder (ASD) Interventions Based on Lattice Computing Techniques.

Recent years have witnessed the proliferation of social robots in various domains including special education. However, specialized tools to assess their effect on human behavior, as well as to holistically design social robot applications, are often missing. In response, this work presents novel tools for analysis of human behavior data regarding robot-assisted special education. The objectives include, first, an understanding of human behavior in response to an array of robot actions and, second, an improved intervention design based on suitable mathematical instruments. To achieve these objectives, Lattice Computing (LC) models in conjunction with machine learning techniques have been employed to construct a representation of a child's behavioral state. Using data collected during real-world robot-assisted interventions with children diagnosed with Autism Spectrum Disorder (ASD) and the aforementioned behavioral state representation, time series of behavioral states were constructed. The paper then investigates the causal relationship between specific robot actions and the observed child behavioral states in order to determine how the different interaction modalities of the social robot affected the child's behavior.

Consensus guidelines for the diagnosis and management of pyridoxine-dependent epilepsy due to α-aminoadipic semialdehyde dehydrogenase deficiency.

Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an autosomal recessive condition due to a deficiency of α-aminoadipic semialdehyde dehydrogenase, which is a key enzyme in lysine oxidation. PDE-ALDH7A1 is a developmental and epileptic encephalopathy that was historically and empirically treated with pharmacologic doses of pyridoxine. Despite adequate seizure control, most patients with PDE-ALDH7A1 were reported to have developmental delay and intellectual disability. To improve outcome, a lysine-restricted diet and competitive inhibition of lysine transport through the use of pharmacologic doses of arginine have been recommended as an adjunct therapy. These lysine-reduction therapies have resulted in improved biochemical parameters and cognitive development in many but not all patients. The goal of these consensus guidelines is to re-evaluate and update the two previously published recommendations for diagnosis, treatment, and follow-up of patients with PDE-ALDH7A1. Members of the International PDE Consortium initiated evidence and consensus-based process to review previous recommendations, new research findings, and relevant clinical aspects of PDE-ALDH7A1. The guideline development group included pediatric neurologists, biochemical geneticists, clinical geneticists, laboratory scientists, and metabolic dieticians representing 29 institutions from 16 countries. Consensus guidelines for the diagnosis and management of patients with PDE-ALDH7A1 are provided.

TCR Sequencing Reveals the Distinct Development of Fetal and Adult Human Vγ9Vδ2 T Cells.

Phosphoantigen-reactive Vγ9Vδ2 T cells represent the main innate human γδ T cell subset and dominate the fetal and adult peripheral blood γδ T cell repertoire. It has been hypothesized that adult blood Vγ9Vδ2 T cells find their origin in the fetus like it is established for mouse innate γδ T cells. To address this issue, we analyzed the CDR3 of the TCR of human blood and thymic Vγ9Vδ2 T cells from fetal until adult life. We first identified key differences in the CDR3 repertoire of fetal and adult blood Vγ9Vδ2 T cells, including in CDR3 features important for phosphoantigen reactivity. Next, we showed that most of these key adult CDR3 features were already present in the postnatal thymus and were further enhanced upon selection in vitro by the microbial-derived phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate. Finally, we demonstrated that the generation of a fetal-type or adult-type Vγ9Vδ2 CDR3 repertoire is determined by the fetal and postnatal nature of the hematopoietic stem and precursor cell. Thus, our data indicate that fetal blood Vγ9Vδ2 T cells find their origin in the fetal thymus whereas adult blood Vγ9Vδ2 T cells are generated to a large degree independently after birth.

Women's experiences of VBAC in Cyprus: a qualitative study.

BACKGROUND AND AIM: In 21st century, there has been an increasing interest in vaginal birth after previous caesarean section (VBAC) in Cyprus, a country with a very high operative birth rate. Research-based evidence of women's VBAC experiences in Cyprus is non-existent, despite its significance for the well-being of mothers and families. The aim of this study is to gain insight into the women's lived experience of VBAC in Cyprus. In this study women's experiences of VBAC are explored for the first time in Cyprus. METHOD: The study is qualitative and exploratory in nature. Data were collected through semi-structured interviews with 10 women, who experienced vaginal birth after a previous caesarean section (VBAC) in Cyprus. A descriptive phenomenological approach was employed for the analysis of data. RESULTS: Analysis of data yielded four major themes: (a) medicalization of childbirth, (b) preparing for a VBAC, (c) birth environment, and (d) healing through VBAC. On the whole, the women interviewed described their previous experience of CS as traumatic, in contrast to vaginal childbirth. VBAC was considered an utterly positive experience that made the women feel empowered and proud of themselves. CONCLUSION: This study offers valuable insight into a newly researched subject in Cyprus, which is necessary for advancing perinatal care in Cyprus. The findings indicate that women need evidence-based information, guidelines on birthing options, good preparation with tailored information and personalized care for a successful vaginal birth after a previous caesarean section. Proper, non-biased, consultations are a main factor that affects women's choice of mode of birth. The introduction of new, women-friendly perinatal strategies that respect and promote childbirth rights is imperative in the case of Cyprus. All women have the right to exercise informed choice and the choice to alternative birthing options.

Triple-Band Single-Layer Rectenna for Outdoor RF Energy Harvesting Applications.

A triple-band single-layer rectenna for outdoor RF energy applications is introduced in this paper. The proposed rectenna operates in the frequency bands of LoRa, GSM-1800, and UMTS-2100 networks. To obtain a triple-band operation, a modified E-shaped patch antenna is used. The receiving module (antenna) of the rectenna system is optimized in terms of its reflection coefficient to match the RF-to-DC rectifier. The final geometry of the proposed antenna is derived by the application of the Moth Search Algorithm and a commercial electromagnetic solver. The impedance matching network of the proposed system is obtained based on a three-step process, including the minimization of the reflection coefficient versus frequency, as well as the minimization of the reflection coefficient variations and the maximization of the DC output voltage versus RF input power. The proposed RF-to-DC rectifier is designed based on the Greinacher topology. The designed rectenna is fabricated on a single layer of FR-4 substrate. Measured results show that our proposed rectenna can harvest RF energy from outdoor (ambient and dedicated) sources with an efficiency of greater than 52%.

Effects of major adipokines and the -420 C > G resistin gene polymorphism on the long-term outcome of patients with acute ischemic stroke.

Aim: The association between adiponectin, leptin, and resistin and the long-term outcome of ischemic stroke are controversial. We aimed to evaluate this relationship. Methods: We prospectively studied 83 patients consecutively hospitalized for acute ischemic stroke (38.6% males, age 79.7 ± 6.3 years). Serum adiponectin, leptin, and resistin levels and the -420C > G polymorphism of the resistin gene were determined at admission. Stroke severity at admission was evaluated with the National Institutes of Health Stroke Scale (NIHSS). One year after discharge, functional status, incidence of cardiovascular events and all-cause mortality were recorded. Functional status was evaluated with the modified Rankin scale (mRS). Results: Patients with the G allele had lower mRS (p < .05) and patients with adverse outcome had higher serum resistin levels (p < .05). The only independent predictor of adverse outcome was mRS at discharge (risk ratio (RR) 2.78, 95% confidence interval (CI) 1.54-5.00; p < .001). Higher adiponectin levels were an independent predictor of cardiovascular morbidity (RR 1.07, 95% CI 1.01-1.14; p < .05). Patients who died had higher serum adiponectin levels than those who survived (p < .05). The only independent predictor of all-cause mortality was NIHSS at admission (RR 1.19, 95% CI 1.04-1.35; p < .01). Conclusions: In patients with acute ischemic stroke, the G allele of the -420C > G polymorphism of the resistin gene promoter is more frequent in those with a more favorable functional outcome at one year after discharge. Patients with higher serum resistin levels appear to have worse long-term functional outcome, while higher serum adiponectin levels are associated with higher incidence of cardiovascular events.

Nitrotriazole-Based Compounds as Antichagasic Agents in a Long-Treatment In Vivo Assay.

3-Nitrotriazole-based compounds belonging to various chemical subclasses were found to be very effective against Chagas disease both in vitro and in vivo after a short administration schedule. In this study, five compounds with specific characteristics were selected to be administered for longer periods of time to mice infected with the virulent Trypanosoma cruzi Y strain to further evaluate their effectiveness as antichagasic agents and whether or not potential adverse effects occur. Benznidazole was included for comparison purposes. Complete parasitemia depletion, weight gain, 100% survival, and a lack of myocardial inflammation were observed with four of the compounds and benznidazole administered intraperitoneally at 15 or 20 mg/kg of body weight/day for 40 days. There was a significant reduction in the number of treatment days (number of doses) necessary to induce parasitemia suppression with all four compounds compared to that required with benznidazole. Partial cures were obtained with only one compound tested at 15 mg/kg/day and on the schedule mentioned above but not with benznidazole. Taken together, our data suggest that these compounds demonstrate potent trypanocidal activity comparable to or better than that of the reference drug, benznidazole, when they are administered at the same dose and on the same schedule.

Blocking LDHA glycolytic pathway sensitizes glioblastoma cells to radiation and temozolomide.

PURPOSE: Up-regulation of lactate dehydrogenase LDHA, is a frequent event in human malignancies and relate to poor postoperative outcome. In the current study we examined the hypothesis that LDHA and anaerobic glycolysis, may contribute to the resistance of glioblastoma to radiotherapy and to temozolomide. METHODS AND MATERIALS: The expression of LDH5 isoenzyme (fully encoded by the LDHA gene) was assessed in human glioblastoma tissues. Experimental in vitro studies involved the T98 and U87 glioblastoma cell lines. Their sensitivity to radiotherapy and to temozolomide, following silencing of LDHA gene or following exposure to the LDHA chemical inhibitor 'oxamate' and to the glycolysis inhibitor '2-deoxy-d-glucose' (2DG), was studied. RESULTS: Glioblastoma tissues showed strong cytoplasmic and nuclear LDH5 expression in 0-90% (median 20%) of the neoplastic cells. T98 and U87 cell lines showed that blocking glycolysis, either with LDHA gene silencing or exposure to oxamate (30 mM) and blockage of glycolysis with 2DG (500 µM), results in enhanced radiation sensitivity, an effect that was more robust in the T98 radioresistant cell line. Furthermore, all three glycolysis targeting methods, significantly sensitized both cell lines to Temozolomide. CONCLUSIONS: The current study provides evidence that a large subgroup of human glioblastomas are highly glycolytic, and that inhibitors of glycolysis, like LDHA targeting agents, may prove of therapeutic importance by enhancing the efficacy of radiotherapy and temozolomide against this lethal disease.