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Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.

Existing literature has established the benefits of recess and physical activity for children. In response, more schools in the United States are implementing, or even requiring, extended recess time for students. With these policy changes, it is important to understand the impact upon students, faculty, and staff. The following study used the shared experience of three elementary schools in the southern United States who recently implemented extended recess. Semi-structured focus groups with administrators, teachers, paraprofessionals, and students from three elementary schools were included in this pilot study. A grounded theory approach was utilized, and findings confirmed the known benefits of recess but also enhanced understanding of the process, which included the challenges involved as well as the importance of stakeholder feedback. Recommendations are included for the professional school counselor which are applicable in both, the United States and internationally.

Background Thalassemia is a hemoglobin synthesis disorder that causes patients to need lifelong blood transfusions, leading to iron overload and alter organ function, including the liver. Hepcidin, produced by the liver, plays a role in iron homeostasis and should be increased in excess iron stores. However, the level decreases in thalassemia due to some factors, such as ineffective erythropoiesis and liver damage. Recent publications revealed that hepcidin could be associated with iron overload and also a marker of liver diseases. Objective To analyse the correlation between hepcidin level, markers of iron overload, and liver damage in beta-thalassemia major. Methods This cross-sectional study included all ?-thalassemia major age 2-18 years admitted to Dr. Mohammad Hoesin Hospital, Palembang, South Sumatera, who underwent blood transfusions from March to August 2022. We measured the level of iron overload markers, hepcidin, liver function test (LFT), and performed liver ultrasonography (USG). Results Of 97 subjects, median hepcidin level was 10.01 ng/mL and 68% of the subjects showed a decrease. The iron overload parameters were evaluated from serum iron levels (P=0.13), ferritin levels (P=0.90), and transferrin saturation (P=0.29) and 24.7% had abnormal liver USG findings. Spearman’s correlation revealed that only direct bilirubin (DB) (r=0.35; P=0.001) and liver USG (r=0.20; P=0.05) had positive correlations with decreased levels of hepcidin. Also, it had 91.7% sensitivity in predicting liver damage from ultrasound. Conclusion The hepcidin level was not significantly associated with iron overload markers.

Purpose: Plantago Major (PM) is widely used for the treatment of different diseases due to several active compounds. Previous studies demonstrated the treatment effect of this plant on lung cancer cell lines. Here, we introduced a patient having lung cancer proved by Computed Tomography (CT) and pathological findings. The treatment effect of PM was assessed and presented based on CT and laboratory examinations for this patient as a first human case study. Materials and Methods: A 64-year-old woman, with gastrointestinal bleeding as well as high Erythrocyte Sedimentation Rate (ESR) and C-Reactive Protein (CRP) levels, was examined by the chest CT scan for suspicious lung cancer. Pathological findings confirmed the non-small cell lung cancer (adenocarcinoma, stage IIA). The patient consumed 150 ml of PM seeds extract (3-4 times daily) orally for about 4 months. Follow-up CT and laboratory examinations were performed after the treatment period to assess the effect of PM. Results: The volume of the tumor was reduced by about 62% (based on CT imaging findings) after the treatment with PM. In addition, the laboratory examinations illustrate that ESR and CRP levels reduced remarkably (from 97 mm/h to 24 mm/h for ESR; from 36.8 mg/L to 1.2 mg/L for CRP) after the treatment. Conclusion: Based on our human study, PM, as a natural compound with antioxidant, anti-inflammatory, and antibiotic characteristics, could have an anti-proliferative effect on non-small cell lung cancer. However, more follow-up examinations on big sample sizes are needed to assess the treatment effect of PM.

Background: Psychological distress among college students is becoming a matter of significant clinical concern. Despite metacognitive beliefs and resting heart rate variability (HRV) are both associated with risk of mood disorders, whether in presence of maladaptive cognitive beliefs, resting HRV can be considered a protective factor for psychological distress in college students has yet to be investigated. Therefore, the present study aimed to test the relationship between maladaptive metacognitions (i.e., positive beliefs about worry; concerns about uncontrollability and danger; lack of cognitive confidence; beliefs about the need to control thoughts; cognitive self-consciousness) and psychological distress among college students, and the possible moderating effects of resting HRV. Methods: Forty college students reporting negative history of neurological/psychiatric diseases and no use of drugs/substances of abuse were recruited at the University of Padova, Italy. Psychological distress and maladaptive metacognitions were assessed using the Depression Anxiety-Stress Scales 21 and the Metacognitions Questionnaire 30, respectively. Moreover, the electrocardiogram was recorded at rest for 5 minutes and HRV was computed as the root mean square of the successive differences in the interbeat intervals (rMSSD). Limitations: Given the small sample size, this study should be used to design larger confirmatory studies. Conclusions: Our findings suggest specific maladaptive metacognitions and high resting HRV as potential psychological vulnerability and psychophysiological protective factors of psychological distress among college students. From a clinical perspective, the present study suggests to implement prevention strategies targeting vagally-mediated HRV in order to reduce the risk of developing clinically significant psychological distress in college students.

IL-37 is a member of the IL-1 superfamily exerting anti-inflammatory functions in a number of diseases. Extracellular IL-37 triggers the inhibitory receptor IL-1R8 that is known to regulate different NK cell pathways and functional activities including their anti-tumor effect. However, the effect of IL-37 on human NK cell functions is still to be unveiled. This study aimed to investigate the functional effect of IL-37 in human NK cells activated with IL-15. We found that IL-37 enhanced both NK cell cytotoxic activity against different tumor cell lines and cytokines production. These effects were associated with increased phosphorylation of ERK and NF-Kb. The improved NK cell activity was also strictly related to a time-dependent GSK3β-mediated degradation of IL-1R8. The enhanced activation profile of IL-37 treated NK cells possibly due to IL-1R8 degradation was confirmed by the results with IL-1R8-silenced NK cells. Lastly, in line with these data, through the analysis of the TNM plot database of a large group of patients, IL-37 mRNA expression was found to be significantly lower in colon and skin cancers than in normal tissues. Colon adenocarcinoma and neuroblastoma patients with higher IL-37 mRNA levels had significantly higher overall survival, suggesting that the presence of IL-37 might be considered an independent positive prognostic factor for this tumor. Our results provide novel information on the mechanisms regulating IL-1R8 function in human NK cells, highlighting the IL-37-IL-1R8 axis as a potential new target to improve the anti-tumor immune response.

The long story of NK cells started about 50 y ago with the first demonstration of a natural cytotoxic activity within an undefined subset of circulating leukocytes, has involved an ever-growing number of researchers, fascinated by the apparently easy-to-reach aim of getting a “universal anti-tumor immune tool”. In fact, in spite of the impressive progress obtained in the first decades, these cells proved far more complex than expected and, paradoxically, the accumulating findings have continuously moved forward the attainment of a complete control of their function for immunotherapy. The refined studies of these latter years have indicated that NK cells can epigenetically calibrate their functional potential, in response to specific environmental contexts, giving rise to extraordinarily variegated subpopulations, comprehensive of memory-like cells, tissue-resident cells, or cells in various differentiation stages, or distinct functional states. In addition, NK cells can adapt their activity in response to a complex body of signals, spanning from the interaction with either suppressive or stimulating cells (myeloid-derived suppressor cells or dendritic cells, respectively) to the engagement of various receptors (specific for immune checkpoints, cytokines, tumor/viral ligands, or mediating antibody-dependent cell-mediated cytotoxicity). According to this picture, the idea of an easy and generalized exploitation of NK cells is changing, and the way is opening toward new carefully designed, combined and personalized therapeutic strategies, also based on the use of genetically modified NK cells and stimuli capable of strengthening and redirecting their effector functions against cancer.

Introduction: Psoriasis is a chronic immune-mediated disease that can be challenging to treat, especially in patients with severe disease or high body weight. Tildrakizumab is a monoclonal antibody which inhibits IL-23, approved for moderate-to-severe psoriasis with a standard 100 mg dose. A 200 mg dose may provide greater efficacy for patients over 90 kg or with high disease burden.Methods: This multicenter, prospective study evaluated the effectiveness and safety of tildrakizumab 200 mg in patients with moderate-to-severe psoriasis, focusing on those with specific challenges: body weight over 90 kg, baseline PASI ≥20, and difficult-to-treat areas. The study also compared bio-naive versus bio-experienced and male versus female patients. Adults received tildrakizumab 200 mg subcutaneously at weeks 0 and 4, then every 12 weeks.Results: Clinical improvements were assessed using PASI, DLQI, genital PASI, and NAPSI scores. After 24 weeks, the mean PASI score dropped from 14.6 to 0.4, with PASI 90 and PASI 100 scores exceeding 80% (100.0% and 80.3%, respectively). DLQI scores improved from 14.2 to 1.8, and significant improvements were seen in genital PASI and NAPSI scores. No significant adverse events occurred.Conclusions: Tildrakizumab 200 has been shown to be an effective therapeutic option, particularly for patients with high body weight, significant disease burden, and involvement of sensitive areas with no new safety signals.

Introduction: Adherence to mesalamine therapy in ulcerative colitis is often inadequate. This affects long-term remission and to some extent the risk of colon cancer. Means for assessing non-adherent behavior are cumbersome, expensive, and/or time consuming. Unless multiple tools are used in association, a proportion of patients with volitional and non-volitional non-adherence is nonetheless undetected. The study was aimed at evaluating to which extent rephrasing a single question on adherence to mesalamine therapy may help identifying patients who are not compliant with medication prescription. Methods: One-hundred and seventy-four inflammatory bowel disease outpatients were asked in two consecutive visits, in random order, if they “regularly assumed the prescribed dose of mesalamine” (adherence-centered question—AQ) or “how often they skipped mesalamine pills” (non-adherence centered question—NQ). Answer concordance was evaluated in relation to clinical and demographic variables. Results: The concordance between AQ and NQ was low (K = 0.22). Lower compliance to therapy was admitted in 37.4% more patients following NQ than AQ. The reported adherence to AQ was invariably higher than that of NQ, irrespective of the variable taken into consideration. The likelihood of non-concordant answers was non-significantly higher in CD patients than in UC and in patients with shorter disease duration than in those with longstanding disease, but the logistic regression model did not identify individual variables responsible for the different answers. Conclusions: Being simple and not requiring additional time expense, centering the question on medication non-adherence identifies a large proportion of patients who would not admit non-adherence or underestimate the number of skipped doses of medication, when directly asked if they are compliant to therapy.

IgM is the major circulating Ig isotype in teleost fish, showing in Antarctic fish unique features such as an extraordinary long hinge region, which plays a crucial role in antibody structure and function. In this work, we describe the replacement of the hinge region of a murine monoclonal antibody (mAb) with the peculiar hinge from Antarctic fish IgM. We use the CRISPR/Cas9 system as a powerful tool for generating the engineered mAb. Then, we assessed its functionality by using an innovative plasmonic substrate based on bimetallic nanoislands (AgAuNIs). The affinity constant of the modified mAb was 2.5-fold higher than that obtained from wild-type mAb against the specific antigen. Here, we show the suitability of the CRISPR/Cas9 method for modifying a precise region in immunoglobulin gene loci. The overall results could open a frontier in further structural modifications of mAbs for biomedical and diagnostic purposes.

Introduction: This was an observational, retrospective, multicenter study, enrolling elderly patients (>65 years old) treated with ixekizumab with a diagnosis of psoriasis (PsO) and/or psoriatic arthritis (PsA) during the period 2020 to 2023. Objectives: We sought to investigate the efficacy of ixekizumab in elderly patients in the treatment of moderate to severe psoriasis. Methods: We included 73 patients with psoriasis (32.9%), psoriatic arthritis (1.4%) and both of them (PsO-PsA 65.8%), attending the outpatient clinics of seven Italian referral center for psoriasis in Lazio region: Policlinico Umberto I Università Roma La Sapienza, Sant’Andrea Università di Roma La Sapienza, Polo Pontino Università Roma La Sapienza, Fondazione Policlinico Universitario A. Gemelli, Università Campus Biomedico Roma, Istituto Dermopatico dell’Immacolata, and Policlinico Tor Vergata. We collected data related to the characteristics of the patients (age, sex, body mass index) and of the disease (age at onset, duration of psoriasis, previous treatments). The severity of psoriasis was measured with the Psoriasis Area and Severity Index (PASI) score at baseline and after 16, 24, 52, 104 and 156 weeks of treatment. Results: PASI90 was achieved by all the patients in week 16 and remained stable until the end of the study. PASI100 has been achieved by 55.1% of patients at weeks 16 and by 81.3% at week 104. A statistically significant difference has been highlighted between baseline and all the other time points (p

Aldo Pezzuto,1 Alberto Ricci,1 Michela D’Ascanio,1 Alba Moretta,1 Giuseppe Tonini,2 Noemi Calabrò,1 Valeria Minoia,1 Alessia Pacini,1 Giuliana De Paolis,1 Eleonora Chichi,1 Elisabetta Carico,3 Antonella Tammaro4 1Department of Cardiovascular and Respiratory Sciences, S Andrea Hospital, Sapienza University, Rome, Italy; 2Oncology Department, Campus Bio-Medico University, Rome, Italy; 3Clinical and Molecular Medicine Department, S Andrea Hospital, Sapienza University, Rome, Italy; 4Department of Neuroscience- NESMOS, S.Andrea Hospital, Sapienza University, Rome, ItalyCorrespondence: Aldo Pezzuto, Email aldo.pezzuto@uniroma1.itBackground: Cigarette smoke exposure is the main preventable cause of chronic obstructive pulmonary disease (COPD). Airflow limitation is closely associated with smoking exposure. Smoking could also interfere with lipid metabolism.Aim: To determine the respiratory functional and metabolic changes after smoking cessation in smokers in the short term.Methods: All patients were current smokers. They were assessed by spirometry and questionnaires such as COPD assessment test(CAT), modified Medical Research Council (mMRC) test for dyspnea, Fagestrom’s test for nicotine dependence. Exhaled CO was detected in order to evaluate smoking exposure and smoking cessation (normal value< 7 ppm). A blood sampling was eventually taken for vitamin D and cholesterol assay. All patients underwent therapy with counselling and varenicline as first-line treatment according to its schedule. Detection time: at baseline and one month after smoking cessation.Results: All patients quit smoking during treatment. The mean age was 62 with a prevalence of males. The analysis revealed the following mean values at baseline: CAT mean score was 15, pack-years 35.5, Fagestrom’s Test mean score 5.0. The West’s value was 8.5, whereas Body mass index (BMI) was 25.5.Cigarette daily consumption mean value was 22.5. The comparison before and at follow up one month after smoking cessation about functional and metabolic parameters, show us the following results: FEV 1 was increased by 200 mL (p< 0.02), FEF 25/75 was improved as well as mMRC test. The eCO was dropped to as low as 8 ppM. Interestingly the vitamin D level was increased from 25 to 28 ng/mL without any support therapy. The cholesterol total level was reduced and CAT value and DLCO were also significantly improved.Conclusion: Quit smoking is useful to improve symptoms, respiratory function and metabolic parameters in the short term.Keywords: airflow limitation, smoking cessation, lipid metabolism

Mycoplasma spp. has been involved in canine infertility, but research on this topic is limited and shows conflicting results, as it has also been isolated from vaginal swabs of healthy dogs. This study aimed to retrospectively research Mycoplasma DNA by a conventional dual Polymerase Chain Reaction from 114 cases of canine abortion, stillbirth, and neonatal mortality that occurred in Central Italy. In addition, four fetal membranes from dystocic dams were analyzed. In total, 7 out of 114 cases (6.14%) and one of the fetal membranes tested positive for Mycoplasma DNA. From five of them (62.5%), other microorganisms were identified, particularly Canid herpesvirus-1 (2/8, 25%) and Escherichia coli, Staphylococcus aureus, and/or Staphylococcus pseudintermedius (3/8, 37.5%), notoriously responsible for infertility in bitches or neonatal mortality. In two different litters, only one puppy of each one tested positive for Mycoplasma DNA. Additionally, Mycoplasma DNA was detected in fetal membranes collected during a cesarean section of a bitch whelping Mycoplasma-negative puppies, supporting that Mycoplasma spp. is part of the normal genital microflora of the female. The detection of Mycoplasma DNA in association with other major pathogens and its detection in the female genital tract without transmission to puppies support the hypothesis that Mycoplasma belongs to the autochthonous genital microflora or, at most, may play a secondary role in canine abortion and neonatal mortality.

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.

Abstract By responding to a host of adverse conditions, ranging from DNA damage to viral infection, transcription factor p53 supports genomic stability, cellular health, and survival. Not surprisingly, tumours across the cancer spectrum carry mutations in p53, misexpress the protein, or dysregulate its activity. Several signalling pathways, many of which comprise oncogenic proteins, converge upon p53 to control its stability and activity. We here present the conserved kinase/ATPase RioK1 as an upstream factor that determines p53 activity at the DNA, RNA, and protein levels. It achieves this task by integrating the regulatory events that act on p53 into a coherent response circuit. We will also discuss how RIOK1 overexpression represents an alternative mechanism for cancers to inactivate p53, and how targeting RioK1 could eradicate malignancies that are driven by a dysregulated RioK1-p53 network.

Background Chronic kidney disease (CKD) has become a global burden on the healthcare system and significantly impacts the quality of life of children with the condition. Objective To assess quality of life in children with CKD as well as its relationship with sociodemographic, medical, and psychosocial factors. Methods This cross-sectional analytic study was conducted from June to November 2021 at Dr. Moh Hoesin Hospital, Palembang. Children with CKD aged 2–18 years were included by consecutive sampling. Parents and patients were asked to complete the PedsQL™ generic score scale version 4.0 questionnaire. Results We assessed quality of life in 112 children with CKD from parents’ and children’s reports in the PedsQL™ questionnaire. Physical and emotional parameters had the lowest scores. Based on parental reports, quality of life was significantly associated with disease severity (P=0.002), behavioral disorders (P=0.007), and sleep disturbances (P=0.001). Based on the children’s reports, the factors significantly associated with quality of life were anemia (P=0.044), sleep disturbances (P=0.024), and behavioral disorders (P=0.002). Almost one-third of children with CKD had general impairment of quality of life, both from parental reports (32.1%) and children’s reports (33.0%). Conclusion Disease severity, anemia, sleep disturbance, and behavioral disorders were all associated with poorer quality of life in children with CKD.

BackgroundIn early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s).MethodsSP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs.ResultsSPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals.ConclusionsTLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID‐19 subjects.

BackgroundNeuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function.MethodsWe studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays.ResultsWe identified mature (CD105+/CD133−) and undifferentiated (CD133+/CD105−) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic.ConclusionsNB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.