Interactions of Graphene Oxide and Few-Layer Graphene with the Blood-Brain Barrier.

Thanks to their biocompatibility and high cargo capability, graphene-based materials (GRMs) might represent an ideal brain delivery system. The capability of GRMs to reach the brain has mainly been investigated in vivo and has highlighted some controversy. Herein, we employed two in vitro BBB models of increasing complexity to investigate the bionano interactions with graphene oxide (GO) and few-layer graphene (FLG): a 2D murine Transwell model, followed by a 3D human multicellular assembloid, to mimic the complexity of the in vivo architecture and intercellular crosstalk. We developed specific methodologies to assess the translocation of GO and FLG in a label-free fashion and a platform applicable to any nanomaterial. Overall, our results show good biocompatibility of the two GRMs, which did not impact the integrity and functionality of the barrier. Sufficiently dispersed subpopulations of GO and FLG were actively uptaken by endothelial cells; however, the translocation was identified as a rare event.

Human RORγt(+)CD34(+) cells are lineage-specified progenitors of group 3 RORγt(+) innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) are defined by the expression of the transcription factor RORγt, which is selectively required for their development. The lineage-specified progenitors of ILC3s and their site of development after birth remain undefined. Here we identified a population of human CD34(+) hematopoietic progenitor cells (HPCs) that express RORγt and share a distinct transcriptional signature with ILC3s. RORγt(+)CD34(+) HPCs were located in tonsils and intestinal lamina propria (LP) and selectively differentiated toward ILC3s. In contrast, RORγt(-)CD34(+) HPCs could differentiate to become either ILC3s or natural killer (NK) cells, with differentiation toward ILC3 lineage determined by stem cell factor (SCF) and aryl hydrocarbon receptor (AhR) signaling. Thus, we demonstrate that in humans RORγt(+)CD34(+) cells are lineage-specified progenitors of IL-22(+) ILC3s and propose that tonsils and intestinal LP, which are enriched both in committed precursors and mature ILC3s, might represent preferential sites of ILC3 lineage differentiation.

Cyclodextrin Polymers as Delivery Systems for Targeted Anti-Cancer Chemotherapy.

In the few last years, nanosystems have emerged as a potential therapeutic approach to improve the efficacy and selectivity of many drugs. Cyclodextrins (CyDs) and their nanoparticles have been widely investigated as drug delivery systems. The covalent functionalization of CyD polymer nanoparticles with targeting molecules can improve the therapeutic potential of this family of nanosystems. In this study, we investigated cross-linked γ- and ß-cyclodextrin polymers as carriers for doxorubicin (ox) and oxaliplatin (Oxa). We also functionalized γ-CyD polymer bearing COOH functionalities with arginine-glycine-aspartic or arginine moieties for targeting the integrin receptors of cancer cells. We tested the Dox and Oxa anti-proliferative activity in the presence of the precursor polymer with COOH functionalities and its derivatives in A549 (lung, carcinoma) and HepG2 (liver, carcinoma) cell lines. We found that CyD polymers can significantly improve the antiproliferative activity of Dox in HepG2 cell lines only, whereas the cytotoxic activity of Oxa resulted as enhanced in both cell lines. The peptide or amino acid functionalized CyD polymers, loaded with Dox, did not show any additional effect compared to the precursor polymer. Finally, studies of Dox uptake showed that the higher antiproliferative activity of complexes correlates with the higher accumulation of Dox inside the cells. The results show that CyD polymers could be used as carriers for repositioning classical anticancer drugs such as Dox or Oxa to increase their antitumor activity.

Inhibitory 2B4 contributes to NK cell education and immunological derangements in XLP1 patients.

X-linked lymphoproliferative disease 1 (XLP1) is an inherited immunodeficiency, caused by mutations in SH2D1A encoding Signaling Lymphocyte Activation Molecule (SLAM)-associated protein (SAP). In XLP1, 2B4, upon engagement with CD48, has inhibitory instead of activating function. This causes a selective inability of cytotoxic effectors to kill EBV-infected cells, with dramatic clinical sequelae. Here, we investigated the NK cell education in XLP1, upon characterization of killer Ig-like receptor (KIR)/KIR-L genotype and phenotypic repertoire of self-HLA class I specific inhibitory NK receptors (self-iNKRs). We also analyzed NK-cell cytotoxicity against CD48+ or CD48- KIR-ligand matched or autologous hematopoietic cells in XLP1 patients and healthy controls. XLP1 NK cells may show a defective phenotypic repertoire with substantial proportion of cells lacking self-iNKR. These NK cells are cytotoxic and the inhibitory 2B4/CD48 pathway plays a major role to prevent killing of CD48+ EBV-transformed B cells and M1 macrophages. Importantly, self-iNKR defective NK cells kill CD48- targets, such as mature DCs. Self-iNKR- NK cells in XLP1 patients are functional even in resting conditions, suggesting a role of the inhibitory 2B4/CD48 pathway in the education process during NK-cell maturation. Killing of autologous mature DC by self-iNKR defective XLP1 NK cells may impair adaptive responses, further exacerbating the patients' immune defect.

Hematopoietic stem cell transplantation: Improving alloreactive Bw4 donor selection by genotyping codon 86 of KIR3DL1/S1.

KIR3DL1 is a natural killer (NK) cell receptor that recognizes the Bw4 epitope of human leukocyte antigen (HLA) class I molecules. Following hematopoietic stem cell transplantation for patients lacking Bw4, KIR3DL1-expressing NK cells from Bw4-positive donors can be alloreactive and eliminate tumor cells. However, KIR3DL1 alleles having T instead of C at nucleotide 320 (encoding leucine 86 instead of serine 86) are not expressed on the cell surface. Thus, not all individuals testing positive for KIR3DL1 are optimal donors for Bw4-negative recipients. Therefore, we developed a method for genotyping codon 86, which was validated by its perfect correlation with NK cell phenotype for 100 donors of diverse KIR3DL1/S1 genotype. We typed 600 donors and found that ∼12.2% had the KIR3DL1 gene, but did not express cell-surface KIR3DL1. By contrast, high-expressing allotypes were identified when haplotypes from four families with duplicated KIR3DL1/S1 genes were characterized at high resolution. Identifying donors who have KIR3DL1 but lack cell-surface KIR3DL1 would refine donor selection. With this technique, the number of individuals identified who may not be optimal donors for Bw4-negative patients increases by threefold, when compared with standard methods. Taken together, we propose that allele typing of killer cell Ig-like receptor (KIR) polymorphisms should become a standard practice when selecting donors.

γδ T-cell reconstitution after HLA-haploidentical hematopoietic transplantation depleted of TCR-αß+/CD19+ lymphocytes.

We prospectively assessed functional and phenotypic characteristics of γδ T lymphocytes up to 7 months after HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) depleted of αß(+) T cells and CD19(+) B cells in 27 children with either malignant or nonmalignant disorders. We demonstrate that (1) γδ T cells are the predominant T-cell population in patients during the first weeks after transplantation, being mainly, albeit not only, derived from cells infused with the graft and expanding in vivo; (2) central-memory cells predominated very early posttransplantation for both Vδ1 and Vδ2 subsets; (3) Vδ1 cells are specifically expanded in patients experiencing cytomegalovirus reactivation and are more cytotoxic compared with those of children who did not experience reactivation; (4) these subsets display a cytotoxic phenotype and degranulate when challenged with primary acute myeloid and lymphoid leukemia blasts; and (5) Vδ2 cells are expanded in vitro after exposure to zoledronic acid (ZOL) and efficiently lyse primary lymphoid and myeloid blasts. This is the first detailed characterization of γδ T cells emerging in peripheral blood of children after CD19(+) B-cell and αß(+) T-cell-depleted haplo-HSCT. Our results can be instrumental to the development of clinical trials using ZOL for improving γδ T-cell killing capacity against leukemia cells. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

IL-1ß inhibits ILC3 while favoring NK-cell maturation of umbilical cord blood CD34(+) precursors.

NK cells are innate lymphocytes characterized by the expression of nuclear factor interleukin 3 regulated (NFIL3 or E4BP4), eomesodermin (EOMES) transcription factors (TFs), and by the ability to exert cytolytic activity and release IFN-γ. In the haploidentical-hematopoietic stem cell transplantation (haplo-HSCT) setting, CD34(+) donor derived NK cells play a major role in the control of leukemic relapses. Therefore, it is important to better define cytokines that influence NK-cell differentiation from CD34(+) precursors. We analyzed the effects of IL-1ß on NK-cell differentiation from umbilical cord blood (UCB) CD34(+) cells. While IL-1ß inhibited CD161(+) CD56(+) cell proliferation, an increased expression of LFA-1, CD94/NKG2A, KIRs, and perforin on CD56(+) cells was detected. In addition, within the CD161(+) CD56(+) IL-1RI(+) LFA-1(-) cell fraction (representing group 3 innate lymphoid cells, ILC3-like cells), a significant increase of EOMES, NKp46, and CD94/NKG2A receptors, cytolytic granules, and IFN-γ was detected. This increase was paralleled by a decrease of related orphan receptors (RORγt) TF, NKp44 expression, and IL-22 production. These data suggest that IL-1ß inhibits ILC3- while favoring NK-cell maturation. Since in haplo-HSCT conditioning regimen, infections or residual leukemia cells may induce IL-1ß production, this may influence the NK/ILC3 development from donor-derived CD34(+) precursors.

XLP1 inhibitory effect by 2B4 does not affect DNAM-1 and NKG2D activating pathways in NK cells.

X-linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM-associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B-cell lymphomas. Here, we show that in the absence of SLAM-associated protein, co-engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM-dependent signaling pathways including activating killer Ig-like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN-γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM-1 and NKG2D triggering receptors. Thus, while CD48(+) B-EBV and lymphoma B cells devoid of NKG2D and DNAM-1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross-talk of inhibitory 2B4 with triggering NK (and T) receptors.

Few-layered graphene increases the response of nociceptive neurons to irritant stimuli.

The unique properties of few-layered graphene (FLG) make it interesting for a variety of applications, including biomedical applications, such as tissue engineering and drug delivery. Although different studies focus on applications in the central nervous system, its interaction with the peripheral nervous system has been so far overlooked. Here, we investigated the effects of exposure to colloidal dispersions of FLG on the sensory neurons of the rat dorsal root ganglia (DRG). We found that the FLG flakes were actively internalized by sensory neurons, accumulated in large intracellular vesicles, and possibly degraded over time, without major toxicological concerns, as neuronal viability, morphology, protein content, and basic electrical properties of DRG neurons were preserved. Interestingly, in our electrophysiological investigation under noxious stimuli, we observed an increased functional response upon FLG treatment of the nociceptive subpopulation of DRG neurons in response to irritants specific for chemoreceptors TRPV1 and TRPA1. The observed effects of FLG on DRG neurons may open-up novel opportunities for applications of these materials in specific disease models.

Terpyridine Glycoconjugates and Their Metal Complexes: Antiproliferative Activity and Proteasome Inhibition.

Metal terpyridine complexes have gained substantial interest in many application fields, such as catalysis and supramolecular chemistry. In recent years, the biological activity of terpyridine and its metal complexes has aroused considerable regard. On this basis, we synthesised new terpyridine derivatives of trehalose and glucose to improve the water solubility of terpyridine ligands and target them in cancer cells through glucose transporters. Glucose derivative and its copper(II) and iron(II) complexes showed antiproliferative activity. Interestingly, trehalose residue reduced the cytotoxicity of terpyridine. Moreover, we tested the ability of parent terpyridine ligands and their copper complexes to inhibit proteasome activity as an antineoplastic mechanism.

Donor selection for adoptive immunotherapy with NK cells in AML patients: Comparison between analysis of lytic NK cell clones and phenotypical identification of alloreactive NK cell repertoire.

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym "NK-AML" (NCT03955848), and "MRD-NK". The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2+ donors and HLA-C1+ patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories.

Why Do So Many People with Type 2 Diabetes Who Take Insulin Have Lipohypertrophy? Fate or Educational Deficiencies?

INTRODUCTION: Cutaneous lipohypertrophy (LH) is a thickened, "rubbery" lesion in the subcutaneous tissue following multiple injections performed at the same site, i.e., an incorrect injection technique. It is widespread, averaging 47% of insulin patients worldwide, and has severe direct and indirect consequences. Direct consequences consist mainly of poor metabolic control and frequent hypoglycemic events (HYPOs), and indirect ones of markedly increased healthcare costs related to hospital access due to acute events and long-term disease complications. This observation also holds for Italy, despite the National Health System organization expecting every patient with diabetes to undergo a series of visits by different care team members, each performing a specific treatment/education task. Indeed, the recent literature points to poor awareness of LH relevance and metabolic consequences among doctors from general and diabetic hospital wards, with educational deficiencies on correct injection practice in nurses too. The aim was to establish if, to what extent, and by whom they had received training on correct insulin injection techniques, and how many initially received notions had persisted over time. METHODS: We investigated the possible causes of such a failure from the point of view of 1160 insulin-requiring subjects with type 2 diabetes (T2DM), reporting for the first time to specialized diabetic structures through a validated questionnaire and, in the same patients, we searched for LH by inspection/palpation according to international guidelines, further confirmed by ultrasound scans. We then analyzed differences in education and injecting behavior between subjects classified as LH+ or LH- depending on the presence or absence of LH lesions. RESULTS: We documented significant educational gaps, with 50% of patients failing to refer to healthcare professionals and relying on their peers with diabetes, thought to be more experienced in 15% of the cases. Seventy-five percent of LH- patients received education from healthcare providers, while 90% of LH+ learned from another patient or could not remember how they knew, and 68% of LH+ versus 52% of LH- (p < 0.01) patients had failed to receive training on injection techniques by healthcare providers. All of this enabled the most disabling features of diabetes from the very beginning of the disease history. CONCLUSIONS: This study documents, from the patients' point of view, that educational gaps are significant and that, even in initially trained subjects, education on correct injection techniques has a fleeting effect if not regularly recalled. Therefore, to rehabilitate LH+ patients as soon as possible and prevent LH- patients from inadvertently slipping into the other group, there is an urgent need to educate doctors and nurses repeatedly on the importance of correctly injecting insulin to improve patients' knowledge and skills.

Optimized spirooxindole-pyrazole hybrids targeting the p53-MDM2 interplay induce apoptosis and synergize with doxorubicin in A549 cells.

Recently, cancer research protocols have introduced clinical-stage spirooxindole-based MDM2 inhibitors. However, several studies reported tumor resistance to the treatment. This directed efforts to invest in designing various combinatorial libraries of spirooxindoles. Herein, we introduce new series of spirooxindoles via hybridization of the chemically stable core spiro[3H-indole-3,2'-pyrrolidin]-2(1H)-one and the pyrazole motif inspired by lead pyrazole-based p53 activators, the MDM2 inhibitor BI-0252 and promising molecules previously reported by our group. Single crystal X-ray diffraction analysis confirmed the chemical identity of a representative derivative. Fifteen derivatives were screened for cytotoxic activities via MTT assay against a panel of four cancer cell lines expressing wild-type p53 (A2780, A549, HepG2) and mutant p53 (MDA-MB-453). The hits were 8h against A2780 (IC50 = 10.3 µM) and HepG2 (IC50 = 18.6 µM), 8m against A549 (IC50 = 17.7 µM), and 8k against MDA-MB-453 (IC50 = 21.4 µM). Further MTT experiments showed that 8h and 8j potentiated doxorubicin activity and reduced its IC50 by at least 25% in combinations. Western blot analysis demonstrated that 8k and 8m downmodulated MDM2 in A549 cells. Their possible binding mode with MDM2 were simulated by docking analysis.

Role of pericytes in blood-brain barrier preservation during ischemia through tunneling nanotubes.

Crosstalk mechanisms between pericytes, endothelial cells, and astrocytes preserve integrity and function of the blood-brain-barrier (BBB) under physiological conditions. Long intercellular channels allowing the transfer of small molecules and organelles between distant cells called tunneling nanotubes (TNT) represent a potential substrate for energy and matter exchanges between the tripartite cellular compartments of the BBB. However, the role of TNT across BBB cells under physiological conditions and in the course of BBB dysfunction is unknown. In this work, we analyzed the TNT's role in the functional dialog between human brain endothelial cells, and brain pericytes co-cultured with human astrocytes under normal conditions or after exposure to ischemia/reperfusion, a condition in which BBB breakdown occurs, and pericytes participate in the BBB repair. Using live time-lapse fluorescence microscopy and laser-scanning confocal microscopy, we found that astrocytes form long TNT with pericytes and endothelial cells and receive functional mitochondria from both cell types through this mechanism. The mitochondrial transfer also occurred in multicellular assembloids of human BBB that reproduce the three-dimensional architecture of the BBB. Under conditions of ischemia/reperfusion, TNT formation is upregulated, and astrocytes exposed to oxygen-glucose deprivation were rescued from apoptosis by healthy pericytes through TNT-mediated transfer of functional mitochondria, an effect that was virtually abolished in the presence of TNT-destroying drugs. The results establish a functional role of TNT in the crosstalk between BBB cells and demonstrate that TNT-mediated mitochondrial transfer from pericytes rescues astrocytes from ischemia/reperfusion-induced apoptosis. Our data confirm that the pericytes might play a pivotal role in preserving the structural and functional integrity of BBB under physiological conditions and participate in BBB repair in brain diseases.

Characterization of KIR+ NK cell subsets with a monoclonal antibody selectively recognizing KIR2DL1 and blocking the specific interaction with HLA-C.

The phenotypic identification of different NK cell subsets allows more in-depth characterization of KIR repertoire and function, which are of potential interest in KIR and disease association studies. KIR genes are highly polymorphic, but a great homology exists among the various sequences and few monoclonal antibodies (mAbs) specifically recognize a single KIR. This is the case of HP-DM1 which was demonstrated by analysis of cell transfectants and epitope mapping to be exclusively KIR2DL1-specific, covering all allotypes identified to date, except for KIR2DL1*022 and *020, and also to react with KIR2DS1*013. Here, we compared in immunofluorescence analyses the staining of HP-DM1 with other available mAbs to precisely identify KIR2DL1+ NK cells in potential donors for αßT/B-depleted haplo-HSCT, with known KIR genotype. HP-DM1 mAb was used in combination with EB6 or 11PB6 (anti-KIR2DL1/S1 and anti-KIR2DL3*005), 143211 (anti-KIR2DL1/S5), and HP-MA4 (anti-KIR2DL1/S1/S3/S5) mAbs, allowing the accurate identification of different KIR+ NK cell subsets. These phenotypic evaluations appeared useful to dissect the expression pattern of various KIR2D in NK cells from KIR2DL3*005+ individuals, particularly if KIR2DS1 is present. HP-DM1 mAb remarkably refined NK cell phenotyping of donors carrying KIR2DS5, either in the centromeric or telomeric region. Functional assays with KIR2DL1+ /S1+ /S5+ NK cells confirmed that only HP-DM1 exclusively reacts with KIR2DL1. Finally, we demonstrated that HP-DM1 mAb blocked KIR2DL1 recognition of C2+ HLA-C. Altogether, the data support that HP-DM1 is a unique reagent valuable for characterizing KIR+ NK cell subsets.

Exploiting Natural Killer Cell Engagers to Control Pediatric B-cell Precursor Acute Lymphoblastic Leukemia.

Natural killer (NK) cells represent a promising cell type in antitumor immunotherapy for efficacy and safety, particularly in the treatment of hematologic malignancies. NK cells have been shown to exert antileukemia activity in the context of haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Products have been developed to boost the activation of NK cells only when cross-linked by tumor cells, avoiding any off-target effect. Here, we tested the in vitro effect of different NK-cell engagers (NKCE), which trigger either NKp46 or NKp30 together with CD16A, and target either CD19 or CD20 to induce killing of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Target cells were NALM-16 and MHH-CALL-4 cell lines and four primary leukemias, while effector cells were resting NK cells derived from healthy donors and pediatric patients with leukemia after αßT/B-depleted haplo-HSCT. The NK cell-resistant MHH-CALL-4 was efficiently killed using all NKCEs. Boosting of NK activity against MHH-CALL-4 was also evident by degranulation and IFNγ production. Because of the lack of CD20 and high expression of CD19 on primary BCP-ALL, we focused on NKCEs targeting CD19. NKp46- and NKp30-based NKCEs displayed similar potency at inducing NK-cell activity, even when challenged with primary BCP-ALL blasts. Their efficacy was shown also using NK cells derived from transplanted patients. NKCE-induced activation against BCP-ALL can override HLA-specific inhibitory interactions, although the strongest response was observed by the alloreactive NK-cell subset. These data support the therapeutic use of NKp46/CD16A/CD19-NKCE to fight refractory/relapsed leukemia in pretransplantation or posttransplantation settings.

The Economic Burden of Insulin Injection-Induced Lipohypertophy. Role of Education: The ISTERP-3 Study.

INTRODUCTION: The history of insulin-induced skin lipohypertrophy (LH) runs parallel to that of insulin's 100 years, and an average of 47% of insulin-treated patients still suffer from it today. The metabolic and economic effects of LH are significant, with hypoglycemia being the most striking. The objective of the study was to perform a 52-week follow-up of 713 insulin-treated patients with type 2 diabetes (T2DM) and LH to detect any differences in the occurrence of hypoglycemic events (HYPOs) and related healthcare costs as well as in LH rates and injection habits between an intensive education intervention group (IG) and control group (CG) provided with a single educational session at the starting point. METHODS: All participants were trained in accurately self-monitoring blood glucose and recording all HYPOs for 6 months, which allowed baseline recordings before they were randomized into the IG, comprising 395 insulin-treated subjects undergoing repeated, structured multimodal education on correct injection techniques as a longstanding behavioral rehabilitation strategy, and the CG, comprising 318 subjects receiving the same structured, multimodal educational session, but only initially. RESULTS: Changes in LH rate and size and in performance were large in the IG and only slight and transient in the CG. A striking difference in the rate of decrease of HYPOs was also apparent between groups. Indeed, estimated costs of health interventions for severe and symptomatic HYPOs, which were on the order of €70,000 and €9300, respectively, in the two groups at baseline decreased by 5.9 times and 13.7 times, respectively, at the end of follow-up in the IG and by only approximately half in the CG. Full details of the changes occurring as a result of intensive education are provided in the text. CONCLUSIONS: The effect of only initial education in the CG was not significant, thus providing evidence of the virtual worthlessness of a single training session on injection techniques, typical of worldwide daily clinical practice, and easily explaining the extremely high prevalence of LH in insulin-treated patients. Conversely, highly positive effects on LH prevalence and size as well as costs expected from decreased HYPO rate were obtained in the IG. To our knowledge, ours is the first 18-month randomized trial in the field. If our experimental model were to be used as an effective, longstanding behavioral rehabilitation strategy and therefore adapted to real-world settings universally, LH prevalence and costs related to their clinical consequences would be drastically reduced. However, only with a strong, relentless commitment of universities, scientific societies, and patient associations can we achieve this ambitious goal, which would provide great institutional savings and improved quality of life for people with diabetes.

The Durability of an Intensive, Structured Education-Based Rehabilitation Protocol for Best Insulin Injection Practice: The ISTERP-2 Study.

INTRODUCTION: Studies on the durability of an intensive, structured education protocol on best insulin injection practice are missing for people with type 2 diabetes mellitus (T2DM). The aim of this study was to assess the durability of an intensive, structured education-based rehabilitation protocol on best insulin injection practice in well-trained subjects from our previous intensive, multimedia intervention study registered as the ISTERP-1 study. A total of 158 subjects with T2DM from the well-trained group of the 6-month-long ISTERP-1 study, all of whom had successfully attained lower glucose levels compared to baseline levels with lower daily insulin doses and with less frequent and severe hypoglycemic episodes, participated in the present investigation involving an additional 6-month follow-up period, called the ISTERP-2 study. METHODS: Participants were randomized into an intervention group and a control group, depending on whether they were provided or not provided with further education refresher courses for 6 months. At the end of the 6 months, the two groups were compared in terms of injection habits, daily insulin dose requirement, number of severe or symptomatic hypoglycemic events, and glycated hemoglobin (HbA1c) levels. RESULTS: Despite being virtually superimposable at baseline, the two groups behaved quite differently during the follow-up. The within-group analysis of observed parameters showed that the subjects in the intervention group maintained and even improved the good behavioral results learned during the ISTERP-1 study by further reducing both the rate of injection technique errors (p < 0.001) and size of lipohypertrophic lesions at injection sites (p < 0.02). Conversely, those in the control group progressively abandoned best practice, except for the use of ice-cold insulin and, consequently, had significantly higher HbA1c levels and daily insulin dose requirements at the end of the follow-up than at baseline (p < 0.05). In addition, as expected from all the above, the rate of hypoglycemic episodes also decreased in the intervention group (p < 0.05), resulting in a significant difference between groups after 6 months (p < 0.02). CONCLUSION: Our data provide evidence that intensive, structured education refresher courses have no outstanding durability, so that repeated refresher courses, at least at 6-month intervals, are needed to have positive effects on people with T2DM, contributing not only to prevention but also to long-term rehabilitation. TRIAL REGISTRATION: Trial Registration no. 118 bis/15.04.2018.

An Anti-inflammatory microRNA Signature Distinguishes Group 3 Innate Lymphoid Cells From Natural Killer Cells in Human Decidua.

Innate lymphoid cells (ILCs) are a heterogeneous subset of lymphocytes deeply implicated in the innate immune responses to different pathogens, in lymphoid organogenesis and in the maintenance of tissue homeostasis. Group 3 innate lymphoid cells (ILC3) have been detected in human decidua, where they play a role in the early inflammatory phase favoring implantation and tissue remodeling as well as in the subsequent regulatory phase preventing fetal rejection and supporting neoangiogenesis. A balance between inflammation and immune tolerance is required to maintain pregnancy, thus maternal immune system must be controlled by finely tuned mechanisms. microRNAs (miRNAs) are small non-coding RNAs with important regulatory roles in immune cells, but their function in decidual ILC3 (dILC3) and in decidual NK (dNK) cells is still undefined. Here, we examined the miRNome by microarray in these cells during the first trimester of pregnancy and compared with miRNA profiles of peripheral blood NK (pbNK) cells from pregnant women. We show that distinct miRNA profiles could clearly distinguish dILC3 from NK cells. Correlation analyses revealed that dNK and pbNK miRNome profiles are more similar to each other as compared to dILC3. In particular, we identified 302 and 279 mature miRNAs differentially expressed in dILC3 as compared to dNK and pbNK, respectively. The expression of miR-574-3p and the miR-99b/let-7e/miR-125a miRNA cluster resulted the most increased in dILC3. Remarkably, gene ontology analysis and pathway enrichments of miRNA targets revealed an involvement of these miRNAs in the promotion of anti-inflammatory responses. In agreement to this finding, we also found a higher expression of the anti-inflammatory miR-146a-5p in dILC3 with respect to NK cells. Overall, our data identified specific miRNA signatures distinguishing dILC3, dNK, and pbNK cells. Our data suggest the existence of a tight epigenetic control mediated by miRNAs in dILC3, potentially acting as a brake to prevent exaggerated inflammatory responses and to maintain the immune homeostasis in the early phases of pregnancy.

Myeloma cells induce the accumulation of activated CD94low NK cells by cell-to-cell contacts involving CD56 molecules.

Natural killer (NK) cells represent innate effector cells potentially able to play a role during the immune response against multiple myeloma (MM). To better define the distribution and the specific properties of NK cell subsets during MM disease, we analyzed their features in the bone marrow and peripheral blood of newly diagnosed MM patients. Our findings revealed that, in both compartments, NK cells were more abundant than in healthy donors. Among total MM-NK cells, a significant increase of CD94lowCD56dim NK cell subset was observed, which already appears in clinical precursor conditions leading to MM, namely monoclonal gammopathy of undetermined significance and smoldering MM, and eventually accumulates with disease progression. Moreover, a consistent fraction of CD94lowCD56dim NK cells was in a proliferation phase. When analyzed for their killing abilities, they represented the main cytotoxic NK cell subset against autologous MM cells. In vitro, MM cells could rapidly induce the expansion of the CD94lowCD56dim NK cell subset, thus reminiscent of that observed in MM patients. Mechanistically, this accumulation relied on cell to cell contacts between MM and NK cells and required both activation via DNAM-1 and homophilic interaction with CD56 expressed on MM cells. Considering the growing variety of combination treatments aimed at enhancing NK cell-mediated cytotoxicity against MM, these results may also be informative for optimizing current immunotherapeutic approaches.