Distinct roles of vaccine-induced SARS-CoV-2-specific neutralizing antibodies and T cells in protection and disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) lack cross-reactivity between SARS-CoV species and variants and fail to mediate long-term protection against infection. The maintained protection against severe disease and death by vaccination suggests a role for cross-reactive T cells. We generated vaccines containing sequences from the spike or receptor binding domain, the membrane and/or nucleoprotein that induced only T cells, or T cells and NAbs, to understand their individual roles. In three models with homologous or heterologous challenge, high levels of vaccine-induced SARS-CoV-2 NAbs protected against neither infection nor mild histological disease but conferred rapid viral control limiting the histological damage. With no or low levels of NAbs, vaccine-primed T cells, in mice mainly CD8+ T cells, partially controlled viral replication and promoted NAb recall responses. T cells failed to protect against histological damage, presumably because of viral spread and subsequent T cell-mediated killing. Neither vaccine- nor infection-induced NAbs seem to provide long-lasting protective immunity against SARS-CoV-2. Thus, a more realistic approach for universal SARS-CoV-2 vaccines should be to aim for broadly cross-reactive NAbs in combination with long-lasting highly cross-reactive T cells. Long-lived cross-reactive T cells are likely key to prevent severe disease and fatalities during current and future pandemics.

Pulsed Electromagnetic Fields: A Novel Attractive Therapeutic Opportunity for Neuroprotection After Acute Cerebral Ischemia.

OBJECTIVES: Acute cerebral ischemia is characterized by several pathological processes evolving during time, which contribute to the final tissue damage. Secondary processes, such as prolonged inflammatory response, impaired mitochondrial function, and oxidative stress, are responsible for the progression of brain injury to the peri-infarct area, called "penumbra." Adenosine has been shown to play a crucial role in regulating the inflammatory cascade following brain ischemia. Pulsed electromagnetic fields (PEMFs) act as modulators of adenosine receptors, increasing the functionality of the endogenous adenosine. In particular, PEMF exposure induces a significant upregulation of A2A and A3 adenosine receptors in different neuronal cell types. Several lines of evidence suggest that PEMF exposure might play a neuroprotective role after ischemic damage. MATERIALS AND METHODS: This review summarizes the current knowledge on the mechanism of action of PEMFs and their biological effects on neuronal damage both in preclinical and clinical studies. RESULTS: PEMFs counteract hypoxia-induced apoptosis and ROS production in neuronal-like cells and exert a strong anti-inflammatory effect on microglial cells. Data from stroke animal models showed that PEMFs exposure is able to reduce the size of the infarct area and decrease the levels of pro-inflammatory mediators. In clinical studies, PEMFs stimulation proved to be safe and well tolerated. Preliminary results on acute ischemic stroke patients showed a dose-dependent reduction in the lesion size. CONCLUSIONS: Altogether, these data demonstrate the efficacy of PEMFs against several mechanisms underlying ischemic damage and suggest that PEMFs might represent a novel noninvasive adjunctive treatment for acute ischemic stroke, providing neuroprotection and reducing functional deficits following ischemia.

Pulsed Electromagnetic Field Stimulation in Osteogenesis and Chondrogenesis: Signaling Pathways and Therapeutic Implications.

Mesenchymal stem cells (MSCs) are the main cell players in tissue repair and thanks to their self-renewal and multi-lineage differentiation capabilities, they gained significant attention as cell source for tissue engineering (TE) approaches aimed at restoring bone and cartilage defects. Despite significant progress, their therapeutic application remains debated: the TE construct often fails to completely restore the biomechanical properties of the native tissue, leading to poor clinical outcomes in the long term. Pulsed electromagnetic fields (PEMFs) are currently used as a safe and non-invasive treatment to enhance bone healing and to provide joint protection. PEMFs enhance both osteogenic and chondrogenic differentiation of MSCs. Here, we provide extensive review of the signaling pathways modulated by PEMFs during MSCs osteogenic and chondrogenic differentiation. Particular attention has been given to the PEMF-mediated activation of the adenosine signaling and their regulation of the inflammatory response as key player in TE approaches. Overall, the application of PEMFs in tissue repair is foreseen: (1) in vitro: to improve the functional and mechanical properties of the engineered construct; (2) in vivo: (i) to favor graft integration, (ii) to control the local inflammatory response, and (iii) to foster tissue repair from both implanted and resident MSCs cells.

Pulsed Electromagnetic Fields Stimulate HIF-1α-Independent VEGF Release in 1321N1 Human Astrocytes Protecting Neuron-Like SH-SY5Y Cells from Oxygen-Glucose Deprivation.

Pulsed electromagnetic fields (PEMFs) are emerging as an innovative, non-invasive therapeutic option in different pathological conditions of the central nervous system, including cerebral ischemia. This study aimed to investigate the mechanism of action of PEMFs in an in vitro model of human astrocytes, which play a key role in the events that occur following ischemia. 1321N1 cells were exposed to PEMFs or hypoxic conditions and the release of relevant neurotrophic and angiogenic factors, such as VEGF, EPO, and TGF-ß1, was evaluated by means of ELISA or AlphaLISA assays. The involvement of the transcription factor HIF-1α was studied by using the specific inhibitor chetomin and its expression was measured by flow cytometry. PEMF exposure induced a time-dependent, HIF-1α-independent release of VEGF from 1321N1 cells. Astrocyte conditioned medium derived from PEMF-exposed astrocytes significantly reduced the oxygen-glucose deprivation-induced cell proliferation and viability decrease in the neuron-like cells SH-SY5Y. These findings contribute to our understanding of PEMFs action in neuropathological conditions and further corroborate their therapeutic potential in cerebral ischemia.

Bone Morphogenetic Protein-2 Signaling in the Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Induced by Pulsed Electromagnetic Fields.

Pulsed electromagnetic fields (PEMFs) are clinically used with beneficial effects in the treatment of bone fracture healing. This is due to PEMF ability to favor the osteogenic differentiation of mesenchymal stem cells (MSCs). Previous studies suggest that PEMFs enhance the osteogenic activity of bone morphogenetic protein-2 (BMP2) which is used in various therapeutic interventions. This study investigated the molecular events associated to the synergistic activity of PEMFs and BMP2 on osteogenic differentiation. To this aim, human MSCs (hMSCs) were exposed to PEMFs (75 Hz, 1.5 mT) in combination with BMP2, upon detection of the minimal dose able to induce differentiation. Changes in the expression of BMP signaling pathway genes including receptors and ligands, as well as in the phosphorylation of BMP downstream signaling proteins, such as SMAD1/5/8 and MAPK, were analyzed. Results showed the synergistic activity of PEMFs and BMP2 on osteogenic differentiation transcription factors and markers. The PEMF effects were associated to the increase in BMP2, BMP6, and BMP type I receptor gene expression, as well as SMAD1/5/8 and p38 MAPK activation. These results increase knowledge concerning the molecular events involved in PEMF stimulation showing that PEMFs favor hMSCs osteogenic differentiation by the modulation of BMP signaling components.

Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis.

Primary Myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by a skewed megakaryopoiesis and an overproduction of proinflammatory and profibrotic mediators that lead to the development of bone marrow (BM) fibrosis. Since we recently uncovered the upregulation of miR-34a-5p in PMF CD34+ hematopoietic progenitor cells (HPCs), in order to elucidate its role in PMF pathogenesis here we unravelled the effects of miR-34a-5p overexpression in HPCs. We showed that enforced expression of miR-34a-5p partially constrains proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF CD34+ cells. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression on HPCs lineage choice, by favouring the megakaryocyte and monocyte/macrophage commitment. Collectively our data unravel the role of miR-34a-5p in HPCs fate decision and suggest that the increased expression of miR-34a-5p in PMF HPCs could be important for the skewing of megakaryopoiesis and the production of monocytes, that are key players in BM fibrosis in PMF patients.

Integrative analysis of copy number and gene expression data suggests novel pathogenetic mechanisms in primary myelofibrosis.

Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, gene expression and copy number signals were integrated and several genomic abnormalities leading to a concordant alteration in gene expression levels were identified. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus was accompanied by a coordinated transcriptional up-regulation in PMF patients. PAOX inhibition resulted in rapid cell death of PMF progenitor cells, while sparing normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterizes PMF patients.

miRNA-mRNA integrative analysis in primary myelofibrosis CD34+ cells: role of miR-155/JARID2 axis in abnormal megakaryopoiesis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34(+) cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34(+) cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41(+) MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF.

Purinergic signaling inhibits human acute myeloblastic leukemia cell proliferation, migration, and engraftment in immunodeficient mice.

Extracellular ATP and UTP nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). In the present study, we show that ATP and UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs. ATP-stimulated leukemic cells, but not normal CD34+ cells, undergo down-regulation of genes involved in cell proliferation and migration, whereas cell-cycle inhibitors are up-regulated. Functionally, ATP induced the inhibition of proliferation and accumulation of AML cells, but not of normal cells, in the G0 phase of the cell cycle. Exposure to ATP or UTP inhibited AML-cell migration in vitro. In vivo, xenotransplantation experiments demonstrated that the homing and engraftment capacity of AML blasts and CD34+CD38- cells to immunodeficient mice BM was significantly inhibited by pretreatment with nucleotides. P2R-expression analysis and pharmacologic profiling suggested that the inhibition of proliferation by ATP was mediated by the down-regulation of the P2X7R, which is up-regulated on untreated blasts, whereas the inhibition of chemotaxis was mainly mediated via P2Y2R and P2Y4R subtypes. We conclude that, unlike normal cells, P2R signaling inhibits leukemic cells and therefore its pharmacologic modulation may represent a novel therapeutic strategy.

FOXP1 and TP63 involvement in the progression of myelodysplastic syndrome with 5q- and additional cytogenetic abnormalities.

BACKGROUND: The progression of low-risk del(5q) myelodysplastic syndrome to acute myeloid leukemia is increased when associated with mutations of TP53, or with additional chromosomal abnormalities. However, to date the prognostic impact and molecular consequences of these rearrangements were poorly investigated. Single additional alterations to del(5q) by balanced chromosome rearrangements were rarely found in myelodysplasia. In particular, balanced alterations involving TP63 and FOXP1 genes were never reported in the literature. CASE PRESENTATION: Here we report on a 79-year woman with an aggressive form of myelodysplastic syndrome with del(5q), no TP53 mutation, and a novel complex rearrangement of chromosome 3 in bone marrow cells. Our results revealed that the FOXP1 and TP63 genes were both relocated along chromosome 3. Strikingly, immunohistochemistry analysis showed altered protein levels, disclosing that this rearrangement triggered the expression of FOXP1 and TP63 genes. FOXP1 was also found activated in other patients with myelodysplasia and acute myeloid leukemia, showing that it is an important, recurrent event. CONCLUSIONS: We document an apparent role of FOXP1 and TP63, up to now poorly documented, in the progression of MDS in our patient who is lacking mutations in the TP53 tumor suppressor gene normally associated with poor outcome in myelodysplastic syndrome with 5q-. Finally, our results may suggest a possible broader role of FOXP1 in the pathogenesis and progression of myelodysplasia and acute myeloid leukemia.

c-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression.

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets, which can account for c-myb knockdown effects. Indeed, chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.

An automated 3D-printed perfusion bioreactor combinable with pulsed electromagnetic field stimulators for bone tissue investigations.

In bone tissue engineering research, bioreactors designed for replicating the main features of the complex native environment represent powerful investigation tools. Moreover, when equipped with automation, their use allows reducing user intervention and dependence, increasing reproducibility and the overall quality of the culture process. In this study, an automated uni-/bi-directional perfusion bioreactor combinable with pulsed electromagnetic field (PEMF) stimulation for culturing 3D bone tissue models is proposed. A user-friendly control unit automates the perfusion, minimizing the user dependency. Computational fluid dynamics simulations supported the culture chamber design and allowed the estimation of the shear stress values within the construct. Electromagnetic field simulations demonstrated that, in case of combination with a PEMF stimulator, the construct can be exposed to uniform magnetic fields. Preliminary biological tests on 3D bone tissue models showed that perfusion promotes the release of the early differentiation marker alkaline phosphatase. The histological analysis confirmed that perfusion favors cells to deposit more extracellular matrix (ECM) with respect to the static culture and revealed that bi-directional perfusion better promotes ECM deposition across the construct with respect to uni-directional perfusion. Lastly, the Real-time PCR results of 3D bone tissue models cultured under bi-directional perfusion without and with PEMF stimulation revealed that the only perfusion induced a ~ 40-fold up-regulation of the expression of the osteogenic gene collagen type I with respect to the static control, while a ~ 80-fold up-regulation was measured when perfusion was combined with PEMF stimulation, indicating a positive synergic pro-osteogenic effect of combined physical stimulations.

A universal SARS-CoV DNA vaccine inducing highly cross-reactive neutralizing antibodies and T cells.

New variants in the SARS-CoV-2 pandemic are more contagious (Alpha/Delta), evade neutralizing antibodies (Beta), or both (Omicron). This poses a challenge in vaccine development according to WHO. We designed a more universal SARS-CoV-2 DNA vaccine containing receptor-binding domain loops from the huCoV-19/WH01, the Alpha, and the Beta variants, combined with the membrane and nucleoproteins. The vaccine induced spike antibodies crossreactive between huCoV-19/WH01, Beta, and Delta spike proteins that neutralized huCoV-19/WH01, Beta, Delta, and Omicron virus in vitro. The vaccine primed nucleoprotein-specific T cells, unlike spike-specific T cells, recognized Bat-CoV sequences. The vaccine protected mice carrying the human ACE2 receptor against lethal infection with the SARS-CoV-2 Beta variant. Interestingly, priming of cross-reactive nucleoprotein-specific T cells alone was 60% protective, verifying observations from humans that T cells protect against lethal disease. This SARS-CoV vaccine induces a uniquely broad and functional immunity that adds to currently used vaccines.

Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34- cell population with intrinsic resistance to imatinib.

We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin(-)CD34(-)) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34(-) cells are leukemic. CML Lin(-)CD34(-) cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin(-)CD34(-) cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34(+) cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin(-)CD34(-) cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin(-)CD34(-) cells in vitro. Moreover, leukemic CD34(-) cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34(-) leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

Reduction of muscle contraction and pain in electroporation-based treatments: An overview.

BACKGROUND: In the first studies of electrochemotherapy (ECT), small cutaneous metastases were treated and only mild or moderate pain was observed; therefore, pain was not considered a significant issue. As the procedure began to be applied to larger cutaneous metastases, pain was reported more frequently. For that reason, reduction of both muscle contractions and pain have been investigated over the years. AIM: To present an overview of different protocols described in literature that aim to reduce muscle contractions and pain caused by the electroporation (EP) effect in both ECT and irreversible EP treatments. METHODS: Thirty-three studies published between January 1999 and November 2020 were included. Different protocol designs and electrode geometries that reduce patient pain and the number of muscle contractions and their intensity were analysed. RESULTS: The analysis showed that both high frequency and bipolar/biphasic pulses can be used to reduce pain and muscle contractions in patients who undergo EP treatments. Moreover, adequate electrode design can decrease EP-related morbidity. Particularly, needle length, diameter and configuration of the distance between the needles can be optimised so that the muscle volume crossed by the current is reduced as much as possible. Bipolar/biphasic pulses with an inadequate pulse length seem to have a less evident effect on the membrane permeability compared with the standard pulse protocol. For that reason, the number of pulses and the voltage amplitude, as well as the pulse duration and frequency, must be chosen so that the dose of delivered energy guarantees EP efficacy. CONCLUSION: Pain reduction in EP-based treatments can be achieved by appropriately defining the protocol parameters and electrode design. Most results can be achieved with high frequency and/or bipolar/biphasic pulses. However, the efficacy of these alternative protocols remains a crucial point to be assessed further.

A Systematic Review about Imaging and Histopathological Findings for Detecting and Evaluating Electroporation Based Treatments Response.

BACKGROUND: Imaging methods and the most appropriate criteria to be used for detecting and evaluating response to oncological treatments depend on the pathology and anatomical site to be treated and on the treatment to be performed. This document provides a general overview of the main imaging and histopathological findings of electroporation-based treatments (Electrochemotherapy-ECT and Irreversible electroporation-IRE) compared to thermal approach, such as radiofrequency ablation (RFA), in deep-seated cancers with a particular attention to pancreatic and liver cancer. METHODS: Numerous electronic datasets were examined: PubMed, Scopus, Web of Science and Google Scholar. The research covered the years from January 1990 to April 2021. All titles and abstracts were analyzed. The inclusion criteria were the following: studies that report imaging or histopathological findings after ablative thermal and not thermal loco-regional treatments (ECT, IRE, RFA) in deep-seated cancers including pancreatic and liver cancer and articles published in the English language. Exclusion criteria were unavailability of full text and congress abstracts or posters and different topic respect to inclusion criteria. RESULTS: 558 potentially relevant references through electronic searches were identified. A total of 38 articles met the inclusion criteria: 20 studies report imaging findings after RFA or ECT or IRE in pancreatic and liver cancer; 17 studies report histopathological findings after RFA or ECT or IRE; 1 study reports both imaging and histopathological findings after RFA or ECT or IRE. CONCLUSIONS: Imaging features are related to the type of therapy administrated, to the timing of re-assessment post therapy and to the imaging technique being used to observe the effects. Histological findings after both ECT and IRE show that the treated area becomes necrotic and encapsulated in fibrous tissue, suggesting that the size of the treated lesion cannot be measured as an endpoint to detect response. Moreover, histology frequently reported signs of apoptosis and reduced vital tissue, implying that imaging criteria, which take into account the viability and not the size of the lesion, are more appropriate to evaluate response to treatment.

Does Capacitively Coupled Electric Fields Stimulation Improve Clinical Outcomes After Instrumented Spinal Fusion? A Multicentered Randomized, Prospective, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Lumbar spinal fusion (LSF) is used to treat lumbar degenerative disorders. Methods to improve the functional recovery of patients undergoing LSF is one of the main goals in daily clinical practice. The objective of this study is to assess whether biophysical stimulation with capacitively coupled electric fields (CCEF) can be used as adjuvant therapy to enhance clinical outcome in LSF-treated patients. METHODS: Forty-two patients undergoing LSF were assessed and randomly allocated to either the active or to the placebo group. Follow-up visits were performed at 1, 3, 6, and 12 months after surgery; long-term follow-up was performed at year 10. Visual analogue scale (VAS), the Oswestry Disability Index (ODI), and the 36-item Short Form Health Survey (SF-36) questionnaire were recorded. RESULTS: This study demonstrates a significant improvement in CCEF-treated patients at 6 and 12 months' follow-up for SF-36, and at 12 months' follow-up for ODI values. Based on SF-36 and ODI scores, we reported a significantly higher percentage of successful treatments at 12 months in the active compared with the placebo group. Moreover, in a subset of patients at 10 years' follow-up, a significant difference was reported in VAS and ODI scores between groups. CONCLUSIONS: The results demonstrate that 3 months of CCEF treatment immediately after surgery is effective in reducing ODI and improving SF-36 score, and that these benefits can be maintained up to 12 months. In a subset of patients, these positive outcomes are retained up to 10 years. LEVEL OF EVIDENCE: I. CLINICAL RELEVANCE: This study suggests that CCEF stimulation can be used as an adjunct to LSF for spine diseases, for increasing overall quality of life and improving patients' functional recovery. CCEF is safe and well tolerated, compatible with activities of daily living.

Role of CD34 antigen in myeloid differentiation of human hematopoietic progenitor cells.

CD34 is a transmembrane protein that is strongly expressed on hematopoietic stem/progenitor cells (HSCs); despite its importance as a marker of HSCs, its function is still poorly understood, although a role in cell adhesion has been demonstrated. To characterize the function of CD34 antigen on human HSCs, we examined, by both inhibition and overexpression, the role of CD34 in the regulation of HSC lineage differentiation. Our results demonstrate that CD34 silencing enhances HSC granulocyte and megakaryocyte differentiation and reduces erythroid maturation. In agreement with these results, the gene expression profile of these cells reveals the upregulation of genes involved in granulocyte and megakaryocyte differentiation and the downregulation of erythroid genes. Consistently, retroviral-mediated CD34 overexpression leads to a remarkable increase in erythroid progenitors and a dramatic decrease in granulocyte progenitors, as evaluated by clonogenic assay. Together, these data indicate that the CD34 molecule promotes the differentiation of CD34+ hematopoietic progenitors toward the erythroid lineage, which is achieved, at least in part, at the expense of granulocyte and megakaryocyte lineages.

Signal control of hematopoietic stem cell fate: Wnt, Notch, and Hedgehog as the usual suspects.

PURPOSE OF REVIEW: Hematopoietic homeostasis depends on appropriate self-renewal and differentiation capacity of hematopoietic stem cells. The characterization of the key extracellular signals that integrate with intracellular molecular machinery to regulate hematopoietic stem cells fate choice is crucial to move toward hematopoietic stem cell clinical application. RECENT FINDINGS: Several factors have been described as positive and negative regulators of hematopoietic stem cell self-renewal and differentiation. Most of the hematopoietic cytokines studied promote either survival or differentiation or both in hematopoietic stem cells ex vivo, whereas morphogens (Wnt, Notch, and Hedgehog) may signify a class of hematopoietic stem cell regulators that support expansion of the hematopoietic stem cell pool by a combination of survival and induced self-renewal. SUMMARY: Although Wnt, Notch, and Hedgehog signaling pathways have been implicated in self-renewal and proliferation in vivo, modulation of these pathways alone does not result in substantive expansion of hematopoietic stem cells ex vivo. In addition to these signaling pathways, Bcl-2 family members may have an important role in inducing survival in hematopoietic stem cells both in vivo and ex vivo. Understanding the complex relationship between these unique signaling pathways is essential to achieve successful ex-vivo expansion toward enhanced hematopoietic stem cell transplantation-based therapies.

Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in K562 cells expressing CALR mutants.

Somatic mutations of calreticulin (CALR) have been described in approximately 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis.