RESUMO
Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, to acquire knowledge in imaging combined with therapeutic applications. Then, we conjugated SiNPs to one of the most used anticancer drugs, doxorubicin (DOX). As an anticancer agent, DOX has high efficacy but also an elevated systemic toxicity, causing multiple side effects. Nanostructures are usually employed to increase the drug circulation time and accumulation in target tissues, reducing undesired cytotoxicity. We tested these functionalized SiNPs (DOX-NPs) on breast cancer cell line MCF-7. We evaluated DOX-NP cytotoxicity, the effect on the cell cycle and on the expression of CD44 antigen, a molecule involved in adhesion and in tumor invasion, comparing DOX-NP to free DOX and stand-alone SiNPs. We found a specific ability to release a minor amount of CD44+ extracellular vesicles (EVs), from both CD81 negative and CD81 positive pools. Modulating the levels of CD44 at the cell surface in cancer cells is thus of great importance for disrupting the signaling pathways that favor tumor progression.
Assuntos
Antineoplásicos , Neoplasias da Mama , Nanopartículas , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Mitocôndrias , Células Mieloides , Nanopartículas/química , Dióxido de Silício/químicaRESUMO
Group 1 innate lymphoid cells include natural killer (NK) cells and ILC1s, which mediate the response to intracellular pathogens. Thymic NK (tNK) cells were described with hybrid features of immature NK cells and ILC1 but whether these cells are related to NK cells or ILC1 has not been fully investigated. We report that murine tNK cells expressed the NK-cell associated transcription factor EOMES and developed independent of the essential ILC1 factor TBET, confirming their placement within the NK lineage. Moreover, tNK cells resemble NK cells rather than ILC1 in their requirements for the E protein transcription factor inhibitor ID2. We provide further insight into the mechanisms governing tNK-cell development by showing that the transcription factor ETS1 prevented tNK cell acquisition of the conventional NK-cell maturation markers CD11b and KLRG1. Our data reveal few ILC1 in the thymus and clarify the identity and developmental requirements of tNK cells.
Assuntos
Células Matadoras Naturais/fisiologia , Linfócitos/fisiologia , Timo/imunologia , Fatores de Transcrição/metabolismo , Animais , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Diferenciação Celular , Linhagem da Célula , Imunidade Inata , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Linfócitos/imunologia , Camundongos , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Proteínas com Domínio T/genética , Timócitos/citologia , Timócitos/fisiologia , Timo/citologia , Fatores de Transcrição/genéticaRESUMO
Atherosclerosis is characterized by a proliferation of vascular smooth muscle cells (VSMCs) and their migration to the intima, which induces thickening of the intima itself, but the mechanism remains poorly understood. Low molecular weight heparin (LMWH) inhibits the proliferation of VSMCs. Previous studies have shown that a LMWH, parnaparin (PNP), acts on the processes of atherogenesis and atheroprogression in experimental animal models. The aim of this study was to investigate the involvement of oxidative stress, inflammation and VSMCs in the regulation of vascular wall homeostasis. We also considered the possibility of restoring vascular pathological changes using PNP treatment. In order to evaluate vascular remodelling in this study we have analysed the morphological changes in aortas of an animal model of atherosclerosis, apolipoprotein E-deficient mice (ApoE-/-) fed with a normal or a western diet without treatment or treated with PNP. We also analysed, by immunohistochemistry, the expression of proteins linked to atherogenesis and atheroprogression - an enzyme involved in oxidative stress, iNOS, examples of inflammatory mediators, such as tumour necrosis factor alpha (TNF-α), interleukins 1 and 6 (IL-1 and IL-6), and markers of VSMC changes, in particular plasminogen activator inhibitor-1 and thrombospondin-1 (PAI-1 and TSP-1). Our results could suggest that PNP downregulates VSMC proliferation and migration, mediated by PAI-1 and TSP-1, and reduces inflammation and oxidative stress in vessels. These data suggested that LMWH, in particular PNP, could be a theoretically practical tool in the prevention of atherosclerotic vascular modification.
Assuntos
Apolipoproteínas E/genética , Aterosclerose/genética , Heparina de Baixo Peso Molecular/metabolismo , Mediadores da Inflamação/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Remodelação Vascular/genética , Animais , Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Proliferação de Células , Modelos Animais de Doenças , Heparina de Baixo Peso Molecular/genética , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patologia , Inflamação , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo , Inibidor 1 de Ativador de Plasminogênio/genética , Trombospondina 1/metabolismo , Túnica Íntima/patologiaRESUMO
The present work argues for the involvement of the zinc chelating ability of some non-steroidal anti-inflammatory drugs as an additive mechanism able to increase their efficacy against COVID-19.
Assuntos
Anti-Inflamatórios não Esteroides , COVID-19 , Humanos , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , ZincoRESUMO
Highly mutable influenza is successfully countered based on individual susceptibility and similar precision-like medicine approach should be effective against SARS-COV-2. Among predictive markers to bring precision medicine to COVID-19, circulating ACE2 has potential features being upregulated in both severe COVID-19 and predisposing comorbidities. Spike SARS-CoVs were shown to induce ADAM17-mediated shedding of enzymatic active ACE2, thus accounting for its increased activity that has also been suggested to induce positive feedback loops leading to COVID-19-like manifestations. For this reason, pre-existing ACE2 activity and inhibition of ACE2/ADAM17 zinc-metalloproteases through zinc chelating agents have been proposed to predict COVID-19 outcome before infection and to protect from COVID-19, respectively. Since most diagnostic laboratories are not equipped for enzymatic activity determination, other potential predictive markers of disease progression exploitable by diagnostic laboratories were explored. Concentrations of circulating albumin, zinc, ACE2 protein and its activity were investigated in healthy, diabetic (COVID-19-susceptible) and SARS-CoV-2-negative COVID-19 individuals. ACE2 both protein levels and activity significantly increased in COVID-19 and diabetic patients. Abnormal high levels of ACE2 characterised a subgroup (16-19%) of diabetics, while COVID-19 patients were characterised by significantly higher zinc/albumin ratios, pointing to a relative increase of albumin-unbound zinc species, such as free zinc ones. Data on circulating ACE2 levels are in line with the hypothesis that they can drive susceptibility to COVID-19 and elevated zinc/albumin ratios support the therapeutic use of zinc chelating inhibitors of ACE2/ADAM17 zinc-metalloproteases in a targeted therapy for COVID-19.
Assuntos
COVID-19 , Humanos , COVID-19/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Sistema Renina-Angiotensina/fisiologia , Enzima de Conversão de Angiotensina 2/genética , Peptidil Dipeptidase A , Medicina de Precisão , Zinco/uso terapêutico , Albuminas/metabolismo , BiomarcadoresRESUMO
Cytotoxic functions and susceptibility to apoptosis are crucial aspects of NK cells suitable to counter cancer after infusion in oncologic patients. To test the feasibility and the usefulness of infusing in vitro generated NK cells, these two features were investigated in NK cells developed in vitro from CD34⺠hematopoietic progenitors. Purified CD34⺠cells were cultured for 15-30 days with FLT-3 ligand (FLT3-L) and IL-15 with or without IL-21. To induce terminal differentiation, NK cells were cultured for further 15 days with IL-15, IL-21, or their combination. A CD56(dim) /CD16⺠NK subset, expressing high level of perforin, granzymes, and LFA-1, appeared early in cultures with FLT3-L, IL-15, and IL-21, but it quickly died, indicating its predisposition to apoptosis. On the contrary, CD56(bright) NK cells generated after 30 days of culture with FLT3-L plus IL-15 did not show a considerable apoptosis, nevertheless only a subset of these cells expressed granzyme-B, perforin, LFA-1, and CD94-CD159a heterodimer, indicating a functional immaturity. Interestingly, further 15 days of culture with IL-21 plus IL-15 did not induce the generation of CD56(dim) cells from the CD56(bright) subset and actually inhibited IL-15-induced maturation/activation of this latter subset. In fact, IL-15 alone upregulated granzyme-B, TRAIL, Fas ligand, CD94-CD159a, LFA-1, CD16, KIRs, and TRAIL-R2 on CD56(bright) NK cells. Our results suggest that during differentiation CD56(bright) NK cells, similarly to mature activated NK cells, become highly cytotoxic and are relatively resistant to apoptosis induced by TNF family members.
Assuntos
Antígenos CD34/metabolismo , Apoptose , Antígeno CD56/metabolismo , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Células Matadoras Naturais/citologia , Adulto , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-15/metabolismo , Células Matadoras Naturais/metabolismo , Regulação para CimaRESUMO
The present work provides arguments for the involvement of anti-vector immunity and of SARS-CoV-2 variants on the efficacy of ChAdOx1 nCoV-19 vaccine. First, it is suggested that anti-vector immunity takes place as homologous vaccination with ChAdOx1 nCoV-19 vaccine is applied and interferes with vaccine efficacy when the interval between prime and booster doses is less than 3 months. Second, longitudinal studies suggest that ChAdOx1 nCoV-19 vaccine provides suboptimal efficacy against SARS-CoV-2 Alpha variant, which appears to have an increased transmissibility among vaccinated people. At the moment, ChAdOx1 nCoV-19 vaccine is able to reduce the severity of symptoms and transmissibility. However, if the vaccinated individuals do not maintain physical preventive measures, they could turn into potential spreaders, thus suggesting that mass vaccination will not quickly solve the pandemic. Possible consequences of SARS-CoV-2 evolution and of repeated anti-SARS-CoV-2 vaccinations are discussed and adoption of an influenza-like vaccination strategy is suggested.
Assuntos
COVID-19 , SARS-CoV-2 , Vacinas contra COVID-19 , ChAdOx1 nCoV-19 , Humanos , Eficácia de VacinasRESUMO
Among the first clinical symptoms of the SARS-CoV-2 infection is olfactory−gustatory deficit; this continues for weeks and, in some cases, can be persistent. We prospectively evaluated 162 patients affected by COVID-19 using a visual analogue scale (VAS) for nasal and olfactory−gustatory symptoms. Patients were checked after 7, 14, 21, 28, 90, and 180 days. A total of 118 patients (72.8%) reported an olfactory VAS < 7 at baseline (group B), and 44 (27.2%) reported anosmia (VAS ≥ 7) (group A) and underwent the Brief Smell Identification Test (B-SIT) and Burghart Taste Strips (BTS) to quantify the deficit objectively and repeated the tests to confirm the sense recovery. Group A patients showed B-SIT anosmia and hyposmia in 44.2% and 55.8% of cases, respectively. A total of 88.6% of group A patients reported ageusia with VAS ≥ 7, and BTS confirmed 81.8% of ageusia and 18.2% of hypogeusia. VAS smell recovery was recorded starting from 14 days, with normalization at 28 days. The 28-day B-SIT score showed normosmia in 90.6% of group A patients. The mean time for full recovery (VAS = 0) was shorter in group B (22.9 days) than in group A (31.9 days). Chemosensory deficit is frequently the first symptom in patients with COVID-19, and, in most cases, recovery occurs after four weeks.
Assuntos
Ageusia , COVID-19 , Transtornos do Olfato , Anosmia/etiologia , COVID-19/complicações , Humanos , Transtornos do Olfato/diagnóstico , SARS-CoV-2 , Olfato , PaladarRESUMO
The article describes the rationale for the administration of zinc-chelating agents in COVID-19 patients. In a previous work I have highlighted that the binding of the SARS-CoV spike proteins to the zinc-metalloprotease ACE2 has been shown to induce ACE2 shedding by activating the zinc-metalloprotease ADAM17, which ultimately leads to systemic upregulation of ACE2 activity. Moreover, based on experimental models, it was also shown the detrimental effect of the excessive systemic activity of ACE2 through its downstream pathways, which leads to "clinical" manifestations resembling COVID-19. In this regard, strong upregulation of circulating ACE2 activity was recently reported in COVID-19 patients, thus supporting the previous hypothesis that COVID-19 may derive from upregulation of ACE2 activity. Based on this, a reasonable hypothesis of using inhibitors that curb the upregulation of both ACE2 and ADAM17 zinc-metalloprotease activities and consequent positive feedback-loops (initially triggered by SARS-CoV-2 and subsequently sustained independently on viral trigger) is proposed as therapy for COVID-19. In particular, zinc-chelating agents such as citrate and ethylenediaminetetraacetic acid (EDTA) alone or in combination are expected to act in protecting from COVID-19 at different levels thanks to their both anticoagulant properties and inhibitory activity on zinc-metalloproteases. Several arguments are presented in support of this hypothesis and based on the current knowledge of both beneficial/harmful effects and cost/effectiveness, the use of chelating agents in the prevention and therapy of COVID-19 is proposed. In this regard, clinical trials (currently absent) employing citrate/EDTA in COVID-19 are urgently needed in order to shed more light on the efficacy of zinc chelators against SARS-CoV-2 infection in vivo.
Assuntos
Tratamento Farmacológico da COVID-19 , Quelantes/farmacologia , Ácido Cítrico/farmacologia , Ácido Edético/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Zinco/metabolismo , Proteína ADAM17/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Anticoagulantes/farmacologia , COVID-19/metabolismo , COVID-19/terapia , Descoberta de Drogas , Humanos , Imunização Passiva/efeitos adversos , SARS-CoV-2/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Soroterapia para COVID-19RESUMO
The article describes the possible pathophysiological origin of COVID-19 and the crucial role of renin-angiotensin system (RAS), providing several "converging" evidence in support of this hypothesis. SARS-CoV-2 has been shown to initially upregulate ACE2 systemic activity (early phase), which can subsequently induce compensatory responses leading to upregulation of both arms of the RAS (late phase) and consequently to critical, advanced and untreatable stages of COVID-19 disease. The main and initial actors of the process are ACE2 and ADAM17 zinc-metalloproteases, which, initially triggered by SARS-CoV-2 spike proteins, work together in increasing circulating Ang 1-7 and Ang 1-9 peptides and downstream (Mas and Angiotensin type 2 receptors) pathways with anti-inflammatory, hypotensive and antithrombotic activities. During the late phase of severe COVID-19, compensatory secretion of renin and ACE enzymes are subsequently upregulated, leading to inflammation, hypertension and thrombosis, which further sustain ACE2 and ADAM17 upregulation. Based on this hypothesis, COVID-19-phase-specific inhibition of different RAS enzymes is proposed as a pharmacological strategy against COVID-19 and vaccine-induced adverse effects. The aim is to prevent the establishment of positive feedback-loops, which can sustain hyperactivity of both arms of the RAS independently of viral trigger and, in some cases, may lead to Long-COVID syndrome.
Assuntos
Proteína ADAM17/biossíntese , Enzima de Conversão de Angiotensina 2/biossíntese , COVID-19/metabolismo , Sistema Renina-Angiotensina , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteína ADAM17/antagonistas & inibidores , Angiotensina I/metabolismo , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Regulação Enzimológica da Expressão Gênica , Humanos , Fragmentos de Peptídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Regulação para Cima , Tratamento Farmacológico da COVID-19RESUMO
Since the end of 2019, the medical-scientific community has been facing a terrible pandemic caused by a new airborne viral agent known as SARS-CoV2. Already in the early stages of the pandemic, following the discovery that the virus uses the ACE2 cell receptor as a molecular target to infect the cells of our body, it was hypothesized that the renin-angiotensin-aldosterone system was involved in the pathogenesis of the disease. Since then, numerous studies have been published on the subject, but the exact role of the renin-angiotensin-aldosterone system in the pathogenesis of COVID-19 is still a matter of debate. RAAS represents an important protagonist in the pathogenesis of COVID-19, providing the virus with the receptor of entry into host cells and determining its organotropism. Furthermore, following infection, the virus is able to cause an increase in plasma ACE2 activity, compromising the normal function of the RAAS. This dysfunction could contribute to the establishment of the thrombo-inflammatory state characteristic of severe forms of COVID-19. Drugs targeting RAAS represent promising therapeutic options for COVID-19 sufferers.
Assuntos
COVID-19/metabolismo , Sistema Renina-Angiotensina , SARS-CoV-2/fisiologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/patologia , Descoberta de Drogas , Humanos , Terapia de Alvo Molecular , Sistema Renina-Angiotensina/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
This article challenges the notion of the randomness of mutations in eukaryotic cells by unveiling stress-induced human non-random genome editing mechanisms. To account for the existence of such mechanisms, I have developed molecular concepts of the cell environment and cell environmental stressors and, making use of a large quantity of published data, hypothesised the origin of some crucial biological leaps along the evolutionary path of life on Earth under the pressure of natural selection, in particular, (1) virus-cell mating as a primordial form of sexual recombination and symbiosis; (2) Lamarckian CRISPR-Cas systems; (3) eukaryotic gene development; (4) antiviral activity of retrotransposon-guided mutagenic enzymes; and finally, (5) the exaptation of antiviral mutagenic mechanisms to stress-induced genome editing mechanisms directed at "hyper-transcribed" endogenous genes. Genes transcribed at their maximum rate (hyper-transcribed), yet still unable to meet new chronic environmental demands generated by "pollution", are inadequate and generate more and more intronic retrotransposon transcripts. In this scenario, RNA-guided mutagenic enzymes (e.g., Apolipoprotein B mRNA editing catalytic polypeptide-like enzymes, APOBECs), which have been shown to bind to retrotransposon RNA-repetitive sequences, would be surgically targeted by intronic retrotransposons on opened chromatin regions of the same "hyper-transcribed" genes. RNA-guided mutagenic enzymes may therefore "Lamarkianly" generate single nucleotide polymorphisms (SNP) and gene copy number variations (CNV), as well as transposon transposition and chromosomal translocations in the restricted areas of hyper-functional and inadequate genes, leaving intact the rest of the genome. CNV and SNP of hyper-transcribed genes may allow cells to surgically explore a new fitness scenario, which increases their adaptability to stressful environmental conditions. Like the mechanisms of immunoglobulin somatic hypermutation, non-random genome editing mechanisms may generate several cell mutants, and those codifying for the most environmentally adequate proteins would have a survival advantage and would therefore be Darwinianly selected. Non-random genome editing mechanisms represent tools of evolvability leading to organismal adaptation including transgenerational non-Mendelian gene transmission or to death of environmentally inadequate genomes. They are a link between environmental changes and biological novelty and plasticity, finally providing a molecular basis to reconcile gene-centred and "ecological" views of evolution.
Assuntos
Meio Ambiente , Edição de Genes , Genoma Humano , Desaminases APOBEC/metabolismo , Sistemas CRISPR-Cas/genética , Humanos , Mutação/genéticaRESUMO
The article describes the rationale for inhibition of the renin-angiotensin system (RAS) pathways as specific targets in patients infected by SARS-CoV-2 in order to prevent positive feedback-loop mechanisms. Based purely on experimental studies in which RAS pathway inhibitors were administered in vivo to humans/rodents, a reasonable hypothesis of using inhibitors that block both ACE and ACE2 zinc metalloproteases and their downstream pathways in COVID-19 patients will be proposed. In particular, metal (zinc) chelators and renin inhibitors may work alone or in combination to inhibit the positive feedback loops (initially triggered by SARS-CoV-2 and subsequently sustained by hypoxia independently on viral trigger) as both arms of renin-angiotensin system are upregulated, leading to critical, advanced and untreatable stages of the disease.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/fisiopatologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/fisiopatologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2 , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Angiotensinas/metabolismo , COVID-19 , Infecções por Coronavirus/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Pandemias , Peptidil Dipeptidase A/efeitos adversos , Peptidil Dipeptidase A/genética , Pneumonia Viral/metabolismo , Sistema Renina-Angiotensina/genética , Sistema Renina-Angiotensina/fisiologia , Fatores de Risco , SARS-CoV-2RESUMO
The NK cell population is characterized by distinct NK cell subsets that respond differently to the various activating stimuli. For this reason, the determination of the optimal cytotoxic activation of the different NK cell subsets can be a crucial aspect to be exploited to counter cancer cells in oncologic patients. To evaluate how the triggering of different combination of activating receptors can affect the cytotoxic responses of different NK cell subsets, we developed a microbead-based degranulation assay. By using this new assay, we were able to detect CD107a+ degranulating NK cells even within the less cytotoxic subsets (i.e., resting CD56bright and unlicensed CD56dim NK cells), thus demonstrating its high sensitivity. Interestingly, signals delivered by the co-engagement of NKp46 with 2B4, but not with CD2 or DNAM-1, strongly cooperate to enhance degranulation on both licensed and unlicensed CD56dim NK cells. Of note, 2B4 is known to bind CD48 hematopoietic antigen, therefore this observation may provide the rationale why CD56dim subset expansion correlates with successful hematopoietic stem cell transplantation mediated by alloreactive NK cells against host T, DC and leukemic cells, while sparing host non-hematopoietic tissues and graft versus host disease. The assay further confirms that activation of LFA-1 on NK cells leads to their granule polarization, even if, in some cases, this also takes to an inhibition of NK cell degranulation, suggesting that LFA-1 engagement by ICAMs on target cells may differently affect NK cell response. Finally, we observed that NK cells undergo a time-dependent spontaneous (cytokine-independent) activation after blood withdrawal, an aspect that may strongly bias the evaluation of the resting NK cell response. Altogether our data may pave the way to develop new NK cell activation and expansion strategies that target the highly cytotoxic CD56dim NK cells and can be feasible and useful for cancer and viral infection treatment.
Assuntos
Imunoterapia , Células Matadoras Naturais/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Transdução de Sinais , Antígeno CD56/metabolismo , Degranulação Celular , Polaridade Celular , Células Cultivadas , Humanos , Interleucina-2/metabolismo , Células Matadoras Naturais/fisiologiaRESUMO
CD56(bright) natural killer (NK) cells, generated in vitro from CD34+ hematopoietic progenitor cells, were characterized after a 30-day culture with flt3 ligand plus IL-15. Virtually, all CD56(bright) cells expressed CD117, CD25, natural cytotoxicity receptors (NCRs), NKG2D, CD161, and CD244, while only a subset expressed CD18-CD11a (LFA-1), and CD94 molecule, defining an immature CD56(bright)/NCRs+/NKG2D+/LFA-1(-)/CD94(-) subset. Another small subset of cells expressing CD94 but not LFA-1 integrin was also identified, suggesting that during NK differentiation LFA-1 might be upregulated later than CD94. To verify this hypothesis in vivo, we evaluated the NK cell expression of LFA-1 in both peripheral and umbilical cord blood samples. Interestingly, in these blood fluids, we have identified a lineage negative CD34(-)/LFA-1(low)/NKp46(dim)/NKG2D(dim)/CD94(-) subset that resembled an immature stage of NK cells present in lymph nodes. Altogether, the results indicate that CD18-CD11a integrin, as well as CD11b in mice, may be a useful marker to identify immature stages of NK cell differentiation.
Assuntos
Antígenos CD/biossíntese , Células Matadoras Naturais/citologia , Antígeno-1 Associado à Função Linfocitária/biossíntese , Subfamília D de Receptores Semelhantes a Lectina de Células NK/biossíntese , Receptores Imunológicos/biossíntese , Animais , Antígeno CD11a/biossíntese , Antígeno CD11b/biossíntese , Antígenos CD18/biossíntese , Adesão Celular , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-15/metabolismo , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Proteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Proteínas Proto-Oncogênicas c-kit/biossíntese , Família de Moléculas de Sinalização da Ativação Linfocitária , Fatores de TempoRESUMO
Lymphoid organs are supplied by many nerve endings associated with different kinds of cells and macrophages. The role of this innervation on the release of locally active molecules is still unclear. Lingual tonsils belong to Waldeyer's Ring, in close association with palatine tonsils and nasopharyngeal (adenoids) tonsils, thus constituting part of NALT (nasal-associated lymphoid tissue) together with the tubal tonsils and lateral pharyngeal bands. In this study, we focused our attention on the expression of some neurotrophins (NTs) and their high- and low-affinity receptors in human lingual tonsils. Light immunohistochemistry showed that human tonsillar samples were generally positive for all the NTs investigated (NGF, BDNF, NT-3, NT-4) and their receptors (TrKA, TrKB, TrKC and p75) with some different expression levels. NGF and TrKC were strongly expressed in macrophages, but weakly in lymphocytes. However, BDNF and TrKB was highly expressed in lymphocytes and weaker in macrophages. The low-affinity receptor for NGF, p75, was mainly moderately expressed in the analysed samples. These results suggest the presence of a pattern of neurotrophin innervation in the human lingual tonsil which may play a role in sustaining inflammatory conditions and in modulating a close interaction between the nervous system and the different immune cellular subtypes.
Assuntos
Tonsila Faríngea/inervação , Tonsila Faríngea/metabolismo , Fatores de Crescimento Neural/biossíntese , Receptores de Fator de Crescimento Neural/biossíntese , Endotélio Vascular/metabolismo , Humanos , Imuno-Histoquímica , Linfócitos/metabolismo , Macrófagos/metabolismoRESUMO
Campylobacter jejuni is a Gram-negative spiral-shaped bacterium, commonly associated with gastroenteritis in humans. It explicates its virulence also by the cytolethal distending toxin (CDT), able to cause irreversible cell cycle arrest. Infection by C. jejuni may result in the development of the Guillainâ»Barré Syndrome, an acute peripheral neuropathy. Symptoms of this disease could be caused by CDT-induced cell death and a subsequent inflammatory response. We tested C. jejuni lysates from different strains on donor monocytes: in fact, monocytes are potent producers of both pro- and anti-inflammatory cytokines, playing a major role in innate immunity and in non-specific host responses. We found, by cytometric and confocal analyses, that mitochondria and lysosomes were differently targeted: The C. jejuni strain that induced the most relevant mitochondrial alterations was the ATCC 33291, confirming an intrinsic apoptotic pathway, whereas the C. jejuni ISS 1 wild-type strain mostly induced lysosomal alterations. Lysates from all strains induced endoplasmic reticulum (ER) stress in monocytes, suggesting that ER stress was not associated with CDT but to other C. jejuni virulence factors. The ER data were consistent with an increase in cytosolic Ca2+ content induced by the lysates. On the contrary, the changes in lysosomal acidic compartments and p53 expression (occurring together from time 0, T0, to 24 h) were mainly due to CDT. The loss of p53 may prevent or impede cell death and it was not observable with the mutant strain. CDT not only was responsible for specific death effects but also seemed to promote an apoptotic stimuli-resisting pathway.
Assuntos
Campylobacter jejuni , Estresse do Retículo Endoplasmático , Monócitos/fisiologia , Morte Celular , Sobrevivência Celular , Humanos , Lisossomos , Mitocôndrias , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Although NK cells are considered part of the innate immune system, a series of evidences has demonstrated that they possess characteristics typical of the adaptive immune system. These NK adaptive features, in particular their memory-like functions, are discussed from an ontogenetic and evolutionary point of view.
Assuntos
Imunidade Adaptativa , Imunidade Inata , Memória Imunológica , Células Matadoras Naturais/imunologia , Animais , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Ativação Linfocitária , Vacinas Virais/imunologia , Viroses/imunologiaRESUMO
BACKGROUND: The possible occurrence of an erroneous immunophenotyping due to interference between monoclonal antibodies (MoAbs) is often overlooked when the epitopes are assumed to be not close to each other. This is particularly important when exploring immune cell populations whose identification is still investigational. The commonly held view is that myeloid derived suppressor cells can be identified as either HLA-DR(neg/dim) cells or interleukin-4 receptor-α (CD124)(+) cells among peripheral blood monocytes. We made the serendipitous observation that the fluorescence signal provided by the PE-CD124 MoAb was attenuated when the PE-CF594-HLA-DR MoAb was added to the staining tube. METHODS: Peripheral blood mononuclear cells from healthy donors were stained with the PE-CD124 MoAb and, as control, PE -CD40, -CD4 and -CD14, and either the PE-CF594-HLA-DR MoAb or its unlabeled form. B cells, which also express CD124, were analyzed for comparison. RESULTS: The PE-CF594-HLA-DR MoAb but not its unlabeled form reduced PE-CD124 MoAb staining on monocytes and B cells. No other monocyte and B cell surface marker staining was affected by the PE-CF594-HLA-DR MoAb. The PE-CF594-HLA-DR MoAb interfered with the PE-CD124 MoAb likely because of steric hindrance by bulky fluorochromes, although a quenching due to fluorescence resonance energy transfer might also cooperate to the PE-CD124 MoAb staining attenuation. CONCLUSIONS: Present observations highlight the importance of interference between MoAbs as a source of error when analyzing multicolor flow cytometry data.