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The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadA&Dgr;351-405, devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadA&Dgr;351-405 cellular effects in monocytes. We show that NadA&Dgr;351-405 (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, preincubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadA&Dgr;351-405 cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadA&Dgr;351-405 /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadA&Dgr;351-405 and inhibited adhesin-induced cytokine/ chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadA&Dgr;351-405 determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadA&Dgr;351-405 alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by anti-TLR2 antibodies. We propose that hsp90-dependent recruitment into an hsp90/hsp70/TLR4 transducing signal complex is necessary for the immunestimulating activity of NadA&Dgr;351-405 anti-MenB vaccine candidate. [ABSTRACT FROM AUTHOR]

Several mammalian 5′-nucleotidases (5-NTs), attached to membranes or present in the cytosol or in mitochondria, remove the phosphate from ribo- and deoxyribonucleotides with different specificities for the sugar and base moieties. Some enzymes probably participate in signaling functions by producing adenosine from AMP. A more general function may be to prevent overproduction of deoxyribonucleotides. 5-NTs may affect the pharmacological activity of nucleoside analogs and also be involved in their mitochondrial toxicity. Here we describe for five cloned 5-NT specific assays that largely rely on new inhibitors for some of the enzymes. The assays can be used to quantitate each enzyme in crude cell extracts. To ascertain their validity we applied each assay to extracts from genetically modified cells that overproduce separately each of the five enzymes. The methodology should be useful in further studies of the physiological function of 5-NTs and their influence on the clinical use of nucleoside analogs. [Copyright &y& Elsevier]

Background: Positron emission tomography (PET) using translocator protein (TSPO) ligands has been used to detect neuroinflammatory processes in neurological disorders, including multiple sclerosis (MS). The aim of this study was to evaluate neuroinflammation in a mouse MS model (EAE) using TSPO-PET with 18F-VC701, in combination with magnetic resonance imaging (MRI).Methods: MOG35-55/CFA and pertussis toxin protocol was used to induce EAE in C57BL/6 mice. Disease progression was monitored daily, whereas MRI evaluation was performed at 1, 2, and 4 weeks post-induction. Microglia activation was assessed in vivo by 18F-VC701 PET at the time of maximum disease score and validated by radioligand ex vivo distribution and immunohistochemistry at 2 and 4 weeks post-immunization.Results: In vivo and ex vivo analyses show that 18F-VC701 significantly accumulates within the central nervous system (CNS), particularly in the cortex, striatum, hippocampus, cerebellum, and cervical spinal cord of EAE compared to control mice, at 2 weeks post-immunization. MRI confirmed the presence of focal brain lesions at 2 weeks post-immunization in both T1-weighted and T2 images. Of note, MRI abnormalities attenuated in later post-immunization phase. Neuropathological analysis confirmed the presence of microglial activation in EAE mice, consistent with the in vivo increase of 18F-VC701 uptake.Conclusion: Increase of 18F-VC701 uptake in EAE mice is strongly associated with the presence of microglia activation in the acute phase of the disease. The combined use of TSPO-PET and MRI provided complementary evidence on the ongoing disease process, thus representing an attractive new tool to investigate neuronal damage and neuroinflammation at preclinical levels. [ABSTRACT FROM AUTHOR]

We conducted a retrospective/prospective worldwide study on patients with neuroendocrine neoplasms (NENs) and a molecularly proven SARS-CoV-2 positivity. Preliminary results regarding 85 patients of the INTENSIVE study have been published in 2021. Now we are reporting the 2-year analysis.Here, we are reporting data from consecutive patients enrolled between 1 June 2020, and 31 May 2022. Among the 118 contacted centers, 25 were active to enroll and 19 actively recruiting at the time of data cut-off for a total of 280 patients enrolled. SARS-CoV-2 positivity occurred in 47.5% of patients in 2020, 35.1% in 2021, and 17.4% in 2022. The median age for COVID-19 diagnosis was 60 years. Well-differentiated tumors, non-functioning, metastatic stage, and gastroenteropancreatic (GEP) primary sites represented most of the NENs. COVID-19-related pneumonia occurred in 22.8% of the total, with 61.3% of them requiring hospitalization; 11 patients (3.9%) needed sub-intensive or intensive care unit therapies and 14 patients died (5%), in 11 cases (3.9%) directly related to COVID-19. Diabetes mellitus and age at COVID-19 diagnosis > 70 years were significantly associated with COVID-19 mortality, whereas thoracic primary site with COVID-19 morbidity. A significant decrease in both hospitalization and pneumonia occurred in 2022 vs 2020. In our largest series of NEN patients with COVID-19, the NEN population is similar to the general population of patients with NEN regardless of COVID-19. However, older age, non-GEP primary sites and diabetes mellitus should be carefully considered for increased COVID-19 morbidity and mortality. Relevant information could be derived by integrating our results with NENs patients included in other cancer patients with COVID-19 registries.

Background: Positron emission tomography (PET) using translocator protein (TSPO) ligands has been used to detect neuroinflammatory processes in neurological disorders, including multiple sclerosis (MS). The aim of this study was to evaluate neuroinflammation in a mouse MS model (EAE) using TSPO-PET with 18 F-VC701, in combination with magnetic resonance imaging (MRI)., Methods: MOG 35-55 /CFA and pertussis toxin protocol was used to induce EAE in C57BL/6 mice. Disease progression was monitored daily, whereas MRI evaluation was performed at 1, 2, and 4 weeks post-induction. Microglia activation was assessed in vivo by 18 F-VC701 PET at the time of maximum disease score and validated by radioligand ex vivo distribution and immunohistochemistry at 2 and 4 weeks post-immunization., Results: In vivo and ex vivo analyses show that 18 F-VC701 significantly accumulates within the central nervous system (CNS), particularly in the cortex, striatum, hippocampus, cerebellum, and cervical spinal cord of EAE compared to control mice, at 2 weeks post-immunization. MRI confirmed the presence of focal brain lesions at 2 weeks post-immunization in both T1-weighted and T2 images. Of note, MRI abnormalities attenuated in later post-immunization phase. Neuropathological analysis confirmed the presence of microglial activation in EAE mice, consistent with the in vivo increase of 18 F-VC701 uptake., Conclusion: Increase of 18 F-VC701 uptake in EAE mice is strongly associated with the presence of microglia activation in the acute phase of the disease. The combined use of TSPO-PET and MRI provided complementary evidence on the ongoing disease process, thus representing an attractive new tool to investigate neuronal damage and neuroinflammation at preclinical levels.

A soluble recombinant form of Neisseria meningitidis adhesin A (NadADelta351-405), proposed as a constituent of anti-meningococcal B vaccines, is here shown to specifically interact with and immune-modulate human monocyte-derived dendritic cells (mo-DCs). After priming with IFN-gamma and stimulation with NadADelta351-405, mo-DCs strongly up-regulated maturation markers CD83, CD86, CD80, and HLA-DR, secreted moderate quantities of TNF-alpha, IL-6, and IL-8, and produced a slight, although significant, amount of IL-12p70. Costimulation of mo-DCs with NadADelta351-405 and the imidoazoquinoline drug R-848, believed to mimic bacterial RNA, increased CD86 in an additive way, but strongly synergized the secretion of IL-12p70, IL-1, IL-6, TNF-alpha, and MIP-1alpha, especially after IFN-gamma priming. CD86/CD80 overexpression correlated with the occupation of high-(kd approximately 80 nM) and low-(kd approximately 4 muM) affinity binding sites for NadADelta351-405. Alternatively, secretion of IL-12p70 and TNF-alpha, IL-6, and IL-8 corresponded to the occupation of high- or low-affinity receptors, respectively. Mo-DCs matured by IFN-gamma and NadADelta351-405 supported the proliferation of naive CD4+ T lymphocytes, inducing the differentiation of both IFN-gamma and IL-4 producing phenotypes. Our data show that NadA not only is a good immunogen but is as well endowed with a proimmune, self-adjuvating, activity.