A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications.

Tumor growth and metastasis strongly rely on cell-cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

The Transcription Regulator Patz1 Is Essential for Neural Stem Cell Maintenance and Proliferation.

Proper regulation of neurogenesis, the process by which new neurons are generated from neural stem and progenitor cells (NS/PCs), is essential for embryonic brain development and adult brain function. The transcription regulator Patz1 is ubiquitously expressed in early mouse embryos and has a key role in embryonic stem cell maintenance. At later stages, the detection of Patz1 expression mainly in the developing brain suggests a specific involvement of Patz1 in neurogenesis. To address this point, we first got insights in Patz1 expression profile in different brain territories at both embryonic and postnatal stages, evidencing a general decreasing trend with respect to time. Then, we performed in vivo and ex vivo analysis of Patz1-knockout mice, focusing on the ventricular and subventricular zone, where we confirmed Patz1 enrichment through the analysis of public RNA-seq datasets. Both embryos and adults showed a significant reduction in the number of Patz1-null NS/PCs, as well as of their self-renewal capability, compared to controls. Consistently, molecular analysis revealed the downregulation of stemness markers in NS/PCs derived from Patz1-null mice. Overall, these data demonstrate the requirement of Patz1 for NS/PC maintenance and proliferation, suggesting new roles for this key transcription factor specifically in brain development and plasticity, with possible implications for neurodegenerative disorders and glial brain tumors.

Vaccination with (1-11)E2 in alum efficiently induces an antibody response to ß-amyloid without affecting brain ß-amyloid load and microglia activation in 3xTg mice.

Immunization against ß-amyloid (Aß) is pursued as a possible strategy for the prevention of Alzheimer's disease (AD). In clinical trials, Aß 1-42 proved poorly immunogenic and caused severe adverse effects; therefore, safer and more immunogenic candidate vaccines are needed. Multimeric protein (1-11)E2 is able to induce an antibody response to Aß, immunological memory, and IL-4 production, with no concomitant anti-Aß T cell response. Antisera recognize Aß oligomers, protofibrils, and fibrils. In this study, we evaluated the effect of prophylactic immunization with three doses of (1-11)E2 in alum in the 3xTg mouse model of AD. Immunization with (1-11)E2 efficiently induced anti-Aß antibodies, but afforded no protection against Aß accumulation and neuroinflammation. The identification of the features of the anti-Aß immune response that correlate with the ability to prevent Aß accumulation remains an open problem that deserves further investigation.

Vaccination against ß-Amyloid as a Strategy for the Prevention of Alzheimer's Disease.

Vaccination relies on the phenomenon of immunity, a long-term change in the immunological response to subsequent encounters with the same pathogen that occurs after the recovery from some infectious diseases. However, vaccination is a strategy that can, in principle, be applied also to non-infectious diseases, such as cancer or neurodegenerative diseases, if an adaptive immune response can prevent the onset of the disease or modify its course. Immunization against ß-amyloid has been explored as a vaccination strategy for Alzheimer's disease for over 20 years. No vaccine has been licensed so far, and immunotherapy has come under considerable criticism following the negative results of several phase III clinical trials. In this narrative review, we illustrate the working hypothesis behind immunization against ß-amyloid as a vaccination strategy for Alzheimer's disease, and the outcome of the active immunization strategies that have been tested in humans. On the basis of the lessons learned from preclinical and clinical research, we discuss roadblocks and current perspectives in this challenging enterprise in translational immunology.

Analysis of the Consolidation Phase of Immunological Memory within the IgG Response to a B Cell Epitope Displayed on a Filamentous Bacteriophage.

Immunological memory can be defined as the ability to mount a response of greater magnitude and with faster kinetics upon re-encounter of the same antigen. We have previously reported that a booster dose of a protein antigen given 15 days after the first dose interferes with the development of memory, i.e., with the ability to mount an epitope-specific IgG response of greater magnitude upon re-encounter of the same antigen. We named the time-window during which memory is vulnerable to disruption a "consolidation phase in immunological memory", by analogy with the memory consolidation processes that occur in the nervous system to stabilize memory traces. In this study, we set out to establish if a similar memory consolidation phase occurs in the IgG response to a B cell epitope displayed on a filamentous bacteriophage. To this end, we have analyzed the time-course of anti-ß-amyloid IgG titers in mice immunized with prototype Alzheimer's Disease vaccine fdAD(2-6), which consists of a fd phage that displays the B epitope AEFRH of ß -amyloid at the N-terminus of the Major Capsid Protein. A booster dose of phage fdAD(2-6) given 15 days after priming significantly reduced the ratio between the magnitude of the secondary and primary IgG response to ß-amyloid. This analysis confirms, in a phage vaccine, a consolidation phase in immunological memory, occurring two weeks after priming.

Identification of a Consolidation Phase in Immunological Memory.

Long lasting antibody responses and immunological memory are the desired outcomes of vaccination. In general, multiple vaccine doses result in enhanced immune responses, a notable exception being booster-induced hyporesponsiveness, which has been observed with polysaccharide and glycoconjugate vaccines. In this study, we analyzed the effect of early booster doses of multimeric protein vaccine (1-11)E2 on recall memory to B epitope 1-11 of ß-amyloid. Mice immunized with a single dose of (1-11)E2 stochastically display, when immunized with a recall dose 9 months later, either memory, i.e., an enhanced response to epitope 1-11, or hyporesponsiveness, i.e., a reduced response. Memory is the most common outcome, achieved by 80% of mice. We observed that a booster dose of vaccine (1-11)E2 at day 15 significantly reduced the ratio between the magnitude of the secondary and primary response, causing an increase of hyporesponsive mice. This booster-dependent disruption of recall memory only occurred in a limited time window: a booster dose at day 21 had no significant effect on the ratio between the secondary and primary response magnitude. Thus, this study identifies a consolidation phase in immunological memory, that is a time window during which the formation of memory is vulnerable, and a disrupting stimulus reduces the probability that memory is achieved.

Alum and squalene-oil-in-water emulsion enhance the titer and avidity of anti-Aß antibodies induced by multimeric protein antigen (1-11)E2, preserving the Igg1-skewed isotype distribution.

The development of active immunotherapy for Alzheimer's disease (AD) requires the identification of immunogens that can ensure a high titer antibody response toward Aß, while minimizing the risks of adverse reactions. Multimeric protein (1-11)E2 induces a robust and persistent antibody response to Aß in mice, when formulated in Freund's adjuvant. The goal of this translational study was to evaluate the immunogenicity of (1-11)E2 formulated in alum (Alhydrogel 2%), or in a squalene oil-in-water emulsion (AddaVax), or without adjuvant. A IgG1-skewed isotype distribution was observed for the anti-Aß antibodies generated in mice immunized with either the non-adjuvanted or the adjuvanted vaccine, indicating that (1-11)E2 induces a Th2-like response in all tested conditions. Both Alhydrogel 2% and AddaVax enhanced the titer and avidity of the anti-Aß response elicited by (1-11)E2. We conclude that (1-11)E2 is a promising candidate for anti-Aß immunization protocols that include alum or squalene-oil-in-water emulsion, or no adjuvant.

A multimeric immunogen for the induction of immune memory to beta-amyloid.

The development of active immunotherapy for Alzheimer's disease (AD) requires the identification of immunogens that can ensure a high titer antibody response toward beta-amyloid, whereas minimizing the risks of a cell-mediated adverse reaction. We describe here two novel anti-beta-amyloid vaccines that consist of 'virus like particles' formed by a domain of the bacterial protein E2 that is able to self-assemble into a 60-mer peptide. Peptides 1-11 and 2-6 of beta-amyloid were displayed as N terminal fusions on the surface of the E2 particles. E2-based vaccines induced a fast-rising, robust and persistent antibody response to beta-amyloid in all vaccinated mice. The immune memory induced by a single administration of vaccine (1-11) E2 can be rapidly mobilized by a single booster injection, leading to a very high serum concentration of anti-beta-amyloid antibodies (above 1 mg ml(-1)). E2 vaccination polarizes the immune response toward the production of the anti-inflammatory cytokine interleukin-4 and does not induce a T cell response to beta-amyloid. Thus, E2-based vaccines are promising candidates for the development of immunotherapy protocols for AD.