HIV-1 Env and HLA-C interaction is crucial in modulating viral infectivity

Introduction HLA-C plays a crucial role in the progression of HIV-1 infection. Host genetic HLA-C variants, appear to be associated with a different ability to control HIV-1 infection. A higher HLA-C expression is associated with a better activation of cytotoxic T lymphocytes (CTLs) and of Killer Immunoglobulin like receptors (KIR) on NK-cells, which lead to a better HIV-1 infection control. Vice-versa, a lower HLA-C expression leads to a rapid progression toward AIDS. In addition, different HLA-C alleles present different binding stabilities to \u3b22microglobulin (\u3b22m)/peptide. Noteworthy, some HLA-C highly expressed/protective alleles are also stably bound to \u3b22m/peptide, while some low expressed/non-protective variants present an unstable bond to \u3b22m/peptide. Finally virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Experiments In the present work, it was first characterized the association between HLA-C and HIV-1 Env. We investigated if HIV-1 infection involves HLA-C free chains or the heterotrimeric complex, and to this purpose the A3.01 cell line and its HIV-1-infected counterpart ACH-2, as well as PM1 cells, were used as in vitro infection model. HEK-293T \u3b22m negative cells, generated using CRISPR/Cas9 system, were used to produce HIV-1 pseudoviruses and to test their infectivity. Then, the proportion between HLA-C associated to \u3b22m and HLA-C presents as free chains on the cell surface was characterized on PBMC from healthy donors, bearing both Stable or Unstable HLA-C alleles. In addition, PBMC were tested for their ability to support HIV-1 infection in vitro. Results HIV-1 infection induces the appearance of HLA-C free chains on the surface of infected cells, which may be responsible for the increased HIV-1 infectivity. HIV-1 Env-pseudotyped viruses produced in the absence of \u3b22m, thus lacking HLA-C on their envelope, were less infectious than those produced in the presence of \u3b22m. By analysing PBMC from healthy donors, differences in HLA-C heterotrimers stability and HLA-C expression levels were found. Finally, it was reported that R5 HIV-1 virions produced by PBMC having Unstable HLA-C alleles were more infectious than those produced by PBMC having the Stable variants. Conclusions The outcome of HIV-1 infection might depend both on the HLA-C surface expression levels and on HLA-C/\u3b22m/peptide binding stability. According to this model, PBMC carrying low expressed/Unstable HLA-C alleles have a high proportion of HLA-C free chains on their surface that raises viral infectivity and, at the same time, a low proportion of HLA-C heterotrimeric complexes which leads to a poor control of HIV-1 infection, and thus to a rapid progression toward AIDS.\uc8 noto che alcuni polimorfismi del sistema HLA giocano un ruolo cruciale nell\u2019eziopatogenesi e nella prognosi di numerose malattie infettive, fra le quali l\u2019AIDS (Sindrome da Immunodeficienza Acquisita). Recenti studi hanno evidenziato una forte correlazione fra i livelli di espressione di HLA-C e il controllo della replicazione del virus dell\u2019immunodeficienza umana (HIV-1). Alti livelli di espressione sono stati correlati con un miglior controllo dell\u2019infezione, mentre bassi livelli sono stati associati con una progressione pi\uf9 rapida della malattia. Inoltre, \ue8 noto che la molecola HLA-C, presente sull\u2019envelope di HIV-1, in associazione con la glicoproteina Env, \ue8 in grado di aumentarne l\u2019infettivit\ue0. Il ruolo protettivo di alti livelli di espressione di HLA-C sembra essere in contraddizione con il ruolo dell\u2019 HLA-C stesso nell\u2019aumentare l\u2019infettivit\ue0 virale quando incorporato nel virione. Ci\uf2 potrebbe essere dovuto alla presenza di diverse conformazioni dell\u2019 HLA-C. \uc8 infatti noto che diverse varianti alleliche dell\u2019HLA-C presentano una diversa stabilit\ue0 di legame con la \u3b22 microglobulina (\u3b22m) e il peptide. In particolare, l\u2019HLA-C pu\uf2 presentarsi associato alla \u3b22m e al peptide, costituendo un complesso che svolge un ruolo chiave nell\u2019attivazione del sistema immunitario, oppure come free chain, dissociato dal complesso. I primi risultati di questo lavoro hanno dimostrato che la proteina Env di HIV-1 \ue8 in grado di associarsi all\u2019HLA-C quando presente nella conformazione di free chain. L\u2019ipotesi testata nello studio prevede l\u2019esistenza di un\u2019associazione fra la suscettibilit\ue0 all\u2019infezione da HIV-1 e le diverse varianti alleliche di HLA-C che possono essere preferibilmente presenti o come complesso trimerico o come free chain. Individui con varianti di HLA-C aventi una forte stabilit\ue0 come trimero completo mostrerebbero una maggiore immunit\ue0 contro HIV-1 e una ridotta infettivit\ue0 virale, mentre soggetti con varianti dell\u2019HLA-C che facilmente si dissociano dalla \u3b22m e dal peptide mostrerebbero una ridotta risposta immunitaria nei confronti di HIV-1 e la produzione di virioni maggiormente infettivi. Nel suo complesso, questo studio fornisce nuove informazioni che potrebbero rivelarsi utili per la progettazione di nuove strategie vaccinali e approcci terapeutici contro HIV-1

Mesenchymal Stromal Cells: From Therapeutic Option to Therapeutic Target

As our understanding of mesenchymal stromal cells (MSC) has evolved, they have come to be recognized as an integral part of the tumor tissue, and the exploitability of their intrinsic features in the field of oncology has reached a standstill. Currently, there are 1621 registered clinical trials (clinicaltrials.gov) on “mesenchymal cells”, and yet none of them are exploring or explored unmodified MSC as a therapeutic option for cancer. Indeed, the therapeutic potential of these cells in oncology relies on their ability to migrate towards sites of injury and inflammation and function as delivery systems for the local release of therapeutics [1], recently reported as also occurring via exosomes [2]. Since the first characterization of these cells in healthy bone marrow (BM) by Friedenstein, MSC have been described in various tissues of healthy and diseased bodies, performing a plethora of functions mostly aimed towards the governance of tissue homeostasis. When considering pathological conditions and cancer, in particular, evidence suggests that the ability of local or recruited MSC to maintain a steady state is compromised, and the cells become integrated into the newly formed organ at the expense of its healthy counterpart. Cancer-associated MSC (CA-MSC) have been shown to promote multi-organ metastasis and to govern tumor immune surveillance [3]. Furthermore, BM-derived cancer-associated fibroblasts (CAFs) promote angiogenesis in breast cancer [4] and exhibit a unique inflammatory profile depending on the location. Indeed, several studies have highlighted that the microenvironment can reprogram stromal cells and inflammatory cytokines released in the tumor microenvironment (TME), ultimately boosting the immune-suppressive properties of MSC and favoring tumor growth [5,6]. This Special Issue, entitled “Role of Mesenchymal Stromal Cells (MSC) in Cancer Progression and Cancer Therapy”, aims to further explore the fate of MSC within malignancies, encompassing the crosstalk between the stroma and the tumor as well as that between the different stromal components. A crucial phase of cancer progression is the ability of the tumor to communicate with its surroundings. To this end, Aasebø and colleagues dissected the bidirectional communication between MSC and acute myeloid leukemia (AML) cells [7]. The authors performed a proteomic analysis of AML cells isolated from 40 patients and evaluated how healthy MSC influence the proteomic pattern of cancer cells. The results showed that, overall, patient heterogeneity was maintained upon challenge with MSC; nevertheless, the authors observed a reduction in patient heterogeneity for a minority of proteins, including extracellular matrix molecules, proteases (i.e., protein modifiers) and soluble adhesion molecules, thus highlighting possible targetable pathways. In line with this study, Fallati and colleagues reviewed the ability of MSC to orchestrate a leukemia-supportive microenvironment [8]. The authors detailed the capacity of the cells to contribute to the different stages of cancer and provided an overview of the potential mechanisms of action. They described the possible contribution of MSC to leukemogenesis and the cells’ ability to generate a leukemia-permissive environment by acting directly on tumor cells and indirectly via the microenvironment. Lastly, the authors reviewed the mechanisms of chemoprotection that involve MSC and are based upon a bi-directional exchange of soluble factors (metabolites, amino acids, etc.), extracellular vesicles (EVs) and nanotube-based connections. The emergence of chemoresistance is, indeed, a major clinical problem for tumors in cases where chemotherapy remains the frontline treatment. Železnik Ramuta and colleagues reviewed 42 studies published between 2001 and 2022 evaluating the role of MSC in chemoresistance [9]. The results highlighted the existence of various mechanisms involved in this specific function of MSC, most of them affecting signaling pathways related to apoptosis and proliferation. Sentek and colleagues illustrated the relevance of the niche to interactions between endogenous lung-resident mesenchymal stem cells (LRMSC) and tumor cells [10]. They described peculiar features of “cancer-educated” LR-MSCs and discussed their potential to differentiate into CAFs and pericytes and, ultimately, favor tumor progression. Papait and colleagues acknowledged the role of CAFs in cancer development, addressing the main open questions regarding these controversial cells [11]. They clarified different aspects related to phenotype identification and subtype specification and discussed some of the most advanced technologies involved in the process. Finally, they reviewed pre-clinical and clinical attempts to target CAFs in various types of cancer. As cancer progression is also defined by stroma-to-stroma interactions, Çakır and colleagues investigated the capacity of melanoma-associated fibroblasts (MAFs) to modulate macrophage functions [12]. The authors observed that the MAFs were able to shape the functional phenotype of macrophages and elicit IL-10 secretory production in these cells via both the cyclo-oxygenase pathway and IDO, thus regulating tumor immunity. With their innate features having been unraveled, more questions will be raised concerning the safety and translational relevance of MSC in cancer. This Special Issue highlights the need for a deeper understanding of the fundamental processes that regulate MSC biology in health and disease to develop a clinically relevant therapeutic strategy that takes into consideration the recipient’s environment as a bi-directional type of communication

Espresso Coffee Mitigates the Aggregation and Condensation of Alzheimer′s Associated Tau Protein

: Espresso coffee is among the most consumed beverages in the world. Recent studies report a protective activity of the coffee beverage against neurodegenerative disorders such as Alzheimer's disease. Alzheimer's disease belongs to a group of disorders, called tauopathies, which are characterized by the intraneuronal accumulation of the microtubule-associated protein tau in fibrillar aggregates. In this work, we characterized by NMR the molecular composition of the espresso coffee extract and identified its main components. We then demonstrated with in vitro and in cell experiments that the whole coffee extract, caffeine, and genistein have biological properties in preventing aggregation, condensation, and seeding activity of the repeat region of tau. We also identified a set of coffee compounds capable of binding to preformed tau fibrils. These results add insights into the neuroprotective potential of espresso coffee and suggest candidate molecular scaffolds for designing therapies targeting monomeric or fibrillized forms of tau

Level set-fitted polytopal meshes with application to structural topology optimization

We propose a method to modify a polygonal mesh in order to fit the zero-isoline of a level set function by extending a standard body-fitted strategy to a tessellation with arbitrarily-shaped elements. The novel level set-fitted approach, in combination with a Discontinuous Galerkin finite element approximation, provides an ideal setting to model physical problems characterized by embedded or evolving complex geometries, since it allows skipping any mesh post-processing in terms of grid quality. The proposed methodology is firstly assessed on the linear elasticity equation, by verifying the approximation capability of the level set-fitted approach when dealing with configurations with heterogeneous material properties. Successively, we combine the level set-fitted methodology with a minimum compliance topology optimization technique, in order to deliver optimized layouts exhibiting crisp boundaries and reliable mechanical performances. An extensive numerical test campaign confirms the effectiveness of the proposed method

HTLV ANTISENSE PROTEINS ROLE IN THE NF-\u3baB MODULATION.

The retrovirus HTLV-1 is the causative agent of adult T-cell leukemia, whereas the genetically related sierotype HTLV-2 is sporadically associated with neurological diseases. The HTLV-1 genome encodes regulatory proteins, such as the oncoprotein Tax and the antisense proteins HBZ, involved into T-cells proliferation and transformation. Tax-1, HBZ, and the HTLV-2 homologs, Tax-2 and APH-2 interact with many host cell factors imparing cell signaling pathways involved in the mechanisms of survival, and proliferation, including the NF-\u3baB pathway. The aim of this study is to investigate the involvement of the regulatory proteins HBZ and APH-2 in the constitutively Tax-mediated NF-\u3baB activation. We demonstrated that HBZ and APH-2 differ in the NF-\u3baB promoter suppression. The APH-2 protein, differently from HBZ, localizes into the cytoplasm in presence of Tax, where it prevents the degradation of the inhibitor I\u3baB, hindering the nuclear translocation of p65. Unlike HBZ, we found that APH-2 interacts with the E3 ubiquitin ligase TRAF3, an upstream inhibitor of the alternative NF-\u3baB pathway. By generating a TRAF3-KO cell line applying the CRISPR/Cas9 technique, we are investigating the HBZ and APH-2 activity on the alternative NF-\u3baB cell signaling. This study may provide insight into the effect of host-viral interactions in human viral oncogenesis

HTLV-1 BASIC LEUCIN ZIPPER FACTOR AND ITS HOMOLOGOUS APH-2 IMPAIR NF-\u3baB ACTIVATION MEDIATED BY THE VIRAL ONCOPROTEIN TAX.

HTLV-1 and HTLV-2 are complex retroviruses which share a similar genomic organization but differ in their pathobiology. HTLV-1, the first human retrovirus discovered, is the causal agent of an aggressive adult T-cell leukemia, whereas HTLV-2 is associated with a few cases of neurological disease. Both virus genomes encode an oncogenic protein, Tax, required for viral replication and capable to induce cell transformation. In addition, HTLV-1 and -2 generate an antisense transcript, named HBZ and APH-2, respectively, crucial for viral infection. Comparative studies between HTLV-1 regulatory proteins, Tax-1 and HBZ, and the HTLV-2 homologs, Tax-2 and APH-2, may highlight the contribution of viral proteins to oncogenesis. The purpose of this study is to investigate the functional role of the viral regulatory proteins HBZ and APH-2 in the NF-\u3baB cell signaling, which is constitutively activated by Tax in infected cells. We demonstrated that APH-2 and HBZ differ in their suppression of the NF-\u3baB promoter activity. Unlike HBZ, the APH-2 protein is recruited by Tax in cytoplasmic structures and prevents the degradation of the inhibitor I\u3baB, impairing p65 nuclear translocation. Furthermore, we found that APH-2, but not HBZ, forms complexes with the adaptor protein TRAF3, an upstream inhibitor of the alternative NF-\u3baB pathway. We generated a TRAF3-KO cell line applying the CRISPR/Cas9 technique, which will allow us to investigate the HBZ and APH-2 role in modulating the alternative NF-\u3baB cell signaling. This study may provide insight into the effect of host-viral interactions in human viral oncogenesis

A CRISPR/Cas9 based approach to study the implication of HTLV regulatory proteins in the NF-κB modulation.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive form of T-cell malignancy with no cure. The HTLV-1 oncoprotein Tax plays a key role in CD4+ T-cell transformation, mainly through constitutive activation of both the canonical and the alternative NF-κB pathways. The HTLV-1 basic zipper protein (HBZ), encoded by the antisense viral genome strand, plays an essential role in the oncogenic process in concert with Tax, mediating T-cell proliferation. Unlike HTLV-1, the genetically related retrovirus HTLV-2 is not associated with ATL diseases. Functional comparisons between HTLV-1 regulatory proteins, Tax-1 and HBZ, and the HTLV-2 homologs, Tax-2 and APH-2, may highlight different mechanisms of their oncogenic potential. The aim of this study is to investigate how the antisense proteins HBZ and APH-2 impaired the NF-κB pathway activation. We found that both HBZ and APH-2 antagonized the NF-κB promoter activity mediated by Tax, but not in the same extent. Analyzing the intracellular distribution of the antisense proteins, we found that APH-2 is retained in cytoplasm complexes, whereas HBZ is mainly distributed into the nucleus. We observed that in presence of APH-2 and Tax-2, the degradation of the IκB-α inhibitor was reduced. Moreover, we found that unlike HBZ, APH-2 formed complexes with an upstream inhibitor of the alternative NF-κB pathway, the TNF receptor-associated factor 3, TRAF3. We generated a TRAF3 knock-out cell line applying the CRISPR/Cas9-mediated genome editing. By luciferase assays, we showed that TRAF3 is not required for Tax mediated NFκB promoter activation. Analyses are in progress to test the inhibitory effect of the antisense HBZ and APH-2 proteins on NF-κB promoter activity in absence of TRAF3. The results of this study may contribute to clarify the effect of the alternative NF-κB viral deregulation pathway in the expression of proinflammatory genes

New Insights into the Runt Domain of RUNX2 in Melanoma Cell Proliferation and Migration

The mortality rate for malignant melanoma (MM) is very high, since it is highly invasive and resistant to chemotherapeutic treatments. The modulation of some transcription factors affects cellular processes in MM. In particular, a higher expression of the osteogenic master gene RUNX2 has been reported in melanoma cells, compared to normal melanocytes. By analyzing public databases for recurrent RUNX2 genetic and epigenetic modifications in melanoma, we found that the most common RUNX2 genetic alteration that exists in transcription upregulation is, followed by genomic amplification, nucleotide substitution and multiple changes. Additionally, altered RUNX2 is involved in unchecked pathways promoting tumor progression, Epithelial Mesenchymal Transition (EMT), and metastasis. In order to investigate further the role of RUNX2 in melanoma development and to identify a therapeutic target, we applied the CRISPR/Cas9 technique to explore the role of the RUNT domain of RUNX2 in a melanoma cell line. RUNT-deleted cells showed reduced proliferation, increased apoptosis, and reduced EMT features, suggesting the involvement of the RUNT domain in different pathways. In addition, del-RUNT cells showed a downregulation of genes involved in migration ability. In an in vivo zebrafish model, we observed that wild-type melanoma cells migrated in 81% of transplanted fishes, while del-RUNT cells migrated in 58%. All these findings strongly suggest the involvement of the RUNT domain in melanoma metastasis and cell migration and indicate RUNX2 as a prospective target in MM therapy

GPR56 as a novel marker identifying the CD56dull CD16+ NK cell subset both in blood stream and in inflamed peripheral tissues

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Extracellular Vesicles From Perinatal Cells for Anti-inflammatory Therapy

Perinatal cells, including cells from placenta, fetal annexes (amniotic and chorionic membranes), umbilical cord, and amniotic fluid display intrinsic immunological properties which very likely contribute to the development and growth of a semiallogeneic fetus during pregnancy. Many studies have shown that perinatal cells can inhibit the activation and modulate the functions of various inflammatory cells of the innate and adaptive immune systems, including macrophages, neutrophils, natural killer cells, dendritic cells, and T and B lymphocytes. These immunological properties, along with their easy availability and lack of ethical concerns, make perinatal cells very useful/promising in regenerative medicine. In recent years, extracellular vesicles (EVs) have gained great interest as a new therapeutic tool in regenerative medicine being a cell-free product potentially capable, thanks to the growth factors, miRNA and other bioactive molecules they convey, of modulating the inflammatory microenvironment thus favoring tissue regeneration. The immunomodulatory actions of perinatal cells have been suggested to be mediated by still not fully identified factors (secretoma) secreted either as soluble proteins/cytokines or entrapped in EVs. In this review, we will discuss how perinatal derived EVs may contribute toward the modulation of the immune response in various inflammatory pathologies (acute and chronic) by directly targeting different elements of the inflammatory microenvironment, ultimately leading to the repair and regeneration of damaged tissues