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Frontiers in Immunology 2023The immune system, composed of organs, tissues, cells, and proteins, is the key to protecting the body from external biological attacks and inflammation. The latter... (Review)
Review
BACKGROUND
The immune system, composed of organs, tissues, cells, and proteins, is the key to protecting the body from external biological attacks and inflammation. The latter occurs in several pathologies, such as cancers, type 1 diabetes, and human immunodeficiency virus infection. Immunophenotyping by flow cytometry is the method of choice for diagnosing these pathologies. Under inflammatory conditions, the peripheral blood mononuclear cells (PBMCs) are partially activated and generate intracellular pathways involving Ca-dependent signaling cascades leading to transcription factor expression. Ca signaling is typically studied by microscopy in cell lines but can present some limitations to explore human PBMCs, where flow cytometry can be a good alternative.
OBJECTIVE
In this review, we dived into the research field of inflammation and Ca signaling in PBMCs. We aimed to investigate the structure and evolution of this field in a physio-pathological context, and then we focused our review on flow cytometry analysis of Ca fluxes in PBMCs.
METHODS
From 1984 to 2022, 3865 articles on inflammation and Ca signaling in PBMCs were published, according to The Clarivate Web of Science (WOS) database used in this review. A bibliometric study was designed for this collection and consisted of a co-citation and bibliographic coupling analysis.
RESULTS
The co-citation analysis was performed on 133 articles: 4 clusters highlighted the global context of Ca homeostasis, including chemical probe development, identification of the leading players in Ca signaling, and the link with chemokine production in immune cell function. Next, the bibliographic coupling analysis combined 998 articles in 8 clusters. This analysis outlined the mechanisms of PBMC activation, from signal integration to cellular response. Further explorations of the bibliographic coupling network, focusing on flow cytometry, revealed 21 articles measuring cytosolic Ca in PBMCs, with only 5 since 2016. This final query showed that Ca signaling analysis in human PBMCs using flow cytometry is still underdeveloped and investigates mainly the cytosolic Ca compartment.
CONCLUSION
Our review uncovers remaining knowledge gaps of intracellular players involved in Ca signaling in PBMCs, such as reticulum and mitochondria, and presents flow cytometry as a solid option to supplement gold-standard microscopy studies.
Topics: Humans; Leukocytes, Mononuclear; Flow Cytometry; Cell Line; Signal Transduction; Inflammation
PubMed: 37901222
DOI: 10.3389/fimmu.2023.1272809 -
MSystems Aug 2023A deep understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions is crucial to developing effective therapeutics and addressing the...
A deep understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions is crucial to developing effective therapeutics and addressing the threat of emerging coronaviruses. The role of noncoding regions of viral RNA (ncrRNAs) has yet to be systematically scrutinized. We developed a method using MS2 affinity purification coupled with liquid chromatography-mass spectrometry and designed a diverse set of bait ncrRNAs to systematically map the interactome of SARS-CoV-2 ncrRNA in Calu-3, Huh7, and HEK293T cells. Integration of the results defined the core ncrRNA-host protein interactomes among cell lines. The 5' UTR interactome is enriched with proteins in the small nuclear ribonucleoproteins family and is a target for the regulation of viral replication and transcription. The 3' UTR interactome is enriched with proteins involved in the stress granules and heterogeneous nuclear ribonucleoproteins family. Intriguingly, compared with the positive-sense ncrRNAs, the negative-sense ncrRNAs, especially the negative-sense of 3' UTR, interacted with a large array of host proteins across all cell lines. These proteins are involved in the regulation of the viral production process, host cell apoptosis, and immune response. Taken together, our study depicts the comprehensive landscape of the SARS-CoV-2 ncrRNA-host protein interactome and unveils the potential regulatory role of the negative-sense ncrRNAs, providing a new perspective on virus-host interactions and the design of future therapeutics. Given the highly conserved nature of UTRs in positive-strand viruses, the regulatory role of negative-sense ncrRNAs should not be exclusive to SARS-CoV-2. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic affecting millions of lives. During replication and transcription, noncoding regions of the viral RNA (ncrRNAs) may play an important role in the virus-host interactions. Understanding which and how these ncrRNAs interact with host proteins is crucial for understanding the mechanism of SARS-CoV-2 pathogenesis. We developed the MS2 affinity purification coupled with liquid chromatography-mass spectrometry method and designed a diverse set of ncrRNAs to identify the SARS-CoV-2 ncrRNA interactome comprehensively in different cell lines and found that the 5' UTR binds to proteins involved in U1 small nuclear ribonucleoprotein, while the 3' UTR interacts with proteins involved in stress granules and the heterogeneous nuclear ribonucleoprotein family. Interestingly, negative-sense ncrRNAs showed interactions with a large number of diverse host proteins, indicating a crucial role in infection. The results demonstrate that ncrRNAs could serve diverse regulatory functions.
Topics: Humans; SARS-CoV-2; COVID-19; 3' Untranslated Regions; 5' Untranslated Regions; RNA, Viral; HEK293 Cells
PubMed: 37314180
DOI: 10.1128/msystems.00135-23 -
Chinese Medical Journal Nov 2023Although antiretroviral therapy (ART) can reduce the viral load in the plasma to undetectable levels in human immunodeficiency virus (HIV)-infected individuals, ART... (Review)
Review
Although antiretroviral therapy (ART) can reduce the viral load in the plasma to undetectable levels in human immunodeficiency virus (HIV)-infected individuals, ART alone cannot completely eliminate HIV due to its integration into the host cell genome to form viral reservoirs. To achieve a functional cure for HIV infection, numerous preclinical and clinical studies are underway to develop innovative immunotherapies to eliminate HIV reservoirs in the absence of ART. Early studies have tested adoptive T-cell therapies in HIV-infected individuals, but their effectiveness was limited. In recent years, with the technological progress and great success of chimeric antigen receptor (CAR) therapy in the treatment of hematological malignancies, CAR therapy has gradually shown its advantages in the field of HIV infection. Many studies have identified a variety of HIV-specific CAR structures and types of cytolytic effector cells. Therefore, CAR therapy may be beneficial for enhancing HIV immunity, achieving HIV control, and eliminating HIV reservoirs, gradually becoming a promising strategy for achieving a functional HIV cure. In this review, we provide an overview of the design of anti-HIV CAR proteins, the cell types of anti-HIV CAR (including CAR T cells, CAR natural killer cells, and CAR-encoding hematopoietic stem/progenitor cells), the clinical application of CAR therapy in HIV infection, and the prospects and challenges in anti-HIV CAR therapy for maintaining viral suppression and eliminating HIV reservoirs.
Topics: Humans; Immunotherapy, Adoptive; HIV Infections; HIV-1
PubMed: 37927030
DOI: 10.1097/CM9.0000000000002904 -
Frontiers in Immunology 2023Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited....
Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-I/TCR CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent and antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-I/TCR CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-I/TCR CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.
Topics: Animals; Humans; Aged; Receptors, Antigen, T-Cell; T-Lymphocytes; Leukemia, Myeloid, Acute; Immunotherapy, Adoptive; Disease Models, Animal; Hematopoietic Stem Cell Transplantation
PubMed: 37795087
DOI: 10.3389/fimmu.2023.1270843 -
Science Advances Jul 2023HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand...
HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.
Topics: HIV Integrase Inhibitors; Oxazines; Mutation; HIV Integrase
PubMed: 37478179
DOI: 10.1126/sciadv.adg5953 -
International Journal of Molecular... Jan 2024The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand...
The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a marker gene into the safe harbor site and a super-exon into in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.
Topics: Humans; CRISPR-Cas Systems; Pluripotent Stem Cells; Induced Pluripotent Stem Cells; Hematopoietic Stem Cells; Alleles
PubMed: 38256061
DOI: 10.3390/ijms25020985 -
Cureus Jul 2023Background Tuberculosis (TB) is an infectious disease caused by the bacterium . It primarily affects the lungs but can also affect other organs, such as the kidneys,...
Background Tuberculosis (TB) is an infectious disease caused by the bacterium . It primarily affects the lungs but can also affect other organs, such as the kidneys, bones, and brain. TB is transmitted through the air when an infected individual coughs, sneezes, or speaks, releasing tiny droplets containing the bacteria. Despite significant efforts to combat TB, challenges such as drug resistance, co-infection with human immunodeficiency virus (HIV), and limited resources in high-burden settings continue to pose obstacles to its eradication. TB remains a significant global health challenge, necessitating accurate and timely detection for effective management. Methods This study aimed to develop a TB detection model using chest X-ray images obtained from Kaggle.com, utilizing Google's Collaboration Platform. Over 1196 chest X-ray images, comprising both TB-positive and normal cases, were employed for model development. The model was trained to recognize patterns within the TB chest X-rays to efficiently recognize TB within patients in order to be treated in time. Results The model achieved an average precision of 0.934, with precision and recall values of 94.1% each, indicating its high accuracy in classifying TB-positive and normal cases. Sensitivity and specificity values were calculated as 96.85% and 91.49%, respectively. The F1 score was also calculated to be 0.941. The overall accuracy of the model was found to be 94%. Conclusion These results highlight the potential of machine learning algorithms for TB detection using chest X-ray images. Further validation studies and research efforts are needed to assess the model's generalizability and integration into clinical practice, ultimately facilitating early detection and improved management of TB.
PubMed: 37559842
DOI: 10.7759/cureus.41583 -
Frontiers in Microbiology 2023Certain viruses called tumor viruses or oncoviruses are capable to change the gene expression pattern of distinct human or animal cell types in tissue culture, resulting... (Review)
Review
Certain viruses called tumor viruses or oncoviruses are capable to change the gene expression pattern of distinct human or animal cell types in tissue culture, resulting in uncontrolled proliferation as well as a change in the social behavior of the infected cells: the oncovirus-transformed, immortalized cells are capable to form malignant neoplasms in suitable animal models. At present, seven human viruses are categorized as causative agents of distinct human malignancies. The genomes of human tumor viruses, typically encode viral oncoproteins and non- translated viral RNAs that affect the gene expression pattern of their target cells or induce genetic and epigenetic alterations contributing to oncogenesis. Recently, the application of chromatin conformation capture technologies and three-dimensional (3D) molecular imaging techniques revealed how the gene products or genomes of certain human tumor viruses interact with and induce alterations in the 3D host genome structure. This Mini Review aims to cover selected aspects of these developments. The papers, discussed briefly, describe how insertion of a novel viral binding site for the 3D genome organizer cellular protein CCCTC-binding factor (CTCF) into the DNA of T cells infected by human T-cell lymphotropic virus type 1 (HTLV-1) may contribute to lymphomagenesis, as well as how integration of high risk human papillomavirus genome into the host cell DNA may facilitate cervical carcinogenesis. Recent results regarding the interactions of cellular genomes with the episomal, chromatinized DNA genomes of oncogenic human herpesvirus, Epstein-Barr virus (EBV) will also be summarized, similarly to available data regarding contacts formed by episomal or integrated hepatitis B virus (HBV) DNA with host chromatin. Finally, a putative mechanism of hepatitis C virus (HCV) induced chromatin alterations will be presented, which may solve the riddle, how a cytoplasmic RNA virus without a viral oncogene could induce malingnant transfrormation of hepatocytes.
PubMed: 37928671
DOI: 10.3389/fmicb.2023.1280210 -
BioRxiv : the Preprint Server For... Mar 2024Glioblastoma (GBM), a universally fatal brain cancer, infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression . Synaptic...
Glioblastoma (GBM), a universally fatal brain cancer, infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression . Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic . The extent of integration of GBM cells into brain-wide neuronal circuitry is not well understood. Here we applied a rabies virus-mediated retrograde monosynaptic tracing approach to systematically investigate circuit integration of human GBM organoids transplanted into adult mice. We found that GBM cells from multiple patients rapidly integrated into brain-wide neuronal circuits and exhibited diverse local and long-range connectivity. Beyond glutamatergic inputs, we identified a variety of neuromodulatory inputs across the brain, including cholinergic inputs from the basal forebrain. Acute acetylcholine stimulation induced sustained calcium oscillations and long-lasting transcriptional reprogramming of GBM cells into a more invasive state via the metabotropic CHRM3 receptor. CHRM3 downregulation suppressed GBM cell invasion, proliferation, and survival in vitro and in vivo. Together, these results reveal the capacity of human GBM cells to rapidly and robustly integrate into anatomically and molecularly diverse neuronal circuitry in the adult brain and support a model wherein rapid synapse formation onto GBM cells and transient activation of upstream neurons may lead to a long-lasting increase in fitness to promote tumor infiltration and progression.
PubMed: 38496540
DOI: 10.1101/2024.03.01.583047 -
NeuroImmune Pharmacology and... Sep 2023Alzheimer's disease (AD) is the most common cause of dementia. While pathologic hallmarks, such as extracellular beta-amyloid plaques, are well-characterized in affected... (Review)
Review
Alzheimer's disease (AD) is the most common cause of dementia. While pathologic hallmarks, such as extracellular beta-amyloid plaques, are well-characterized in affected individuals, the pathogenesis that causes plaque formation and eventual cognitive decline is not well understood. A recent resurgence of the decades-old "infectious hypothesis" has garnered increased attention on the potential role that microbes may play in AD. In this theory, it is thought that pathogens such as viruses may act as seeds for beta-amyloid aggregation, ultimately leading to plaques. Interest in the infectious hypothesis has also spurred further investigation into additional characteristics of viral infection that may play a role in AD progression, such as neuroinflammation, latency, and viral DNA integration. While a flurry of research in this area has been recently published, with herpesviruses being of particular interest, the role of pathogens in AD remains controversial. In this review, the insights gained thus far into the possible role of herpesviruses in AD are summarized. The challenges and potential future directions of herpesvirus research in AD and dementia are also discussed.
PubMed: 38013835
DOI: 10.1515/nipt-2023-0011