scholarly journals Immunological Characterization of HIV and SARS-CoV-2 Coinfected Young Individuals

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3187
Author(s):  
Claudia Vanetti ◽  
Daria Trabattoni ◽  
Marta Stracuzzi ◽  
Antonella Amendola ◽  
Clara Fappani ◽  
...  
Keyword(s):  
Immune Response ◽  
General Population ◽  
Crucial Role ◽  
Rna Virus ◽  
Hiv Positive ◽  
Specific Antibodies ◽  
Increased Risk ◽  
Set Up

While the risk of SARS-CoV-2 infection and/or COVID-19 disease progression in the general population has been largely assessed, its impact on HIV-positive individuals remains unclear. We present clinical and immunological data collected in a cohort of HIV-infected young individuals during the first wave of COVID-19 pandemic. SARS-CoV-2 RNA, virus-specific antibodies, as well as the expression of factors involved in the anti-viral immune response were analyzed. Moreover, we set up an in vitro coinfection assay to study the mechanisms correlated to the coinfection process. Our results did not show any increased risk of severe COVID-19 in HIV-positive young individuals. In those subjects who contracted SARS-CoV-2 infection, an increase in IL-10 expression and production was observed. Furthermore, in the in vitro coinfection assay, we revealed a reduction in SARS-CoV-2 replication associated to an upregulation of IL-10. We speculate that IL-10 could play a crucial role in the course of SARS-CoV-2 infection in HIV-positive individuals. These results might help defining clinical management of HIV/SARS-CoV-2 co-infected young individuals, or putative indications for vaccination schedules in this population.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Antibodies Induced by Lipoarabinomannan in Bovines: Characterization and Effects on the Interaction betweenMycobacterium aviumSubsp.paratuberculosisand MacrophagesIn Vitro

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Ana Jolly ◽  
Silvia Beatriz Colavecchia ◽  
Bárbara Fernández ◽  
Eloy Fernández ◽  
Silvia Leonor Mundo
Keyword(s):  
Immune Response ◽  
Mycobacterium Avium ◽  
Active Role ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Specific Antibodies ◽  
Humoral Immune ◽  
Bovine Macrophage

Lipoarabinomannan (LAM) is a major glycolipidic antigen on the mycobacterial envelope. The aim of this study was to characterize the humoral immune response induced by immunization with a LAM extract in bovines and to evaluate the role of the generated antibodies in thein vitroinfection of macrophages withMycobacterium aviumsubsp.paratuberculosis(MAP). Sera from fourteen calves immunized with LAM extract or PBS emulsified in Freund's Incomplete Adjuvant and from five paratuberculosis-infected bovines were studied. LAM-immunized calves developed specific antibodies with IgG1 as the predominant isotype. Serum immunoglobulins were isolated and their effect was examined in MAP ingestion and viability assays using a bovine macrophage cell line. Our results show that the antibodies generated by LAM immunization significantly increase MAP ingestion and reduce its intracellular viability, suggesting an active role in this model.

scholarly journals Screening Assays for Epigenetic Targets Using Native Histones as Substrates

2011 ◽  
Vol 17 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Alexander-Thomas Hauser ◽  
Elisabeth-Maria Bissinger ◽  
Eric Metzger ◽  
Antje Repenning ◽  
Uta-Maria Bauer ◽  
...  
Keyword(s):  
In Vitro Assays ◽  
The Past ◽  
Chemistry Research ◽  
Core Histones ◽  
Homogeneous Assay ◽  
Screening Assays ◽  
Set Up ◽  
Identification And Characterization

In the past years, a lot of attention has been given to the identification and characterization of selective and potent inhibitors of chromatin-modifying enzymes to better understand their specific role in transcriptional regulation. As aberrant histone methylation is involved in different pathological processes, the search for methyltransferase and demethylase inhibitors has emerged as a crucial issue in current medicinal chemistry research. High-throughput in vitro assays are important tools for the identification of new methyltransferase or demethylase inhibitors. These usually use oligopeptide substrates derived from histone sequences, although in many cases, they are not good substrates for these enzymes. Here, the authors report about the setup and establishment of in vitro assays that use native core histones as substrates, enabling an assay environment that better resembles native conditions. They have applied these substrates for the known formaldehyde dehydrogenase assay for the histone demethylase LSD1 and have established two new antibody-based assays. For LSD1, a heterogeneous assay format was set up, and a homogeneous assay was used for the characterization of the arginine methyltransferase PRMT1. Validation of the system was achieved with reference inhibitors in each case.

scholarly journals EFFECTS OF MODERATELY VIRULENT AFRICAN SWINE FEVER VIRUS ON INTERLEUKIN-10 PRODUCTION

2019 ◽  
pp. 23-28 ◽  
Author(s):  
A. S. Pershin ◽  
I. V. Shevchenko ◽  
A. S. Igolkin ◽  
Ye. V. Aronova ◽  
N. N. Vlasova
Keyword(s):  
Immune Response ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Noticeable Effect ◽  
Specific Antibodies ◽  
Virulent Virus ◽  
Virus Isolates ◽  
Highly Virulent

A characteristic feature of African swine fever virus (ASFV) is the ability to escape from host immune response, affecting macrophages and replicating in them. Besides, ASFV - specific antibodies do not completely neutralize the virus. Cytokines are important factors for various viral infection pathologies. The virulence of ASFV isolates may depend on the capacity to regulate cytokine expression by macrophages. Thus, when comparing in vitro and in vivo cytokine production by macrophages, it was established that infection with low virulent virus isolates leads to an immune response with a predominance of cytokines involved in cellular immunity, such as INF-α and IL-12p40, as compared with infection with highly virulent isolates. The aim of this paper was to study the effect of African swine fever virus on the production of IL-10, a pleiotropic cytokine that inhibits synthesis of cytokines and shows a strong antiinflammatory effect. For this, 12 piglets were experimentally infected intramuscularly with a continuous cell culture-adapted ASFV isolate Vero25 at a dose of 10 HAdU per animal followed by control infection of surviving animals with the reference virus isolate Arm 07 at a dose of 1,000 HAdU per animal. Temperature measurements were taken and blood sampling to obtain serum was conducted during the experiment. IL-10 amount in blood sera was determined using Invitrogen test systems (Thermo Fisher, USA). A higher IL-10 level (15.8–173 pg/ml) was observed in blood sera of dead animals infected with a moderately virulent virus, as compared with surviving pigs (4–5 pg/ml). No correlation between the speed of appearance of specific antibodies and IL-10 serum levels has been established. No noticeable effect of the IL-10 serum level prior to infection on the survival rate of animals has been observed. Further studies are needed to establish a causal relationship, including study of the expression of various cytokines during infection with both low- and highly virulent virus isolates.

scholarly journals Development and characterization of a novel human 3D model of bone metastasis from breast carcinoma in vitro cultured

2021 ◽  
Vol 4 (s1) ◽  
Author(s):  
Deyanira Contartese ◽  
Francesca Salamanna ◽  
Veronica Borsari ◽  
Stefania Pagani ◽  
Maria Sartori ◽  
...  
Keyword(s):  
Bone Metastasis ◽  
Breast Carcinoma ◽  
3D Model ◽  
Ex Vivo ◽  
Ex Vivo Model ◽  
Fresh Tissue ◽  
Vertebral Bone ◽  
Set Up

Breast cancer frequently metastasizes to the skeleton causing significant morbidity. Here, we set-up a novel and advanced ex vivo model by using fresh tissue from human vertebral bone metastasis from breast carcinoma patients able to retain the tumor microenvironment and tumor cells heterogeneity.

scholarly journals Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Hirokazu Kimura ◽  
Raymond M Paranal ◽  
Neha Nanda ◽  
Laura D Wood ◽  
James R Eshleman ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Ductal Adenocarcinoma ◽  
Proliferation Assay ◽  
Variants Of Uncertain Significance ◽  
Pathogenic Variants ◽  
Deleterious Alleles ◽  
Increased Risk ◽  
Uncertain Significance

Pathogenic germline CDKN2A variants are associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC). CDKN2A variants of uncertain significance (VUSs) are reported in up to 4.3% of patients with PDAC and result in significant uncertainty for patients and their family members as an unknown fraction are functionally deleterious, and therefore, likely pathogenic. Functional characterization of CDKN2A VUSs is needed to reclassify variants and inform clinical management. 29 germline CDKN2A VUSs previously reported in patients with PDAC or in ClinVar were evaluated using a validated in vitro cell proliferation assay. 12 of the 29 CDKN2A VUSs were functionally deleterious (11 VUSs) or potentially functionally deleterious (1 VUS) and were reclassified as likely pathogenic variants. Thus, over 40% of CDKN2A VUSs identified in patients with PDAC are functionally deleterious and likely pathogenic. When incorporating VUSs found to be functionally deleterious, and reclassified as likely pathogenic, the prevalence of pathogenic/likely pathogenic CDKN2A in patients with PDAC reported in the published literature is increased to up to 4.1% of patients, depending on family history. Therefore, CDKN2A VUSs may play a significant, unappreciated role in risk of pancreatic cancer. These findings have significant implications for the counselling and care of patients and their relatives.

Isolation and characterization of porcine macrophages and their inflammatory and fusion responses in different stiffness environments

2021 ◽  
Author(s):  
Vijaykumar Meli ◽  
Ryan Donahue ◽  
Jarrett M Link ◽  
Jerry C Hu ◽  
Kyriacos A. Athanasiou ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Immune Response ◽  
Medical Devices ◽  
Host Immune Response ◽  
In Vitro Studies ◽  
Essential Step ◽  
Isolation And Characterization

Evaluating the host immune response to biomaterials is an essential step in the development of medical devices and tissue engineering strategies. To aid in this process, in vitro studies, whereby...

scholarly journals Characterization of a Novel 5′ Subgenomic RNA3a Derived from RNA3 of Brome Mosaic Bromovirus

2006 ◽  
Vol 80 (24) ◽  
pp. 12357-12366 ◽  
Author(s):  
Rafal Wierzchoslawski ◽  
Anna Urbanowicz ◽  
Aleksandra Dzianott ◽  
Marek Figlerowicz ◽  
Jozef J. Bujarski
Keyword(s):  
Movement Protein ◽  
Rna Virus ◽  
Minus Strand ◽  
Cloning And Sequencing ◽  
Genomic Rnas ◽  
Brome Mosaic Bromovirus ◽  
Cellular Mrnas ◽  
In Vitro Systems

ABSTRACT The synthesis of 3′ subgenomic RNA4 (sgRNA4) by initiation from an internal sg promoter in the RNA3 segment was first described for Brome mosaic bromovirus (BMV), a model tripartite positive-sense RNA virus (W. A. Miller, T. W. Dreher, and T. C. Hall, Nature 313:68-70, 1985). In this work, we describe a novel 5′ sgRNA of BMV (sgRNA3a) that we propose arises by premature internal termination and that encapsidates in BMV virions. Cloning and sequencing revealed that, unlike any other BMV RNA segment, sgRNA3a carries a 3′ oligo(A) tail, in which respect it resembles cellular mRNAs. Indeed, both the accumulation of sgRNA3a in polysomes and the synthesis of movement protein 3a in in vitro systems suggest active functions of sgRNA3a during protein synthesis. Moreover, when copied in the BMV replicase in vitro reaction, the minus-strand RNA3 template generated the sgRNA3a product, likely by premature termination at the minus-strand oligo(U) tract. Deletion of the oligo(A) tract in BMV RNA3 inhibited synthesis of sgRNA3a during infection. We propose a model in which the synthesis of RNA3 is terminated prematurely near the sg promoter. The discovery of 5′ sgRNA3a sheds new light on strategies viruses can use to separate replication from the translation functions of their genomic RNAs.

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