direct-stochastic-optical-reconstruction-microscopy-extracellular

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

The University of North Carolina at Chapel Hill School of Medicine

In HIV-infected populations in developed countries, the most recent published cancer incidence trend analyses are only updated through 2008. We assessed changes in the distribution of cancer types and incidence trends among HIV-infected patients in North Carolina up until 2011.

Recent cancer incidence trends in an observational clinical cohort of HIV-infected patients in the US, 2000 to 2011.

The most common human papillomavirus (HPV) genotypes isolated from cervical cancer in select African countries are HPV-16, HPV-18, HPV-35, and HPV-45, but the most common genotypes in Zambia are unknown. The overall objective of this study was to assess the potential impact of current HPV vaccines in preventing cervical cancer in Zambia, by determining the combined prevalence of HPV-16 and/or HPV-18 in invasive cervical cancer (ICC) and high-grade pre-cancer [cervical intraepithelial neoplasia 2 or 3 (CIN2/3)] cases.

Identification of human papillomaviruses from formalin-fixed, paraffin-embedded pre-cancer and invasive cervical cancer specimens in Zambia: a cross-sectional study.

Endemic Burkitt lymphoma (eBL) is primarily a childhood cancer in parts of Africa and Brazil. Classic studies describe eBL as a homogeneous entity based on t(8;14) IgH-Myc translocation and clinical response to cytotoxic therapy. By contrast, sporadic BL (sBL) in Western countries is considered more heterogeneous, and affects both children and adults. It is overrepresented in AIDS patients. Unlike diffuse large B cell lymphoma (DLBCL), molecular subtypes within BL have not been well defined. We find that differential IgM positivity can be used to describe two subtypes of pediatric Burkitt lymphoma both in a high incidence region (Brazil), as well as in a sporadic region (US), suggesting the phenotype is not necessarily geographically isolated. Moreover, we find that IgM positivity also distinguishes between early and late onset BL in the standard Eμ-Myc mouse model of BL. This suggests that the t(8;14) translocation not only can take place before, but also after isotype switch recombination, and that IgM-negative, t(8;14) positive lymphomas in children should nevertheless be considered BL.

Differential IgM expression distinguishes two types of pediatric Burkitt lymphoma in mouse and human.

Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product. Here, we present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From our results, we propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods.

Large-scale, cross-flow based isolation of highly pure and endocytosis-competent extracellular vesicles.

Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV). PEL has a highly active mTOR pathway, which makes mTOR a potential therapeutic target. MLN0128 is an ATP-competitive inhibitor of mTOR that has entered clinical trials for solid tumors. Our results demonstrated that MLN0128 has a greater effect on inhibiting proliferation than the allosteric mTOR inhibitor rapamycin. MLN0128 has ∼30 nM 50% inhibitory concentration (IC) across several PEL cell lines, including PEL that is resistant to conventional chemotherapy. MLN0128 induced apoptosis in PEL, whereas rapamycin induced G arrest, consistent with a different mechanism of action. MLN0128 inhibited phosphorylation of mTOR complex 1 and 2 targets, while rapamycin only partially inhibited mTOR complex 1 targets. PEL xenograft mouse models treated with MLN0128 showed reduced effusion volumes in comparison to the vehicle-treated group. Rapamycin-resistant (RR) clones with an IC for rapamycin 10 times higher than the parental IC emerged consistently after rapamycin exposure as a result of transcriptional adaptation. MLN0128 was nevertheless capable of inducing apoptosis in these RR clones. Our results suggest that MLN0128 might offer a new approach to the treatment of chemotherapy-resistant PEL. Primary effusion lymphoma (PEL) is an aggressive and incurable malignancy, which is usually characterized by lymphomatous effusions in body cavities without tumor masses. PEL has no established treatment and a poor prognosis, with a median survival time shorter than 6 months. PEL usually develops in the context of immunosuppression, such as HIV infection or post-organ transplantation. The optimal treatment for PEL has not been established, as PEL is generally resistant to traditional chemotherapy. The molecular drivers for PEL are still unknown; however, PEL displays a constitutively active mammalian target of rapamycin (mTOR) pathway, which is critical for metabolic and cell survival mechanisms. Therefore, the evaluation of novel agents targeting the mTOR pathway could be clinically relevant for the treatment of PEL.

Targeting mTOR with MLN0128 Overcomes Rapamycin and Chemoresistant Primary Effusion Lymphoma.

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in  arise quickly, while mutations in the  locus emerge later during fibroblast passage and disrupt formation of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As CMV replicates in the context of host antibodies in vivo, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and acquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. Additional mutations resulting in single amino acid changes also occurred in  encoding glycoprotein M,  encoding a subunit of the helicase/primase complex, and  encoding the Immediate Early 2 protein. An epitheliotropic RL13+/UL131A+ virus was isolated by limiting dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, production of virus stocks by culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than previously available.

Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components During Fibroblast Passage of Human Cytomegalovirus.

KSHV-associated inflammatory cytokine syndrome (KICS) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV). KICS is associated with high-level, systemic replication of KSHV. This study characterized the clinical and virologic features of a KICS patient over time. Additionally, it compared the cytokine profiles of the KICS case to Kaposi's sarcoma (KS) (n = 11) and non-KS (n = 6) cases. This KICS case presented with elevated levels of KSHV and IL-10, as expected. Surprisingly, this case did not have elevated levels of IL-6 or human immunodeficiency virus 1 (HIV-1). Nevertheless, treatment with anti-IL6 receptor antibody (tocilizumab) reduced KSHV viral load and IL-10. The KSHV genome sequence showed no significant changes over time, except in ORF24. Phylogenetic analysis established this isolate as belonging to KSHV clade A and closely related to other US isolates. These findings suggest IL-10 as potential biomarker and therapy target for KICS.

Runaway Kaposi Sarcoma-associated herpesvirus replication correlates with systemic IL-10 levels.

Kaposi Sarcoma (KS) is among the most angiogenic cancers in humans and an AIDS-defining condition. KS-associated herpesvirus (KSHV) is necessary for KS development, as is vascular endothelial growth factor (VEGF-A). DLX1008 is a novel anti-VEGF-A antibody single-chain variable fragment (scFv) with low picomolar affinity for VEGF-A. In vivo imaging techniques were used to establish the efficacy of DLX1008 and to establish the mechanism of action; this included non-invasive imaging by ultrasound and optical fluorescence, verified by post-mortem histochemistry. The results showed that DLX1008 was efficacious in a KS mouse model. The NSG mouse xenografts suffered massive internal necrosis or involution, consistent with a lack of blood supply. We found that imaging by ultrasound was superior to external caliper measurements in the validation of the angiogenesis inhibitor DLX1008. Further development of DLX1008 against VEGF-dependent sarcomas is warranted.

DLX1008 (brolucizumab), a single-chain anti-VEGF-A antibody fragment with low picomolar affinity, leads to tumor involution in an in vivo model of Kaposi Sarcoma.

The fields of extracellular vesicles (EV) and virus infections are marred in a debate on whether a particular mRNA or non-coding RNA (i.e., miRNA) is packaged into a virus particle or copurifying EV and similarly, whether a particular mRNA or non-coding RNA is contained in meaningful numbers within an EV. Key in settling this debate, is whether the purification methods are adequate to separate virus particles, EV and contaminant soluble RNA and RNA:protein complexes. Differential centrifugation/ultracentrifugation and precipitating agents like polyethylene glycol are widely utilized for both EV and virus purifications. EV are known to co-sediment with virions and other particulates, such as defective interfering particles and protein aggregates. Here, we discuss how encased RNAs from a heterogeneous mixture of particles can be distinguished by different purification methods. This is particularly important for subsequent interpretation of whether the RNA associated phenotype is contributed solely by virus or EV particles or a mixture of both. We also discuss the discrepancy of miRNA abundance in EV from different input material.

Purification Methods and the Presence of RNA in Virus Particles and Extracellular Vesicles.

Viruses are obligate intracellular parasites that usurp cellular signaling networks to promote pathogen spread and disease progression. Signaling through extracellular vesicles (EVs) is an emerging field of study in the virus-host interaction network. EVs relay information both locally and distally through incorporated contents, typically without tripping innate immune sensors. Therefore, this extracellular signaling axis presents itself as a tantalizing target for promoting a favorable niche for the pathogen(s) takeover of the host, particularly for chronic infections. From the incorporation of virus-encoded molecules such as micro RNAs and proteins/enzymes to the envelopment of entire infectious particles, evolutionary distinct viruses have shown a remarkable ability to converge on this means of communication. In this review, we will cover the recent advances in this field and explore how EV can be used as potential biomarkers for chronic, persistent, or latent virus infections.

Extracellular vesicles in virus infection and pathogenesis.

Characterizing the asymptomatic spread of SARS-CoV-2 is important for understanding the COVID-19 pandemic. This study was aimed at determining asymptomatic spread of SARS-CoV-2 in a suburban, Southern U.S. population during a period of state restrictions and physical distancing mandates. This is one of the first published seroprevalence studies from North Carolina and included multicenter, primary care, and emergency care facilities serving a low-density, suburban and rural population since description of the North Carolina state index case introducing the SARS-CoV-2 respiratory pathogen to this population. To estimate point seroprevalence of SARS-CoV-2 among asymptomatic individuals over time, two cohort studies were examined. The first cohort study, named ScreenNC, was comprised of outpatient clinics, and the second cohort study, named ScreenNC2, was comprised of inpatients unrelated to COVID-19. Asymptomatic infection by SARS-CoV-2 (with no clinical symptoms) was examined using an Emergency Use Authorization (EUA)-approved antibody test (Abbott) for the presence of SARS-CoV-2 IgG. This assay as performed under CLIA had a reported specificity/sensitivity of 100%/99.6%. ScreenNC identified 24 out of 2,973 (0.8%) positive individuals among asymptomatic participants accessing health care during 28 April to 19 June 2020, which was increasing over time. A separate cohort, ScreenNC2, sampled from 3 March to 4 June 2020, identified 10 out of 1,449 (0.7%) positive participants. This study suggests limited but accelerating asymptomatic spread of SARS-CoV-2. Asymptomatic infections, like symptomatic infections, disproportionately affected vulnerable communities in this population, and seroprevalence was higher in African American participants than in White participants. The low, overall prevalence may reflect the success of shelter-in-place mandates at the time this study was performed and of maintaining effective physical distancing practices among suburban populations. Under these public health measures and aggressive case finding, outbreak clusters did not spread into the general population.

SARS-CoV-2 Seroprevalence among a Southern U.S. Population Indicates Limited Asymptomatic Spread under Physical Distancing Measures.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constantly evolving. Prior studies focused on high-case-density locations, such as the northern and western metropolitan areas of the United States. This study demonstrates continued SARS-CoV-2 evolution in a suburban southern region of the United States by high-density amplicon sequencing of symptomatic cases. 57% of strains carry the spike D614G variant, which is associated with higher genome copy numbers, and its prevalence expands with time. Four strains carry a deletion in a predicted stem loop of the 3' UTR. The data are consistent with community spread within local populations and the larger continental United States. The data instill confidence in current testing sensitivity and validate "testing by sequencing" as an option to uncover cases, particularly nonstandard coronavirus disease 2019 (COVID-19) clinical presentations. This study contributes to the understanding of COVID-19 through an extensive set of genomes from a non-urban setting and informs vaccine design by defining D614G as a dominant and emergent SARS-CoV-2 isolate in the United States.

Article	Year
Recent cancer incidence trends in an observational clinical cohort of HIV-infected patients in the US, 2000 to 2011. Infectious agents and cancer\| PubMed ID: 23705808	2013
Identification of human papillomaviruses from formalin-fixed, paraffin-embedded pre-cancer and invasive cervical cancer specimens in Zambia: a cross-sectional study. Virology journal\| PubMed ID: 25591541	2015
Differential IgM expression distinguishes two types of pediatric Burkitt lymphoma in mouse and human. Oncotarget\| PubMed ID: 27566574	2016
Large-scale, cross-flow based isolation of highly pure and endocytosis-competent extracellular vesicles. Journal of extracellular vesicles\| PubMed ID: 30533204	2018
Targeting mTOR with MLN0128 Overcomes Rapamycin and Chemoresistant Primary Effusion Lymphoma. mBio\| PubMed ID: 30782662	2019
Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components During Fibroblast Passage of Human Cytomegalovirus. Viruses\| PubMed ID: 30841507	2019
Runaway Kaposi Sarcoma-associated herpesvirus replication correlates with systemic IL-10 levels. Virology\| PubMed ID: 31629226	2020
DLX1008 (brolucizumab), a single-chain anti-VEGF-A antibody fragment with low picomolar affinity, leads to tumor involution in an in vivo model of Kaposi Sarcoma. PloS one\| PubMed ID: 32407363	2020
Purification Methods and the Presence of RNA in Virus Particles and Extracellular Vesicles. Viruses\| PubMed ID: 32825599	2020
Extracellular vesicles in virus infection and pathogenesis. Current opinion in virology\| PubMed ID: 32846272	2020
SARS-CoV-2 Seroprevalence among a Southern U.S. Population Indicates Limited Asymptomatic Spread under Physical Distancing Measures. mBio\| PubMed ID: 32994333	2020
High-Density Amplicon Sequencing Identifies Community Spread and Ongoing Evolution of SARS-CoV-2 in the Southern United States. Cell reports\| PubMed ID: 33113345	2020

Dirk P. Dittmer

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