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.
Kaposi sarcoma (KS) is among the most common childhood malignancies in central, eastern, and southern Africa. Although its unique clinical features have been established, biological mechanisms ...related to the causative agent, KS‐associated herpes‐virus (KSHV), have yet to be explored in children. We performed a prospective observational pilot study to explore associations between KSHV viral load (VL), human interleukin‐6 (IL‐6) and IL‐10 levels, and clinical characteristics of 25 children with KS in Lilongwe, Malawi from June 2013–August 2015. The median age was 6.4 years. Lymphadenopathy was the most common site of KS involvement (64%), followed by skin and oral mucosa (44% each), woody edema (12%), and pulmonary (8%). Baseline samples for plasma KSHV VL, IL‐6 and IL‐10 analyses were available for 18/25 patients (72%) at time of KS diagnosis. KSHV VL was detectable at baseline in 12/18 (67%) patients, the median baseline IL‐6 level was 8.53 pg/mL (range 4.31–28.33), and the median baseline IL‐10 level was 19.53 pg/mL (range 6.91–419.69). Seven (39%) patients presented with an IL‐6 level > 10 pg/mL (exceeding twice the upper limit of normal). Detectable KSHV VL was significantly associated with lymphadenopathic KS (p = 0.004), while having undetectable KSHV VL was associated with a higher likelihood of presenting with hyperpigmented skin lesions (p = 0.01). Detectable KSHV VL and elevated IL‐6 levels are present in a subset of children with KS. Lytic activation of KSHV and associated elevation in KSHV VL may contribute to the unique clinical manifestations of pediatric KS in KSHV‐endemic regions of Africa.
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Kaposi sarcoma is among the most common childhood malignancies in regions endemic for Kaposi sarcoma‐associated herpesvirus (KSHV), but virological characteristics of the disease remain undefined. Here the authors studied 25 children with Kaposi sarcoma in southeastern Africa and established links between distinct clinical patterns and elevated KSHV viral load and interleukin‐6 levels. The results points to a role of KSHV lytic activation and a potential KSHV inflammatory cytokine syndrome in the lymphadenopathic form of childhood Kaposi sarcoma.
Primary effusion lymphoma (PEL) is a B cell lymphoma that is always associated with Kaposi’s sarcoma-associated herpesvirus (KSHV) and in many cases also with Epstein–Barr virus (EBV); however, the ...requirement for EBV coinfection is not clear. Here, we demonstrate that adding exogenous EBV to KSHV⁺ single-positive PEL leads to increased KSHV genome maintenance and KSHV latency-associated nuclear antigen (LANA) expression. To show that EBV was necessary for naturally coinfected PEL, we nucleofected KSHV⁺/EBV⁺ PEL cell lines with an EBV-specific CRISPR/Cas9 plasmid to delete EBV and observed a dramatic decrease in cell viability, KSHV genome copy number, and LANA expression. This phenotype was reversed by expressing Epstein–Barr nuclear antigen 1 (EBNA-1) in trans, even though EBNA-1 and LANA do not colocalize in infected cells. This work reveals that EBV EBNA-1 plays an essential role in the pathogenesis of PEL by increasing KSHV viral load and LANA expression.
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.
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.
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•NGS of SARS-CoV-2 from a rural/suburban area shows local spread as an epidemic driver•The D614G spike mutant is observed in >50% of cases•Deletion in the 3′ UTR of SARS-CoV-2 is identified•Targeted NGS has 100% specificity and is as sensitive as qPCR
McNamara et al. use next-generation sequencing (NGS) with a high-density tiling array across SARS-CoV-2 to find a deletion and document how the D614G spike protein mutation rapidly swept through a rural/suburban population. D614G is associated with slightly higher viral loads.
Variants of concern (VOC) in SARS-CoV-2 refer to viruses whose viral genomes differ from the ancestor virus by ≥3 single-nucleotide variants (SNVs) and that show the potential for higher ...transmissibility and/or worse clinical progression. VOC have the potential to disrupt ongoing public health measures and vaccine efforts. Still, too little is known regarding how frequently new viral variants emerge and under what circumstances. We report a study to determine the degree of SARS-CoV-2 sequence evolution in 94 patients and to estimate the frequency at which highly diverse variants emerge. Two cases accumulated ≥9 SNVs over a 2-week period and one case accumulated 23 SNVs over 3 weeks, including three nonsynonymous mutations in the spike protein (D138H, E554D, D614G). The remainder of the infected patients did not show signs of intra-host evolution. We estimate that in as much as 2% of hospitalized COVID-19 cases, variants with multiple mutations in the spike glycoprotein emerge in as little as 1 month of persistent intra-host virus replication. This suggests the continued local emergence of variants with multiple nonsynonymous SNVs, even in patients without overt immune deficiency. Surveillance by sequencing for (i) viremic COVID-19 patients, (ii) patients suspected of reinfection, and (iii) patients with diminished immune function may offer broad public health benefits.
New SARS-CoV-2 variants can potentially disrupt ongoing public health measures and vaccine efforts. Still, little is known regarding how frequently new viral variants emerge and under what circumstances. Based on this study, we estimate that in hospitalized COVID-19 cases, variants with multiple mutations may emerge locally in as little as 1 month, even in patients without overt immune deficiency. Surveillance by sequencing for continuously shedding patients, patients suspected of reinfection, and patients with diminished immune function may offer broad public health benefits.
Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of ...virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus BLT mice and T cell-only mice ToM) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice MoM) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.
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.
•IL10 and IL-6 drive KSHV-associated inflammatory cytokine syndrome (KICS).•High-level herpesvirus replication yields full-length genome sequences from routine clinical samples.•KICS and KSHV replication can occur independently of AIDS and HIV replication.
This study compared the mutation profile and tumor mutational burden (TMB) in women with HIV (WWH) diagnosed with lung adenocarcinoma (n = 8) or breast ductal neoplasm (n = 13) who were enrolled into ...the Women's Interagency HIV Study (WIHS).
Previous studies tended to focus on single institutions based on sample availability. This study is based on a representative, multicenter cohort that represents the racial and ethnic composition of women with HIV in the United States.
The study sequenced the complete human exome of n = 26 cancer samples from HIV-positive women, using Ion torrent next-generation sequencing. The study cohort was compared with a HIV-negative cohort obtained from the Genomic Data Commons Data Portal of the NCI.
There were no differences in known cancer mutations between breast cancer and lung cancer that developed in WWH and those that developed in HIV-negative (HIV-) women; however, WWH presented a significantly higher TMB in comparison to HIV- patients. Seventy-five percent of lung cancers and 61% of breast cancers were defined as TMB-high (more than 10 mutation/mb of DNA).
This study affirms the recommendation that WWH be included in clinical trials of novel treatments for these cancers. Although these data are preliminary, the high TMB in WLHV suggests, paradoxically, that this immune challenged population may benefit greatly from immune checkpoint inhibitor therapies.
Abstract
We describe 7 human immunodeficiency virus–infected Malawian children with Kaposi sarcoma who met criteria for Kaposi sarcoma herpesvirus (KSHV) inflammatory cytokine syndrome. Each ...presented with persistent fevers, bulky lymphadenopathy, massive hepatosplenomegaly, and severe cytopenias. Plasma analyses were performed in 2 patients, both demonstrating extreme elevations of KSHV viral load and interleukin 6.
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