Delivery of Anticancer Drugs Using Microbubble-Assisted Ultrasound in a 3D Spheroid Model.

Tumor spheroids are promising three-dimensional (3D) in vitro tumor models for the evaluation of drug delivery methods. The design of noninvasive and targeted drug methods is required to improve the intratumoral bioavailability of chemotherapeutic drugs and reduce their adverse off-target effects. Among such methods, microbubble-assisted ultrasound (MB-assisted US) is an innovative modality for noninvasive targeted drug delivery. The aim of the present study is to evaluate the efficacy of this US modality for the delivery of bleomycin, doxorubicin, and irinotecan in colorectal cancer (CRC) spheroids. MB-assisted US permeabilized the CRC spheroids to propidium iodide, which was used as a drug model without affecting their growth and viability. Histological analysis and electron microscopy revealed that MB-assisted US affected only the peripheral layer of the CRC spheroids. The acoustically mediated bleomycin delivery induced a significant decrease in CRC spheroid growth in comparison to spheroids treated with bleomycin alone. However, this US modality did not improve the therapeutic efficacy of doxorubicin and irinotecan on CRC spheroids. In conclusion, this study demonstrates that tumor spheroids are a relevant approach to evaluate the efficacy of MB-assisted US for the delivery of chemotherapeutics.

Pathology Assessments of Multiple Organs in Fatal COVID-19 in Intensive Care Unit vs. Non-intensive Care Unit Patients.

Purpose: The objective of the present study was to provide a detailed histopathological description of fatal coronavirus disease 2019 (COVID 19), and compare the lesions in Intensive Care Unit (ICU) and non-ICU patients. Methods: In this prospective study we included adult patients who died in hospital after presenting with confirmed COVID-19. Multiorgan biopsies were performed. Data generated with light microscopy, transmission electron microscopy (TEM) and RT-PCR assays were reviewed. Results: 20 patients were enrolled in the study and the main pulmonary finding was alveolar damage, which was focal in 11 patients and diffuse in 8 patients. Chronic fibrotic and inflammatory lesions were observed in 18 cases, with acute inflammatory lesions in 12 cases. Diffuse lesions, collapsed alveoli and dystrophic pneumocytes were more frequent in the ICU group (62.5%, vs. 25%; 63%, vs. 55%; 87.5%, vs. 54%). Acute lesions (82%, vs. 37.5%; p = 0.07) with neutrophilic alveolitis (63.6% vs. 0%, respectively; p = 0.01) were observed more frequently in the non-ICU group. Viral RNA was detected in 12 lung biopsies (60%) up to 56 days after disease upset. TEM detected viral particles in the lung and kidney biopsy samples up to 27 days after disease upset. Furthermore, abundant networks of double-membrane vesicles (DMVs, a hallmark of viral replication) were observed in proximal tubular epithelial cells. Conclusion: Lung injury was different in ICU and non-ICU patients. Extrapulmonary damage consisting in kidney and myocardial injury were more frequent in ICU patients. Our TEM experiments provided the first description of SARS-CoV-2-induced DMVs in kidney biopsy samples-a sign of intense viral replication in this organ.

Crystalline podocytopathy and tubulopathy linked to kappa light chain deposits in a context of smoldering multiple myeloma.

A 42-year-old man with smoldering immunoglobulin G kappa multiple myeloma showed a heavy proteinuria composed of free light chain, prompting performance of a kidney biopsy. Electron microscopy revealed numerous rhomboid-shaped crystals labelled by the anti-kappa in immunogold, notably in the cytoplasm of podocytes, establishing the diagnosis of crystalline podocytopathy. This case illustrates a rare form of monoclonal gammopathy of renal significance, and highlights the key role of electron microscopy and immunogold to better elucidate the location and composition of crystals.

Hepatitis B Virus Entry into Cells.

Hepatitis B virus (HBV), an enveloped partially double-stranded DNA virus, is a widespread human pathogen responsible for more than 250 million chronic infections worldwide. Current therapeutic strategies cannot eradicate HBV due to the persistence of the viral genome in a special DNA structure (covalently closed circular DNA, cccDNA). The identification of sodium taurocholate co-transporting polypeptide (NTCP) as an entry receptor for both HBV and its satellite virus hepatitis delta virus (HDV) has led to great advances in our understanding of the life cycle of HBV, including the early steps of infection in particular. However, the mechanisms of HBV internalization and the host factors involved in this uptake remain unclear. Improvements in our understanding of HBV entry would facilitate the design of new therapeutic approaches targeting this stage and preventing the de novo infection of naïve hepatocytes. In this review, we provide an overview of current knowledge about the process of HBV internalization into cells.

Clinicopathologic features of infection-related glomerulonephritis with IgA deposits: a French Nationwide study.

BACKGROUND: Infection-related glomerulonephritis with IgA deposits (IRGN-IgA) is a rare disease but it is increasingly reported in the literature. Data regarding epidemiology and outcome are lacking, especially in Europe. We aimed to assess the clinical, pathologic and outcome data of IRGN-IgA. METHODS: Clinical and outcome data from patients from 11 French centers over the 2007-2017 period were collected retrospectively. We reviewed pathologic patterns and immunofluorescence of renal biopsies and evaluated C4d expression in IRGN-IgA. We analyzed the correlation between histological presentation and outcome. RESULTS: Twenty-seven patients (23 men, mean age: 62 ± 15 years) were included. Twenty-one (78%) had Staphylococcus aureus infection and twelve (44%) were diabetic. At the time of biopsy, 95.2% had haematuria, 48.1% had a serum creatinine level of > 4 mg/dL, and 16% had hypocomplementemia. The most common pathologic presentation included mesangial (88.9%) and endocapillary proliferative glomerulonephritis (88.9%) with interstitial fibrosis and tubular atrophy (IF/TA) (85.1%). Diffuse and global glomerular C4d expression was found in 17.8%, mostly in biopsies with acute or subacute patterns, and was associated with a short delay between infection and renal biopsy compared to segmental and focal staining. After median follow-up of 13.2 months, 23.1% died, 46.2% had persistent renal dysfunction and 15.4% reached end-stage renal disease. Renal outcome was correlated to IF/TA severity. CONCLUSIONS: Infection-related glomerulonephritis with IgA deposits is usually associated with Staphylococcus infections and mainly affects adult men. This entity has a poor prognosis which is correlated to interstitial fibrosis and tubular atrophy severity.

Hepatitis B virus entry into HepG2-NTCP cells requires clathrin-mediated endocytosis.

Hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with 250 million individuals chronically infected. Many stages of the HBV infectious cycle have been elucidated, but the mechanisms of HBV entry remain poorly understood. The identification of the sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor and the establishment of NTCP-overexpressing hepatoma cell lines susceptible to HBV infection opens up new possibilities for investigating these mechanisms. We used HepG2-NTCP cells, and various chemical inhibitors and RNA interference (RNAi) approaches to investigate the host cell factors involved in HBV entry. We found that HBV uptake into these cells was dependent on the actin cytoskeleton and did not involve macropinocytosis or caveolae-mediated endocytosis. Instead, entry occurred via the clathrin-mediated endocytosis pathway. HBV internalisation was inhibited by pitstop-2 treatment and RNA-mediated silencing (siRNA) of the clathrin heavy chain, adaptor protein AP-2 and dynamin-2. We were able to visualise HBV entry in clathrin-coated pits and vesicles by electron microscopy (EM) and cryo-EM with immunogold labelling. These data demonstrating that HBV uses a clathrin-mediated endocytosis pathway to enter HepG2-NTCP cells increase our understanding of the complete HBV life cycle.

BK Polyomavirus Hijacks Extracellular Vesicles for En Bloc Transmission.

Most people are asymptomatic carriers of the BK polyomavirus (BKPyV), but the mechanisms of persistence and immune evasion remain poorly understood. Furthermore, BKPyV is responsible for nephropathies in kidney transplant recipients. Unfortunately, the sole therapeutic option is to modulate immunosuppression, which increases the risk of transplant rejection. Using iodixanol density gradients, we observed that Vero and renal proximal tubular epithelial infected cells release two populations of infectious particles, one of which cosediments with extracellular vesicles (EVs). Electron microscopy confirmed that a single vesicle could traffic tens of viral particles. In contrast to naked virions, the EV-associated particles (eBKPyVs) were not able to agglutinate red blood cells and did not use cell surface sialylated glycans as an attachment factor, demonstrating that different entry pathways were involved for each type of infectious particle. However, we also observed that naked BKPyV and eBKPyV were equally sensitive to neutralization by the serum of a seropositive patient or commercially available polyvalent immunoglobulin preparations, which occurred at a postattachment step, after endocytosis. In conclusion, our work shows a new mechanism that likely plays a critical role during the primary infection and in the persistence, but also the reactivation, of BKPyV.IMPORTANCE Reactivation of BKPyV is responsible for nephropathies in kidney transplant recipients, which frequently lead to graft loss. The mechanisms of persistence and immune evasion used by this virus remain poorly understood, and a therapeutic option for transplant patients is still lacking. Here, we show that BKPyV can be released into EVs, enabling viral particles to infect cells using an alternative entry pathway. This provides a new view of BKPyV pathogenesis. Even though we did not find any decreased sensitivity to neutralizing antibodies when comparing EV-associated particles and naked virions, our study also raises important questions about developing prevention strategies based on the induction or administration of neutralizing antibodies. Deciphering this new release pathway could enable the identification of therapeutic targets to prevent BKPyV nephropathies. It could also lead to a better understanding of the pathophysiology of other polyomaviruses that are associated with human diseases.

Histogenesis of Merkel Cell Carcinoma: A Comprehensive Review.

Merkel cell carcinoma (MCC) is a primary neuroendocrine carcinoma of the skin. This neoplasia features aggressive behavior, resulting in a 5-year overall survival rate of 40%. In 2008, Feng et al. identified Merkel cell polyomavirus (MCPyV) integration into the host genome as the main event leading to MCC oncogenesis. However, despite identification of this crucial viral oncogenic trigger, the nature of the cell in which MCC oncogenesis occurs is actually unknown. In fact, several hypotheses have been proposed. Despite the large similarity in phenotype features between MCC tumor cells and physiological Merkel cells (MCs), a specialized subpopulation of the epidermis acting as mechanoreceptor of the skin, several points argue against the hypothesis that MCC derives directly from MCs. Alternatively, MCPyV integration could occur in another cell type and induce acquisition of an MC-like phenotype. Accordingly, an epithelial as well as a fibroblastic or B-cell origin of MCC has been proposed mainly based on phenotype similarities shared by MCC and these potential ancestries. The aim of this present review is to provide a comprehensive review of the current knowledge of the histogenesis of MCC.

The Hepatitis C Virus-Induced Membranous Web in Liver Tissue.

Host cell membrane rearrangements induced by the hepatitis C virus (HCV) have been exclusively studied in vitro. These studies have shown that HCV induces double-membrane vesicles (DMVs), which probably serve to separate replication sites from the cytoplasmic sensors of the innate immune response. We report for the first time the observation of HCV-induced membrane rearrangements in liver biopsy specimens from patients chronically infected with HCV. Unlike observations performed in vitro, the membranous web detected in liver tissue seems essentially made of clusters of single-membrane vesicles derived from the endoplasmic reticulum and close to lipid droplets. This suggests that the DMVs could be a hallmark of laboratory-adapted HCV strains, possibly due to their ability to achieve a high level of replication. Alternatively, the concealment of viral RNA in DMVs may be part of innate immune response mechanisms particularly developed in hepatoma cell lines cultured in vitro. In any case, this constitutes the first report showing the differences in the membranous web established by HCV in vitro and in vivo.

[An atypical case of lipoid proteinosis]. / Un cas atypique de protéinose lipoïde.

The lipoid proteinosis is a rare autosomic recessive genodermatosis characterized histologically by deposits of hyaline-like eosinophilic material of characteristic distribution. We herein report the case of a 56-year-old man admitted for progressive aggravated dementia associated with a late-onset dysphonia. Histologic examination of cutaneous and laryngeal biopsies showed deposits of an amorphous and eosinophilic material arranged around vessels, and adnexal structures, stained by PAS and congo red negative. The detection of a mutation in the ECM1 gene confirmed the diagnosis of lipoid proteinosis of atypical clinical presentation.

The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion.

Hepatitis C virus (HCV) assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD) surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER) membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis.

Sequence and functional analysis of the envelope glycoproteins of hepatitis C virus variants selectively transmitted to a new host.

Hepatitis C virus (HCV) remains a challenging public health problem worldwide. The identification of viral variants establishing de novo infections and definition of the phenotypic requirements for transmission would facilitate the design of preventive strategies. We explored the transmission of HCV variants in three cases of acute hepatitis following needlestick accidents. We used single-genome amplification of glycoprotein E1E2 gene sequences to map the genetic bottleneck upon transmission accurately. We found that infection was likely established by a single variant in two cases and six variants in the third case. Studies of donor samples showed that the transmitted variant E1E2 amino acid sequences were identical or closely related to those of variants from the donor virus populations. The transmitted variants harbored a common signature site at position 394, within hypervariable region 1 of E2, together with additional signature amino acids specific to each transmission pair. Surprisingly, these E1E2 variants conferred no greater capacity for entry than the E1E2 derived from nontransmitted variants in lentiviral pseudoparticle assays. Mutants escaping the antibodies of donor sera did not predominate among the transmitted variants either. The fitness parameters affecting the selective outgrowth of HCV variants after transmission in an immunocompetent host may thus be more complex than those suggested by mouse models. Human antibodies directed against HCV envelope effectively cross-neutralized the lentiviral particles bearing E1E2 derived from transmitted variants. These findings provide insight into the molecular mechanisms underlying HCV transmission and suggest that viral entry is a potential target for the prevention of HCV infection.

Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection.

Like most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses.

Ultrastructural and quantitative analysis of the lipid droplet clustering induced by hepatitis C virus core protein.

Hepatitis C virus (HCV) release is linked to the formation of lipid droplet (LD) clusters in the perinuclear area of infected cells, induced by the core protein. We used electron microscopy (EM) to monitor and compare the number and size of LD in cells producing the mature and immature forms of the HCV core protein, and 3D EM to reconstruct whole cells producing the mature core protein. Only the mature protein coated the LD and induced their clustering and emergence from endoplasmic reticulum membranes enriched in this protein. We found no particular association between LD clusters and the centrosome in reconstructed cells. The LD clustering induced by the mature core protein was associated with an increase in LD synthesis potentially due, at least in part, to the ability of this protein to coat the LD. These observations provide useful information for further studies of the mechanisms involved in HCV-induced steatosis.

Hepatitis C virus entry depends on clathrin-mediated endocytosis.

Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.

Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus.

Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported "membranous web." However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle.

High density lipoproteins facilitate hepatitis C virus entry through the scavenger receptor class B type I.

The scavenger receptor class B type I (SR-BI) has recently been shown to interact with hepatitis C virus (HCV) envelope glycoprotein E2, suggesting that it might be involved at some step of HCV entry into host cells. However, due to the absence of a cell culture system to efficiently amplify HCV, it is not clear how SR-BI contributes to HCV entry. Here, we sought to determine how high density lipoproteins (HDLs), the natural ligand of SR-BI, affect HCV entry. By using the recently described infectious HCV pseudotyped particles (HCVpps) that display functional E1E2 glycoprotein complexes, we showed that HDLs are able to markedly enhance HCVpp entry. We did not find any evidence of HDL association with HCVpps, suggesting that HCVpps do not enter into target cells using HDL as a carrier to bind to its receptor. Interestingly, lipid-free apoA-I and apoA-II, the major HDL apolipoproteins, were unable to enhance HCVpp infectivity. In addition, drugs inhibiting HDL cholesteryl transfer (block lipid transport (BLT)-2 and BLT-4) reduced HDL enhancement of HCVpp entry, suggesting a role for lipid transfer in facilitating HCVpp entry. Importantly, silencing of SR-BI expression in target cells by RNA interference markedly reduced HDL-mediated enhancement of HCVpp entry. Finally, enhancement of HCVpp entry was also suppressed when the SR-BI binding region on HCV glycoprotein E2 was deleted. Altogether, these data indicate that HDL-mediated enhancement of HCVpp entry involves a complex interplay between SR-BI, HDL, and HCV envelope glycoproteins, and they highlight the active role of HDLs in HCV entry.

Hepatitis C virus-like particle budding: role of the core protein and importance of its Asp111.

In the absence of a hepatitis C virus (HCV) culture system, the use of a Semliki Forest virus replicon expressing genes encoding HCV structural proteins that assemble into HCV-like particles provides an opportunity to study HCV morphogenesis. Using this system, we showed that the HCV core protein constitutes the budding apparatus of the virus and that its targeting to the endoplasmic reticulum by means of the signal sequence of E1 protein is essential for budding. In addition, the aspartic acid at position 111 in the HCV core protein sequence was found to be crucial for virus assembly, demonstrating the usefulness of this system for mapping amino acids critical to HCV morphogenesis.

Hepatitis C virus-like particle morphogenesis.

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.