Genetic and Molecular Characterization of the Immortalized Murine Hepatic Stellate Cell Line GRX.

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells.

A serum-stable RNA aptamer specific for SARS-CoV-2 neutralizes viral entry.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.

Septins are critical regulators of osteoclastic bone resorption.

Septins are known to play key roles in supporting cytoskeletal stability, vesicular transport, endo-/exocytosis, stabilizing cellular membranes and forming diffusion barriers. Their function in mammalian cells is poorly investigated. The osteoclast offers an interesting tool to investigate septins because all cellular activities septins were reported to be involved in are critical for osteoclasts. However, the existence of septins in osteoclasts has not even been reported. Here we show that the SEPT9 gene and Septin 9 (SEPT9) protein are expressed and synthesized during differentiation of human osteoclasts. Pharmacological stabilization of septin filaments dose dependently inhibits bone resorption of human osteoclasts in vitro suggesting a role for septins in bone resorption. Attesting to this, conditional deletion of Sept9 in mice leads to elevated levels of trabecular bone and diminished femoral growth in vivo. Finally, systematic interrogation of the spatial organization of SEPT9 by confocal microscopy reveals that SEPT9 is closely associated to the structures known to be critical for osteoclast activity. We propose that septins in general and SEPT9 in particular play a previously unappreciated role in osteoclastic bone resorption.

The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses.

Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Krüppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.

Anti-inflammatory effect of a retrovirus-derived immunosuppressive peptide in mouse models.

BACKGROUND: Short dimeric or mulitmeric peptides derived from a highly conserved stretch of amino acids from gammaretroviral envelope proteins has been found to have immunosuppressive properties in vitro. Here we test the hypothesis that such immunosuppressive peptides may serve as immunomodulatory reagents for treatment of inflammatory disorders. RESULTS: The anti-inflammatory effect of a synthetic retrovirus-derived immunosuppressive peptide of 17 amino acids was tested in two murine skin inflammation models, a TPA-induced acute toxic contact eczema model and an oxazolone-induced allergic contact dermatitis. Overall, mice (n = 24) treated with a topically applied cream containing the dimeric immunosuppressive peptide exhibited a reduction of 28.8% in ear thickness (range 20.1-42.5), whereas the application of a scrambled peptide dimer or a monomer of the immunosuppressive peptide remained without effect (p = 0.028). Furthermore, ear biopsies from mice treated with the dimeric immunosuppressive peptide showed a significant reduction in mRNA of the pro-inflammatory cytokines TNF-α, IL-17C, and IL-6 as well as the chemokine CXCL2 compared to mice treated with control peptides. CONCLUSION: Using two murine skin inflammation models, we show that an immunosuppressive retroviral peptide is capable of reducing inflammatory disorders. The results indicate that virus-derived immunosuppressive peptides capable of down-regulating several proinflammatory cytokines may represent a novel class of drugs for the treatment of excess inflammation.

Restriction genes for retroviruses influence the risk of multiple sclerosis.

We recently described that the autoimmune, central nervous system disease, multiple sclerosis (MS), is genetically associated with the human endogenous retroviral locus, HERV-Fc1, in Scandinavians. A number of dominant human genes encoding factors that restrict retrovirus replication have been known for a long time. Today human restriction genes for retroviruses include amongst others TRIMs, APOBEC3s, BST2 and TREXs. We have therefore looked for a role of these retroviral restriction genes in MS using genetic epidemiology. We here report that markers in two TRIMs, TRIM5 and TRIM22 and a marker in BST2, associated statistically with the risk of getting MS, while markers in or near APOBEC3s and TREXs showed little or no effect. This indicates that the two TRIMs and BST2 influence the risk of disease and thus supports the hypothesis of a viral involvement.

Endogenous retroviruses and multiple sclerosis-new pieces to the puzzle.

The possibility that retroviruses play a role in multiple sclerosis (MS) has long been considered; accumulating findings suggest this to be most likely in the form of human endogenous retroviruses (HERVs). A genetic test series of fifty endogenous retroviral loci for association with MS in Danes showed SNP markers near a specific endogenous retroviral locus, HERV-Fc1 located on the X-chromosome, to be positive. Bout Onset MS was associated with the HERV-Fc1 locus, while a rarer form, Primary Progressive MS, was not. Moreover, HERV-Fc1 Gag RNA in plasma was increased 4-fold in patients with recent history of attacks, relative to patients in a stable state and to healthy controls.Finally, genetic variations in restriction genes for retroviruses influence the risk of MS, providing further support for a role of retroviral elements in disease.We speculate that endogenous retroviruses may activate the innate immune system in a variety of ways, involving the host proteins, TRIMs, TLRs, TREXs and STING. Observations in HIV-positive patients suggest that antiretroviral drugs can curb MS. Thus, these new findings regarding the etiology and pathogenesis of MS, suggest alternative ways to challenge autoimmune diseases.

No additional copies of HERV-Fc1 in the germ line of multiple sclerosis patients.

BACKGROUND: Human endogenous retroviruses (HERVs) are suspected to play a role in the development of multiple sclerosis (MS). This suspicion has in part been based on increased expression of viral RNA or proteins or antibodies targeting retroviral products in MS patients. Recently, our group provided genetic evidence for association between the endogenous retrovirus HERV-Fc1 and MS, suggesting that HERV-Fc1 plays a role in this multifactorial disease. We have found increased expression of HERV-Fc1 in MS patients suffering from recent attack, but the underlying mechanism for association is still unknown. FINDINGS: Evidence from animal models indicates that ERV implication in the pathogenesis of diseases can be a result of extra copies of the virus in the germ line. Therefore, we investigated the genome of 81 individuals, 74 patients with MS and 7 healthy controls, by means of Southern blotting, for presence of extra HERV-Fc1 copies. The known insertion at the Xq21.33 position was readily detectable, but no additional insertions in other genomic contexts could be identified in any studied individuals. This substantiates our previous copy-number PCR findings of a 2:1 ratio of HERV-Fc1 DNA between women and men, as expected from the X-chromosome location; there was no difference between patient and control individuals. CONCLUSIONS: No additional germ line copies of HERV-Fc1 could be identified, precluding such copies to underlie the association between this provirus and multiples sclerosis.

Septin9 is involved in septin filament formation and cellular stability.

Septin9 (Sept9) is a member of the filament-forming septin family of structural proteins and is associated with a variety of cancers and with hereditary neuralgic amyotrophy. We have generated mice with constitutive and conditional Sept9 knockout alleles. Homozygous deletion of Sept9 results in embryonic lethality around day 10 of gestation whereas mice homozygous for the conditional allele develop normally. Here we report the consequences of homozygous loss of Sept9 in immortalized murine embryonic fibroblasts. Proliferation rate was not changed but cells without Sept9 had an altered morphology compared to normal cells, particularly under low serum stress. Abnormal, fragmented, and multiple nuclei were more frequent in cells without Sept9. Cell migration, as measured by gap-filling and filter-invasion assays, was impaired, but individual cells did not move less than wild-type cells. Sept9 knockout cells showed a reduced resistance to hypo-osmotic stress. Stress fiber and vinculin staining at focal adhesion points was less prominent. Long septin filaments stained for Sept7 disappeared. Instead, staining was found in short, often curved filaments and rings. Furthermore, Sept7 was no longer localized to the mitotic spindle. Together, these data reveal the importance of Sept9 for septin filament formation and general cell stability.

Induction of humoral and cellular immune responses against the HIV-1 envelope protein using γ-retroviral virus-like particles.

This study evaluates the immunogenicity of the HIV envelope protein (env) in mice presented either attached to γ-retroviral virus-like-particles (VLPs), associated with cell-derived microsomes or as solubilized recombinant protein (gp160). The magnitude and polyfunctionality of the cellular immune response was enhanced when delivering HIV env in the VLP or microsome form compared to recombinant gp160. Humoral responses measured by antibody titres were comparable across the groups and low levels of antibody neutralization were observed. Lastly, we identified stronger IgG2a class switching in the two particle-delivered antigen vaccinations modalities compared to recombinant gp160.

Infliximab induces clonal expansion of γδ-T cells in Crohn's disease: a predictor of lymphoma risk?

BACKGROUND: Concominant with the widespread use of combined immunotherapy in the management of Crohn's disease (CD), the incidence of hepato-splenic gamma-delta (γδ)-T cell lymphoma has increased sharply in CD patients. Malignant transformation of lymphocytes is believed to be a multistep process resulting in the selection of malignant γδ-T cell clones. We hypothesised that repeated infusion of anti-TNF-α agents may induce clonal selection and that concurrent treatment with immunomodulators further predisposes patients to γδ-T cell expansion. METHODOLOGY/PRINCIPAL FINDINGS: We investigated dynamic changes in the γδ-T cells of patient with CD following treatment with infliximab (Remicade®; n=20) or adalimumab (Humira®; n=26) using flow cytometry. In patients with a high γδ-T cell level, the γδ-T cells were assessed for clonality. Of these 46 CD patients, 35 had a γδ-T cells level (mean 1.6%) comparable to healthy individuals (mean 2.2%), and 11 CD patients (24%) exhibited an increased level of γδ-T cells (5-15%). In the 18 patients also receiving thiopurines or methotrexate, the average baseline γδ-T cell level was 4.4%. In three male CD patients with a high baseline value, the γδ-T cell population increased dramatically following infliximab therapy. A fourth male patient also on infliximab monotherapy presented with 20% γδ-T cells, which increased to 25% shortly after treatment and was 36% between infusions. Clonality studies revealed an oligoclonal γδ-T cell pattern with dominant γδ-T cell clones. In support of our clinical findings, in vitro experiments showed a dose-dependent proliferative effect of anti-TNF-α agents on γδ-T cells. CONCLUSION/SIGNIFICANCE: CD patients treated with immunomodulators had constitutively high levels of γδ-T cells. Infliximab exacerbated clonal γδ-T cell expansion in vivo and induced γδ-T cell proliferation in vitro. Overall, young, male CD patients with high baseline γδ-T cell levels may be at an increased risk of developing malignant γδ-T cell lymphomas following treatment with anti-TNF-α agents.

The etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus HERV-Fc1.

We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.

Expression level, tissue distribution pattern, and prognostic impact of vascular endothelial growth factors VEGF and VEGF-C and their receptors Flt-1, KDR, and Flt-4 in different subtypes of non-Hodgkin lymphomas.

The aim of the study was to investigate the expression of angio- and lymphangiogenic molecules (vascular endothelial growth factors VEGF and VEGF-C and their receptors Flt-1, KDR, and Flt-4) in non-Hodgkin lymphomas (NHL) treated in the pre-rituximab era. Pre-therapeutic lymph-node biopsies from 155 patients with NHL (64 follicular lymphomas (FLs), 47 de novo diffuse large B-cell lymphomas (DLBCL) and 44 peripheral T-cell lymphomas (PTCL)) were stained by in situ hybridization and immunohistochemistry. Tumor cell expression of VEGF, VEGF-C and their receptors was detected in most of the analyzed biopsies. In FL, diffuse intratumoral VEGF staining correlated with shorter overall survival (OS) (p = 0.008) and diffuse KDR staining was associated with a higher risk of histologic transformation (p = 0.05). In DLBCL, high KDR expression predicted poor treatment response (p = 0.03) and had a significant adverse impact on OS (p < 0.001). In PTCL, diffuse tissue distribution of VEGF mRNA correlated with an unfavorable 5-year OS (p = 0.004).

Shielding of sleeping beauty DNA transposon-delivered transgene cassettes by heterologous insulators in early embryonal cells.

The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector-containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5'-HS4 chicken beta-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer.

Extracellular matrix remodelling during cell adhesion monitored by the quartz crystal microbalance.

A cell's ability to remodel adsorbed protein layers on surfaces is influenced by the nature of the protein layer itself. Remodelling is often required to accomplish cellular adhesion and extracellular matrix formation which forms the basis for cell spreading, increased adhesion and expression of different phenotypes. The adhesion of NIH3T3 (EGFP) fibroblasts to serum protein (albumin or fibronectin) precoated tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces was examined using the quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy. The cells were either untreated or treated with cycloheximide to examine the contribution of endogenous protein production during cell adhesion to the QCM-D response over a period of 2h. Following adsorption of albumin onto Ta and PS(ox) there was no difference detected between the response to seeding untreated and cycloheximide treated cells. The QCM-D was able to detect differences in the untreated cellular responses to fibronectin versus serum precoated Ta and PS(ox) substrates, while cycloheximide treatment of the cells produced the same QCM-D response for fibronectin and serum precoatings on each of the materials. This confirmed that the process of matrix remodelling by the cells is dependent on the underlying substrate and the preadsorbed proteins and that the QCM-D response is dominated by changes in the underlying protein layer. Changes in dissipation correspond to the development of the actin cytoskeleton as visualised by actin staining.

Mutation of C/EBPalpha predisposes to the development of myeloid leukemia in a retroviral insertional mutagenesis screen.

The CCAAT enhancer binding protein alpha (C/EBPalpha) is an important myeloid tumor suppressor that is frequently mutated in human acute myeloid leukemia (AML). We have previously shown that mice homozygous for the E2F repression-deficient Cebpa(BRM2) allele develop nonfatal AML with long latency and incomplete penetrance, suggesting that accumulation of secondary mutations is necessary for disease progression. Here, we use SRS19-6-driven retroviral insertional mutagenesis to compare the phenotypes of leukemias arising in Cebpa(+/+), Cebpa(+/BRM2), and Cebpa(BRM2/BRM2) mice, with respect to disease type, latency of tumor development, and identity of the retroviral insertion sites (RISs). Both Cebpa(+/BRM2) and Cebpa(BRM2/BRM2) mice preferentially develop myeloid leukemias, but with differing latencies, thereby demonstrating the importance of gene dosage. Determination of RISs led to the identification of several novel candidate oncogenes, some of which may collaborate specifically with the E2F repression-deficient allele of Cebpa. Finally, we used an in silico pathway analysis approach to extract additional information from single RISs, leading to the identification of signaling pathways which were preferentially deregulated in a disease- and/or genotype-specific manner.

Cysteine 138 mutation in HIV-1 Nef from patients with delayed disease progression.

BACKGROUND: The nef gene from HIV-1 has been shown to be an important pathogenic factor when considering development of AIDS. Detection of nef variants with an effect on immune modulation is important to understand HIV-1 pathogenesis and has possible impact on treatment strategies. METHODS: The nef gene of HIV-1 isolates from patients in a long-term non-progressor (LTNP) cohort and a slow-progressor (SP) cohort (n = 11) was analysed and compared with isolates from a control patient group of progressors (n = 18). Most of the patients with delayed disease progression had extensive medical records, providing an insight into the LTNP disease profile and allowing for the stratification of patients based on their CD4 cell decline. RESULTS: In sequences from nine patients, most of the functional domains of HIV-1 Nef appeared intact, and no major deletions were observed to possibly account for an effect on the delayed disease status. However, the results demonstrate a high incidence of a single amino acid polymorphism (cysteine 138) in HIV-1 Nef. The allelic frequency of cysteine 138 between the delayed disease progression group and the progressor group was found to be statistically significant (P = 0.0139). The phylogeny of isolates was investigated and the variants harbouring the cysteine 138 mutation clustered independently. CONCLUSION: The present study describes a viral genetic polymorphism related to AIDS disease progression. The polymorphism (cysteine 138) has previously been reported to confer decreased viral replication (Premkumar DR, et al. AIDS Res Hum Retroviruses 1996; 12(4): 337-45). A sequence database search for comparative mutations revealed a high frequency of cysteine 138 in patients with reported SP AIDS.

Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors and cytokines can induce and maintain lineage-specific differentiation. Due to anatomical and physiological similarities to humans, porcine research models have been proven valuable for the preclinical testing of tissue engineering protocols in large animals. The aim of this study was to evaluate optimized viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells. MATERIALS AND METHODS: MSCs were purified from bone marrow aspirates from the proximal tibiae of four 3-month-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno-associated virus (rAAV)-mediated and retroviral gene delivery. Each method for gene delivery was optimized. Gene transfer efficiency was compared on the basis of eGFP expression as assessed by fluorescence microscopy and fluorescence-activated flow cytometry. BMP-2 gene expression and osteogenic differentiation were evaluated by realtime quantitative RT-PCR and histochemical detection of alkaline phosphatase activity, respectively. RESULTS: Non-viral gene delivery methods resulted in transient eGFP expression by less than 2% of the cells. Using high titer rAAV-based vector up to 90% of the cells were transiently transduced. The efficiency of rAAV-mediated gene delivery was proportional to the rAAV vector titer applied. Retroviral gene delivery resulted in long-term transgene expression of porcine MSCs. A 26-fold increase in percentage of eGFP expressing cells (1.7%+/-0.2% versus 44.1% +/-5.0%, mean +/-SD) and a 68-fold increase in mean fluorescence intensity (327.4+/-56.6 versus 4.8+/-1.3) was observed by centrifugation of retroviral particles onto the target cell layer. Porcine MSCs that were BMP-2 transduced by optimized retroviral gene delivery demonstrated a significant increase in BMP-2 gene expression and showed increased osteogenic differentiation. Retrovirally transduced porcine MSCs were furthermore tested free of replication-competent viruses. DISCUSSION: The non-viral gene transfer methods applied were significantly less efficient compared to the viral methods tested. However, due to advantages with respect to safety issues and ease of handling, improvement of non-viral gene delivery to primary MSCs deserves further attention. The high efficiency of rAAV-mediated gene delivery observed at high titers can be explained by the ability of rAAV vector to transduce nondividing cells and by its tropism towards porcine MSCs. rAAV-mediated gene delivery resulted in transient transgene expression due to lack of stable AAV genome integration. MLV-mediated retroviral gene delivery can be considered a safe method for long-term transgene expression by porcine MSCs, and is therefore particularly attractive for advanced tissue engineering strategies requiring extended transgene expression.