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High-mobility group box 1 (HMGB1) is a protein that binds to DNA and participates in various cellular processes, including DNA repair, transcription, and inflammation. It is also associated with cancer progression and therapeutic resistance. Despite its known role in promoting tumor growth and immune evasion in the tumor microenvironment, the contribution of HMGB1 to the development of Kaposi’s sarcoma (KS) is not well understood. We investigated the effect of HMGB1 on KS pathogenesis using immortalized human endothelial cells infected with Kaposi’s sarcoma-associated human herpes virus (KSHV). Our results showed that a higher amount of HMGB1 was detected in the supernatant of KSHV-infected cells compared to that of mock-infected cells, indicating that KSHV infection induced the secretion of HMGB1 in human endothelial cells. By generating HMGB1 knockout clones from immortalized human endothelial cells using CRISPR/Cas9, we elucidated the role of HMGB1 in KSHV-infected endothelial cells. Our findings indicate that the absence of HMGB1 did not induce lytic replication in KSHV-infected cells, but the cell viability of KSHV-infected cells was decreased in both 2D and 3D cultures. Through the antibody array for cytokines and growth factors, CXCL5, PDGF-AA, G-CSF, Emmprin, IL-17A, and VEGF were found to be suppressed in HMGB1 KO KSHV-infected cells compared to the KSHV-infected wild-type control. Mechanistically, phosphorylation of p38 would be associated with transcriptional regulation of CXCL5, PDGF-A and VEGF. These observations suggest that HMGB1 may play a critical role in KS pathogenesis by regulating cytokine and growth factor secretion and emphasize its potential as a therapeutic target for KS by modulating the tumor microenvironment.

Extracellular vesicles (EVs) contain a variety of biomolecules and provide information about the cells that produce them. EVs from cancer cells found in urine can be used as biomarkers to detect cancer, enabling early diagnosis and treatment. The potential of alpha-2-macroglobulin (A2M) and clusterin (CLU) as novel diagnostic urinary EV (uEV) biomarkers for bladder cancer (BC) was demonstrated previously. To validate the diagnostic value of these proteins in uEVs in a large BC cohort, urine handling conditions before uEV isolation should be optimized during sample transportation from medical centers. In this study, we analyzed the uEV protein quantity, EV particle number, and uEV-A2M/CLU after urine storage at 20°C and 4°C for 0-6 days, each. A2M and CLU levels in uEVs were relatively stable when stored at 4°C for a maximum of three days and at 20°C for up to 24 h, with minimal impact on analysis results. Interestingly, pre-processing to remove debris and cells by centrifugation and filtration of urine did not show any beneficial effects on the preservation of protein biomarkers of uEVs during storage. Here, the importance of optimizing shipping conditions to minimize the impact of pre-analytical handling on the uEVs protein biomarkers was emphasized. These findings provide insights for the development of clinical protocols that use uEVs for diagnostic purposes.

Extracellular vesicles (EVs) derived from urine are promising tools for the diagnosis of urogenital cancers. Urinary EVs (uEVs) are considered potential biomarkers for bladder cancer (BC) because urine is in direct contact with the BC tumor microenvironment and thus reflects the current state of the disease. However, challenges associated with the effective isolation and analysis of uEVs complicate the clinical detection of uEV-associated protein biomarkers. Herein, we identified uEV-derived alpha-2-macroglobulin (a2M) as a novel diagnostic biomarker for BC through comparative analysis of uEVs obtained from patients with BC pre- and post-operation using an antibody array. Furthermore, enzyme-linked immunosorbent assay of uEVs isolated from patients with BC (n=60) and non-cancer control subjects (n=23) validated the significant upregulation of a2M expression in patient uEVs (p

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman disease. In studies of KSHV, efficient virus production and isolation are essential. Reactivation of KSHV can be initiated by treating latently infected cells with chemicals, such as 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. These chemicals have been used as tools to induce lytic replication and viral production in KSHV-producing cell lines. Dimethyl sulfoxide (DMSO) is an organosulfur compound that is frequently used as an aprotic solvent similar to water. In experiments exploring signaling pathways in KSHV-infected cells, DMSO treatment alone as a vehicle affected the lytic gene expression of KSHV. However, to the best of our knowledge, the effects of DMSO on KSHV-producing cells have not yet been reported. Therefore, in this study, we investigated whether DMSO could be used as a reagent to enhance viral production during lytic replication in KSHV-producing cells and assessed the underlying mechanisms. The effects of DMSO on KSHV production were analyzed in iSLK BAC16 cells, which have been widely used for recombinant KSHV production. We found that the production of KSHV virions was significantly increased by treatment with DMSO during the induction of lytic replication. Mechanistically, lytic genes of KSHV were enhanced by DMSO treatment, which was correlated with virion production. Additionally, DMSO induced the phosphorylation of JNK during lytic replication, and inhibition of JNK abolished the effects of DMSO on lytic replication and virion production. Our findings showed that additional treatment with DMSO during the induction of lytic replication significantly improved the yield of KSHV production.

Summary: Despite the worldwide effect of the coronavirus disease 2019 (COVID-19) pandemic, the underlying mechanisms of fatal viral pneumonia remain elusive. Here, we show that critical COVID-19 is associated with enhanced eosinophil-mediated inflammation when compared to non-critical cases. In addition, we confirm increased T helper (Th)2-biased adaptive immune responses, accompanying overt complement activation, in the critical group. Moreover, enhanced antibody responses and complement activation are associated with disease pathogenesis as evidenced by formation of immune complexes and membrane attack complexes in airways and vasculature of lung biopsies from six fatal cases, as well as by enhanced hallmark gene set signatures of Fcγ receptor (FcγR) signaling and complement activation in myeloid cells of respiratory specimens from critical COVID-19 patients. These results suggest that SARS-CoV-2 infection may drive specific innate immune responses, including eosinophil-mediated inflammation, and subsequent pulmonary pathogenesis via enhanced Th2-biased immune responses, which might be crucial drivers of critical disease in COVID-19 patients.

Vaginal dysbiosis, such as bacterial vaginosis (BV) and aerobic vaginitis (AV), is an important cause of premature birth in pregnant women. However, there is very little research on vaginal microbial distribution in AV compared to that in BV. This study aimed to analyze the composition of the vaginal microbiota of pregnant women with AV using microbial community analysis and identify the causative organism using each criterion of the AV scoring system. Also, we compared the quantification of aerobic bacteria using quantitative polymerase chain reaction (qPCR) and their relative abundances (RA) using metagenomics. This prospective case–control study included 228 pregnant Korean women from our previous study. A wet mount test was conducted on 159 women to diagnose AV using the AV scoring system. Vaginal samples were analyzed using metagenomics, Gram staining for Nugent score determination, conventional culture, and qPCR for Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae. The relative abundances (RAs) of eleven species showed significant differences among the three groups (Normal flora (NF), mild AV, and moderate AV). Three species including Lactobacillus crispatus were significantly lower in the AV groups than in the NF group, while eight species were higher in the AV groups, particularly moderate AV. The decrease in the RA of L. crispatus was common in three criteria of the AV scoring system (Lactobacillary, WBC, and background flora grades), while it did not show a significant difference among the three grade groups of the toxic leukocyte criterion. Also, the RAs of anaerobes, such as Gardnerella and Megasphaera, were higher in the AV groups, particularly moderate AV, while the RAs of aerobes were very low (RA < 0.01). Therefore, qPCR was performed for aerobes (Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae); however, their quantification did not show a higher level in the AV groups when compared to that in the NF group. Therefore, AV might be affected by the RA of Lactobacillus spp. and the main anaerobes, such as Gardnerella spp. Activation of leukocytes under specific conditions might convert them to toxic leukocytes, despite high levels of L. crispatus. Thus, the pathogenesis of AV can be evaluated under such conditions.

Purpose: CD81 is a prognostic biomarker for high-grade bladder cancer (BC). In this study, we aimed to determine the functional mechanisms underlying the role of CD81 in BC progression. Materials and Methods: In two invasive BC cell lines (T24, J82), CD81 expression was suppressed by the transfection of lentiviral vectors including CD81-specific shRNAs, and then the migration and invasion of BC cells was analyzed. Enzymatic activity of matrix metalloproteinases (MMPs) was also analyzed by collagen-zymography. The expression of MMPs was confirmed by western blotting using culture supernatants from each cell line. Signaling pathways related to MMPs were investigated using various antibodies. Results: CD81 was successfully knocked down by shRNAs in T24 and J82 cell lines. While the migration of BC cells was not affected after the knockdown of CD81, the invasive activity was significantly increased in both cell lines. Zymography produced distinct bands using supernatants from CD81-knockdown cells, whereas only faint bands were observed with empty vector-transfected cells. We also observed an increased expression of MMPs, specifically MMP2 and 9, in the conditioned media from CD81-knockdown cells by western blotting. Mechanistically, the phosphorylation of extracellular signal-regulated kinase (ERK) was associated with the invasive activity of BC cells, while U0126 (an ERK inhibitor) reduced the invasive activity of CD81-knockdown BC cells. Conclusions: Taken together, CD81 suppression promotes the invasive property of BC cells through MMP signaling via ERK phosphorylation. Our results suggest that the regulation of CD81 expression may have some therapeutic potential in BC.

S100A8 and S100A9 function as essential factors in inflammation and also exert antitumor or tumorigenic activity depending on the type of cancer. Chronic eosinophilic leukemia (CEL) is a rare hematological malignancy having elevated levels of eosinophils and characterized by the presence of the FIP1L1-PDGFRA fusion gene. In this study, we examined the pro-apoptotic mechanisms of S100A8 and S100A9 in FIP1L1-PDGFRα+ eosinophilic cells and hypereosinophilic patient cells. S100A8 and S100A9 induce apoptosis of the FIP1L1-PDGFRα+ EoL-1 cells via TLR4. The surface TLR4 expression increased after exposure to S100A8 and S100A9 although total TLR4 expression decreased. S100A8 and S100A9 suppressed the FIP1L1-PDGFRα-mediated signaling pathway by downregulating FIP1L1-PDGFRα mRNA and protein expression and triggered cell apoptosis by regulating caspase 9/3 pathway and Bcl family proteins. S100A8 and S100A9 also induced apoptosis of imatinib-resistant EoL-1 cells (EoL-1-IR). S100A8 and S100A9 blocked tumor progression of xenografted EoL-1 and EoL-1-IR cells in NOD-SCID mice and evoked apoptosis of eosinophils derived from hypereosinophilic syndrome as well as chronic eosinophilic leukemia. These findings may contribute to a progressive understanding of S100A8 and S100A9 in the pathogenic and therapeutic mechanism of hematological malignancy.

In recent years, porcine circovirus type 2d (PCV2d) has achieved a dominant position worldwide. Various PCV2d capsid-based vaccines have been used to alleviate concerns regarding the emergence of the variant. This study aimed to determine the dosage of recombinant PCV2d capsid protein to induce protective efficacy against experimental challenge with a virulent PCV2d strain. Conventional 3-week-old pigs were intramuscularly inoculated with different doses of the protein (60, 20, 10 and 2 µg). Four weeks after vaccination, all pigs were challenged with pathogenic PCV2d (SNU140003), which was isolated from a farm severely experiencing PCV2-associated disease in Korea. Vaccination with greater than 10 µg of the capsid protein caused a significant (p < 0.05) reduction in PCV2d viremia, lymphoid lesions and lymphoid PCV2 antigen levels in vaccinated challenged pigs compared to unvaccinated challenged pigs. The vaccination also resulted in significantly higher (p < 0.05) titers of neutralizing antibodies against PCV2d. However, the pigs vaccinated with 2 µg had significantly lower neutralizing antibody titers than the other vaccinated groups. They showed a similar level of challenged PCV2d in serum and lymphoid lesion score compared to unvaccinated challenged pigs. The difference in efficacy among the vaccinated groups indicates that there may be a baseline dosage to induce sufficient neutralizing antibodies to prevent viral replication in pigs. In conclusion, at least 10 µg dosage of capsid protein is essential for stable protective efficacy against PCV2d in a pig model.

Extracellular vesicles (EVs) are small vesicles secreted from cells. They have crucial biological functions in intercellular communications and may even be biomarkers for cancer. The various methods used to isolate EVs from body fluid and cell culture supernatant have been compared in prior studies, which determined that the component yield and physical properties of isolated EVs depend largely on the isolation method used. Several novel and combined methods have been recently developed, which have not yet been compared to the established methods. Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed. We investigated the properties of EVs isolated from non-infected and Kaposi's sarcoma-associated herpesvirus-infected human umbilical vein endothelial cells using each method and determined the yields of DNA, RNA, and proteins using quantitative polymerase chain reaction and bicinchoninic acid assays. Additionally, we determined whether the biological activity of EVs correlated with the quantity or physical properties of the EVs isolated using different methods. We found that Exodisc was the most suitable method for obtaining large quantities of EVs, which might be useful for biomarker investigations, and that the EVs separated using Exodisc exhibited the highest complement activation activity. However, we also found that the functional properties of EVs were best maintained when differential centrifugation was used. Effective isolation is necessary to study EVs as tools for diagnosing cancer and our findings may have relevant implications in the field of oncology by providing researchers with data to assist their selection of a suitable isolation method.

Kaposi's Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, which is the most common cancer in acquired immune deficiency syndrome patients. KSHV contains a variety of immunoregulatory proteins. There have been many studies on the modulation of antiviral response by these immunoregulatory proteins of KSHV. However, the antiviral effects of extracellular vesicles (EVs) during de novo KSHV infection have not been investigated to our best knowledge. In this study, we showed that KSHV-infected cells induce interferon-stimulated genes (ISGs) response but not type I interferon in uninfected bystander cells using EVs. mRNA microarray analysis showed that ISGs and IRF-activating genes were prominently activated in EVs from KSHV-infected cells (KSHV EVs)-treated human endothelial cells, which were validated by RT-qPCR and western blot analysis. We also found that this response was not associated with cell death or apoptosis by virus infection. Mechanistically, the cGAS-STING pathway was linked with these KSHV EVs-mediated ISGs expressions, and mitochondrial DNA on the surface of KSHV EVs was one of the causative factors. Besides, KSHV EVs-treated cells showed lower infectivity for KSHV and viral replication activity than mock EVs-treated cells. Our results indicate that EVs from KSHV-infected cells could be an initiating factor for the innate immune response against viral infection, which may be critical to understanding the microenvironment of virus-infected cells.

Kaposi’s sarcoma-associated herpesvirus (KSHV) modulates the immune response to allow the virus to establish persistent infection in the host and facilitate the development of KSHV-associated cancer. The complement system has a central role in the defense against pathogens. Hence, KSHV has adopted an evasion strategy for complement attack using the viral protein encoded by KSHV open reading frame 4. However, despite this defense mechanism, the complement system appears to become activated in KSHV-infected cells as well as in the region surrounding Kaposi’s sarcoma tumors. Given that the complement system can affect cell fate as well as the inflammatory microenvironment, complement activation is likely associated with KSHV pathogenesis. A better understanding of the interplay between KSHV and the complement system may, therefore, translate into the development of novel therapeutic interventions for KSHV-associated tumors. In this review, the mechanisms and functions of complement activation in KSHV-infected cells are discussed.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

ABSTRACT Kaposi’s sarcoma (KS), a highly angiogenic and invasive tumor often involving different organ sites, including the oral cavity, is caused by infection with Kaposi’s sarcoma-associated herpesvirus (KSHV). Diverse cell markers have been identified on KS tumor cells, but their origin remains an enigma. We previously showed that KSHV could efficiently infect, transform, and reprogram rat primary mesenchymal stem cells (MSCs) into KS-like tumor cells. In this study, we showed that human primary MSCs derived from diverse organs, including bone marrow (MSCbm), adipose tissue (MSCa), dental pulp, gingiva tissue (GMSC), and exfoliated deciduous teeth, were permissive to KSHV infection. We successfully established long-term cultures of KSHV-infected MSCa, MSCbm, and GMSC (LTC-KMSCs). While LTC-KMSCs had lower proliferation rates than the uninfected cells, they expressed mixtures of KS markers and displayed differential angiogenic, invasive, and transforming phenotypes. Genetic analysis identified KSHV-derived microRNAs that mediated KSHV-induced angiogenic activity by activating the AKT pathway. These results indicated that human MSCs could be the KSHV target cells in vivo and established valid models for delineating the mechanism of KSHV infection, replication, and malignant transformation in biologically relevant cell types. IMPORTANCE Kaposi’s sarcoma is the most common cancer in AIDS patients. While KSHV infection is required for the development of Kaposi’s sarcoma, the origin of KSHV target cells remains unclear. We show that KSHV can efficiently infect human primary mesenchymal stem cells of diverse origins and reprogram them to acquire various degrees of Kaposi’s sarcoma-like cell makers and angiogenic, invasive, and transforming phenotypes. These results indicate that human mesenchymal stem cells might be the KSHV target cells and establish models for delineating the mechanism of KSHV-induced malignant transformation.

High-grade (HG) bladder cancers (BCs) are genetically unstable and have an unpredictable course. The identification of prognostic factors in HG non-muscle invasive BC (NMIBC) is crucial for improving patients' quality of life and preventing BC-specific mortality. Here, we used an antibody microarray (AbM) to identify novel candidate biomarkers in primary HG NMIBC and validated the prognostic significance of the candidate biomarkers. Three pairs of tissue samples from primary HG NMIBC and normal urothelium were analyzed using an AbM kit containing 656 antibodies, and differentially expressed proteins were identified. Among the 42 upregulated and 14 downregulated proteins with statistical significance in BC tissues, CREB-binding protein and CD81 were selected as representative upregulated and downregulated candidate biomarkers, respectively. We then validated the expression of these candidate biomarkers in primary human urothelial cells and BC cell lines by western blotting and immunofluorescence assays, and the results were consistent with the AbM expression profiles. Additionally, Kaplan-Meier survival using immunohistochemical data from an independent primary HG NMIBC cohort comprising 113 patients showed that expression of the 2 biomarkers was significantly associated with recurrence-free and progression-free survival. In multivariate analysis, the 2 biomarkers remained significant predictors for recurrence-free survival. Taken together, our findings suggest that expression of CREB-binding protein and CD81 in BC tissue specimens may have prognostic value in patients with primary HG NMIBC.

During evolution, herpesviruses have developed numerous, and often very ingenious, strategies to counteract efficient host immunity. Specifically, Kaposi's sarcoma-associated herpesvirus (KSHV) eludes host immunity by undergoing a dormant stage, called latency wherein it expresses a minimal number of viral proteins to evade host immune activation. Here, we show that during latency, KSHV hijacks the complement pathway to promote cell survival. We detected strong deposition of complement membrane attack complex C5b-9 and the complement component C3 activated product C3b on Kaposi's sarcoma spindle tumor cells, and on human endothelial cells latently infected by KSHV, TIME-KSHV and TIVE-LTC, but not on their respective uninfected control cells, TIME and TIVE. We further showed that complement activation in latently KSHV-infected cells was mediated by the alternative complement pathway through down-regulation of cell surface complement regulatory proteins CD55 and CD59. Interestingly, complement activation caused minimal cell death but promoted the survival of latently KSHV-infected cells grown in medium depleted of growth factors. We found that complement activation increased STAT3 tyrosine phosphorylation (Y705) of KSHV-infected cells, which was required for the enhanced cell survival. Furthermore, overexpression of either CD55 or CD59 in latently KSHV-infected cells was sufficient to inhibit complement activation, prevent STAT3 Y705 phosphorylation and abolish the enhanced survival of cells cultured in growth factor-depleted condition. Together, these results demonstrate a novel mechanism by which an oncogenic virus subverts and exploits the host innate immune system to promote viral persistent infection.

We present a rare case of urothelial carcinoma in situ (CIS), which invades the prostate and seminal vesicle (SV). A 70-year-old man underwent transurethral resection of bladder (TURB), and the pathologic examination revealed multiple CIS. Although the patient received intravesical bacillus Calmette-Guerin (BCG) therapy following TURB, recurrence of CIS was confirmed in the bladder and left distal ureter at 3 months following BCG. Radical cystectomy was performed due to BCG-refractory CIS. Microscopically, CIS was found throughout the mucosa of the bladder, left ureter, prostatic duct, and both SVs. Next-generation sequencing revealed significant differences in tumor clonality between bladder and SV CIS cells. Among 101 (bladder CIS) and 95 (SV CIS) somatic mutations, only two were shared, and only one gene (ARHGAP23) was common exon coding region gene. In conclusion, multicentric genetic changes, in line with the field-cancerization effect, may result in SV involvement by CIS of the bladder. [ABSTRACT FROM AUTHOR]

The demyelinating diseases of the central nervous system involve myelin abnormalities, oligodendrocyte damage, and consequent glia activation. Neurotoxicant cuprizone (CPZ) was used to establish a mouse model of demyelination. However, the effects of CPZ on microRNA (miRNA) expression and behavior have not been clearly reported. We analyzed the behavior of mice administered a diet containing 0.2% CPZ for 6 weeks, followed by 6 weeks of recovery. Rotarod analysis demonstrated that the treated group had poorer motor coordination than control animals. This effect was reversed after 6 weeks of CPZ withdrawal. Open-field tests showed that CPZ-treated mice exhibited significantly increased anxiety and decreased exploratory behavior. CPZ-induced demyelination was observed to be alleviated after 4 weeks of CPZ treatment, according to luxol fast blue (LFB) staining and myelin basic protein (MBP) expression. miRNA expression profiling showed that the expression of 240 miRNAs was significantly changed in CPZ-fed mice compared with controls. Furthermore, miR-155-5p and miR-20a-5p upregulations enhanced NgR induction through Smad 2 and Smad 4 suppression in demyelination. Taken together, our results demonstrate that CPZ-mediated demyelination induces behavioral deficits with apparent alterations in miRNA expression, suggesting that differences in miRNA expression in vivo may be new potential therapeutic targets for remyelination. [ABSTRACT FROM AUTHOR]

Abstract  Botulinum neurotoxin serotype B (BoNT/B)-specific Fab was expressed in a cell-free protein synthesis system derived from an E. coli extract. The cell-free synthesized antibody fragment was found to be effective in neutralizing the toxicity of BoNT/B in animal studies. Expression of functional Fab required an appropriately controlled and stably maintained redox potential. Under an optimized redox condition, the cell extract, whose disulfide reducing activity had been exhausted, could generate bio-functional Fab molecules. Use of a cell extract enriched with molecular chaperones (GroEL/ES) and disulfide bond isomerases were effective in obtaining larger quantities of functional Fab. Under the optimized reaction conditions, approximately 30 μg of functional Fab was obtained after purification from 1 mL reaction mixture. [ABSTRACT FROM AUTHOR]

A monoclonal antibody to the light chain of botulinum neurotoxin type B (BoNT/B) was generated and its protective activity was evaluated in vivo. A chimeric rabbit/human Fab library was generated using bone marrow and spleen cDNAs of rabbits immunized with the BoNT/B light chain, and three monoclonal antibodies specific to the catalytic domain of BoNT/B were isolated. One of these clones, BCXRH1, was specific to a conformation-dependent epitope, and partially neutralized the BoNT/B complex in vivo. [ABSTRACT FROM AUTHOR]

Objective: Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus implicated in the development of Kaposi’s sarcoma, primary effusion lymphoma and some forms of multicentric Castleman’s disease. The KSHV open reading frame (ORF) 36 encodes a viral serine/threonine protein kinase. The aim of this study was to characterize the cellular function of the ORF36 protein. Methods: The expression kinetics of the ORF36 protein and its localization were determined. The wild-type ORF36 and its mutant proteins were subjected to in vitro kinase assay. Cell morphology change by ORF36 protein was studied. The focal adhesion kinase (FAK) tyrosine phosphorylation and cleavage were determined when ORF36 was expressed. Results: ORF36 protein expressed in the late phase during the KSHV reactivation. The C-terminal domain of ORF36 protein was important for kinase activity. Moreover, the ORF36 protein altered cell morphology to a round shape, similar to the phenotype of FAK-deficient cells. The kinase activity of ORF36 protein was required for the inhibition of cell spreading. Interestingly, ORF36 protein colocalized with FAK, suppressed its tyrosine phosphorylation and promoted FAK cleavage. Conclusion: Our results collectively demonstrate that the KSHV ORF36 protein is a viral protein kinase that inhibits cell spreading and FAK activation. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The World Health Organization declared that a new strain of novel swine-origin influenza A (H1N1) virus was responsible for the pandemic infection in June 2009. We report a case of encephalitis diagnosed as the H1N1 virus infection. We describe a 17-year-old patient who had a seizure attack, diagnosed with a H1N1 virus infection via real time reverse-transcriptase polymerase chain reaction (RT-PCR). The H1N1 virus infection can be causative of the encephalitis, as with other influenza virus infections. Careful monitoring is essential for reducing complications. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma. The open reading frame (K9) of KSHV encodes viral interferon regulatory factor 1 (vIRF1), which functions as a repressor of interferon-mediated signal transduction. The amino-terminal region of vIRF1 displays significant homology to the DNA-binding domain of cellular interferon regulatory factors, supporting the theory that the protein interacts with specific DNA sequences. Here, we identify the consensus sequence of vIRF1-binding sites from a pool of random oligonucleotides. Moreover, our data show that vIRF1 interacts with the K3:viral dihydrofolate reductase:viral interleukin 6 promoter region in the KSHV genome. [ABSTRACT FROM AUTHOR]

Multiple host proteins affect the gene expression of Kaposi's sarcomaassociated herpesvirus (KSHV) during latent and lytic replication. High-mobility group box 1 (HMGB1) serves as a highly conserved chromosomal protein inside the cell and a prototypical damage-associated molecular pattern molecule outside the cell. HMGB1 has been shown to play a pathogenic role in viral infectious diseases and to regulate the lytic replication of KSHV. However, its functional effects on the KSHV life cycle in KSHV-infected cells have not been fully elucidated. Here, we explored the role of intracellular and extracellular HMGB1 in KSHV virion production by employing CRISPR/Cas9-mediated HMGB1 knockout in the KSHV-producing iSLK BAC16 cell line. Intracellular HMGB1 formed complexes with various proteins, and the abundance of HMGB1-interacting proteins changed during latent and lytic replication. Moreover, extracellular HMGB1 was found to enhance lytic replication by phosphorylating JNK. Of note, the expression of viral genes was attenuated during lytic replication in HMGB1 knockout iSLK BAC16 cells, with significantly decreased production of infectious virions compared to that of wild-type cells. Collectively, our results demonstrate that HMGB1 is an important cellular cofactor that affects the generation of infectious KSHV progeny during lytic replication. [ABSTRACT FROM AUTHOR]

Neisseria elongata, a common oral bacterium, has been recognized as a cause of infections such as infective endocarditis, septicemia, and osteomyelitis. Neisseria-induced infective endocarditis, although infrequently reported, typically arises after dental procedures. Without antibiotic therapy, its complications can be severe. We report the case of a 27-year-old man who presented with fever, severe dyspnea, and a leg abscess from cellulitis. An echocardiogram showed a vegetation-like echogenic structure on the septal leaflet of the patient's native tricuspid valve, and an insignificant Gerbode defect. Three blood cultures grew gram-negative, antibiotic-susceptible coccobacilli that were confirmed to be N. elongata. Subsequent DNA sequencing conclusively isolated N. elongata subsp nitroreducens as the organism responsible for the infective endocarditis. The patient recovered after 21 days of antibiotic therapy. In addition to the patient's unusual case, we discuss the nature and isolation of N. elongata and its subspecies. [ABSTRACT FROM AUTHOR]