A Unique Case of Supernumerary Teeth Erupting Inside a Maxillary Sinus Osteoma.

Introduction: Ectopic foreign bodies in the maxillary sinus occur rarely. Ectopic tooth eruption rarely occurs in the orbit, nasal cavity, maxillary sinus, and elsewhere. Ectopic eruption of teeth in the maxillary sinus is most commonly associated with wisdom teeth and is rarely associated with supernumerary teeth. This rare phenomenon may be accompanied by chronic recurrent sinusitis with headaches and facial pain. However, fibro-osseous lesions in the paranasal sinuses are discovered incidentally on X-ray images and are often asymptomatic. Osteoma is the most common fibro-osseous lesion that develops in the paranasal and nasal sinuses. Osteomas rarely cause serious symptoms such as orbital lesions and intracranial invasion. Case Presentation: We report a rare case of exostosis containing supernumerary teeth within the maxillary sinus. A characteristic pedicled bone lesion with a clear border on computed tomography was the undefined orthopantomogram radiopacity in the maxillary sinus, and the lesion contained supernumerary teeth. As the patient had chronic nasal congestion, the tumor was surgically removed. Pathologically, the surgical specimen revealed an osteoma. The patient's symptoms of chronic sinusitis disappeared. Because the patient had no history of midface trauma or surgery, the supernumerary teeth were speculated to have migrated during a reactive osteogenic process caused by chronic sinusitis. Conclusions: A foreign body in the maxillary sinus can be easily diagnosed by computed tomography. Surgical removal is recommended if the foreign body is symptomatic or occupies more than half of the maxillary sinus. This can help resolve chronic sinusitis symptoms and prevent serious complications in the future.

Metabolomic profiling of cancer-related fatigue involved in cachexia and chemotherapy.

Patients with advanced cancer are frequently burdened with a severe sensation of fatigue called cancer-related fatigue (CRF). CRF is induced at various stages and treatments, such as cachexia and chemotherapy, and reduces the overall survival of patients. Objective and quantitative assessment of CRF could contribute to the diagnosis and prediction of treatment efficacy. However, such studies have not been intensively performed, particularly regarding metabolic profiles. Here, we conducted plasma metabolomics of 15 patients with urological cancer. The patients with and without fatigue, including those with cachexia or chemotherapy-induced fatigue, were compared. Significantly lower concentrations of valine and tryptophan were observed in fatigued patients than in non-fatigued patients. In addition, significantly higher concentrations of polyamine pathway metabolites were observed in patients with fatigue and cachexia than in those without cachexia. Patients with exacerbated fatigue due to chemotherapy showed significantly decreased cysteine and methionine metabolism before chemotherapy compared with those without fatigue exacerbation. These findings suggest that plasma metabolic profiles could help improve the diagnosis and monitoring of CRF.

Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.

Morphological Evidence for Novel Roles of Microtubules in Macrophage Phagocytosis.

Although the phagocytic activity of macrophages has long been studied, the involvement of microtubules in the process is not well understood. In this study, we improved the fixation protocol and revealed a dynamically rearranging microtubule network in macrophages, consisting of a basal meshwork, thick bundles at the cell edge, and astral microtubules. Some astral microtubules extended beneath the cell cortex and continued to form bundles at the cell edge. These microtubule assemblies were mutually exclusive of actin accumulation during membrane ruffling. Although the stabilization of microtubules with paclitaxel did not affect the resting stage of the macrophages, it reduced the phagocytic activity and membrane ruffling of macrophages activated with serum-MAF, which induced rapid phagocytosis. In contrast, the destabilization of microtubules with nocodazole enhanced membrane ruffling and the internalization of phagocytic targets suggesting an inhibitory effect of the microtubule network on the remodeling of the actin network. Meanwhile, the microtubule network was necessary for phagosome maturation. Our detailed analyses of cytoskeletal filaments suggest a phagocytosis control system involving Ca2+ influx, the destabilization of microtubules, and activation of actin network remodeling, followed by the translocation and acidification of phagosomes on the microtubule bundles.

Complete response to second-line chemotherapy with sunitinib of a gastrointestinal stromal tumor: A case report.

Gastrointestinal stromal tumors (GISTs) are a type of sarcoma, and the most common mesenchymal tumor of the gastrointestinal tract. Systemic chemotherapy is recommended for unresectable or metastatic GISTs. Imatinib is an oral multitargeted receptor tyrosine kinase inhibitor that is effective as adjuvant chemotherapy for primary high-risk cases, and as palliative chemotherapy for unresectable or metastatic cases. For imatinib-resistant cases, second-line chemotherapy with sunitinib is recommended due to significantly longer median progression-free survival and higher response rates compared with a placebo. A 54-year-old woman presented with persistent upper abdominal pain and anorexia. An upper gastrointestinal endoscopy and computed tomography revealed a submucosal tumor of the stomach with no apparent metastases. The patient underwent total radical gastrectomy, and was diagnosed histologically with high-risk GIST for recurrence, therefore, the patient received adjuvant chemotherapy with imatinib. However, multiple liver and lymph node metastases were detected, and the patient received sunitinib therapy. After four cycles of sunitinib, the liver and lymph node metastases disappeared, and a complete response (CR) was achieved. To date, there have been no cases of CR in the prospective clinical trials examining the effects of sunitinib, or in case reports worldwide. Therefore, this is a very rare case report of a patient with metastatic GISTs who achieved CR with sunitinib as second-line chemotherapy.

Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae.

BACKGROUND: Yeast is recognized as a generally safe microorganism and is utilized for the production of pharmaceutical products, including vaccines. We previously showed that expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts released Gag virus-like particles (VLPs) extracellularly, suggesting that the production system could be used in vaccine development. In this study, we further establish HIV-1 genome packaging into Gag VLPs in a yeast cell system. RESULTS: The nearly full-length HIV-1 genome containing the entire 5' long terminal repeat, U3-R-U5, did not transcribe gag mRNA in yeast. Co-expression of HIV-1 Tat, a transcription activator, did not support the transcription. When the HIV-1 promoter U3 was replaced with the promoter for the yeast glyceraldehyde-3-phosphate dehydrogenase gene, gag mRNA transcription was restored, but no Gag protein expression was observed. Co-expression of HIV-1 Rev, a factor that facilitates nuclear export of gag mRNA, did not support the protein synthesis. Progressive deletions of R-U5 and its downstream stem-loop-rich region (SL) to the gag start ATG codon restored Gag protein expression, suggesting that a highly structured noncoding RNA generated from the R-U5-SL region had an inhibitory effect on gag mRNA translation. When a plasmid containing the HIV-1 genome with the R-U5-SL region was coexpressed with an expression plasmid for Gag protein, the HIV-1 genomic RNA was transcribed and incorporated into Gag VLPs formed by Gag protein assembly, indicative of the trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system. The concentration of HIV-1 genomic RNA in Gag VLPs released from yeast was approximately 500-fold higher than that in yeast cytoplasm. The deletion of R-U5 to the gag gene resulted in the failure of HIV-1 RNA packaging into Gag VLPs, indicating that the packaging signal of HIV-1 genomic RNA present in the R-U5 to gag region functions similarly in yeast cells. CONCLUSIONS: Our data indicate that selective trans-packaging of HIV-1 genomic RNA into Gag VLPs occurs in a yeast cell system, analogous to a mammalian cell system, suggesting that yeast may provide an alternative packaging system for lentiviral RNA.

Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment.

BACKGROUND: It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV. RESULTS: We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-alpha stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-alpha, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane. CONCLUSION: Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.

Dominant negative inhibition of human immunodeficiency virus particle production by the nonmyristoylated form of gag.

Myristoylation of human immunodeficiency virus (HIV) Gag protein is essential for membrane targeting of Gag and production of viral particles. We show here that coexpression of wild-type and nonmyristoylated forms of HIV Gag resulted in severe inhibition of viral particle production, indicating that the nonmyristoylated counterpart had a dominant negative effect on particle release. When coexpressed, the nonmyristoylated Gag partially incorporated into membrane and lipid raft fractions, likely through coassembly with the wild-type Gag. The membrane and raft associations of the wild-type Gag appeared unaffected, and yet particle production was severely impaired. When viral particles produced from the coexpressing cells were analyzed, the wild-type Gag was more abundant than the nonmyristoylated Gag. Confocal microscopy showed that both forms of Gag were diffusely distributed in the cytoplasm of coexpressing cells but that a portion of the wild-type Gag population was accumulated in EEA1- and CD63-positive endosomes. The intracellular accumulation of Gag was more frequently observed at late time points. The Gag accumulation was also observed on the cell surface protrusion. Electron microscopy of the coexpressing cells revealed budding arrest phenotypes, including the occurrence of interconnected virions on the plasma membrane, and intracellular budding. We also show that the inhibition of particle production and the Gag accumulation to endosomes were suppressed when the nucleocapsid (NC) domain was deleted from the nonmyristoylated Gag, although the NC-deleted Gag was still capable of coassembly. Overall, our data indicate that coassembly with the nonmyristoylated Gag impairs HIV particle release, a phenomenon that may involve NC-mediated Gag-Gag interaction.

Defect of human immunodeficiency virus type 2 Gag assembly in Saccharomyces cerevisiae.

We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.

Establishment of Vero E6 cell clones persistently infected with severe acute respiratory syndrome coronavirus.

Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus.

Human immunodeficiency virus type 1 Gag assembly through assembly intermediates.

Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37 degrees C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30 degrees C resulted in the production of only the 60 S form of Gag multimer, and crescent-shaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37 degrees C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30 degrees C reaction produced only the 60 S multimers. However, when the 60 S multimers were cross-linked so as not to allow conformational changes, in vitro assembly reactions at 37 degrees C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37 degrees C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.

Differential susceptibility of cells expressing allogeneic MHC or viral antigen to killing by antigen-specific CTL.

CD8(+) cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8(+) T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus-infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8-knockout mice. To address this controversy, we generated H-2(d)-specific CD8(+) CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H-2(d) cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL-mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H-2(d) targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective-interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus-specific CTLs. The Sendai virus-infected Meth A and KLN205 lines were able to block the lysis of Sendai virus-infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.

Influenza A virus-infected hosts boost an invasive type of Streptococcus pyogenes infection in mice.

The apparent worldwide resurgence of invasive Streptococcus pyogenes infection in the last two decades remains unexplained. At present, animal models in which toxic shock-like syndrome or necrotizing fasciitis is induced after S. pyogenes infection are not well developed. We demonstrate here that infection with a nonlethal dose of influenza A virus 2 days before intranasal infection with a nonlethal dose of S. pyogenes strains led to a death rate of more than 90% in mice, 10% of which showed necrotizing fasciitis. Infection of lung alveolar epithelial cells by the influenza A virus resulted in viral hemagglutinin expression on the cell surface and promoted internalization of S. pyogenes. However, treatment with monoclonal antibodies to hemagglutinin markedly decreased this internalization. Our results indicate that prior infection with influenza A virus induces a lethal synergism, resulting in the induction of invasive S. pyogenes infection in mice.

HIV type 1 Gag virus-like particle budding from spheroplasts of Saccharomyces cerevisiae.

Expression of retroviral Gag protein in yeast has previously shown Gag targeting to the plasma membrane but little or no production of Gag virus-like particles (VLPs). Here we show that, after removal of the cell wall, the expression of HIV type 1 Gag protein in Saccharomyces cerevisiae spheroplasts allowed simultaneous budding of VLPs from the plasma membrane. Our data show that (i) the VLPs released from yeast spheroplasts were spherical and had morphological features, such as membrane apposed electron-dense layers, characteristic of the immature form of HIV particles; (ii) the VLPs were completely enclosed in the plasma membrane derived from yeast, which is denser than that of higher eukaryotic cells; (iii) the VLP Gag shells remained intact after treatment of nonionic detergent; and (iv) the VLPs were released soon after removal of the cell wall and accumulated up to 300 microg/liter of culture. Our results also show that VLP production was abolished by amino acid substitution of the Gag N-terminal myristoylglycine and impaired when Gag C-terminal deletions were extended beyond the nucleocapsid domain. These results were consistent with those obtained previously in higher eukaryotic expression systems, suggesting that similar Gag domains were used for VLP assembly. We suggest that the system described here offers significant advantages for studying host factors required for VLP budding. The system also may be available for production of vector virus-free VLPs for practical applications such as vaccine development.