Oral rehabilitation and associated quality of life following mandibular reconstruction with free fibula flap: a cross-sectional study.

Introduction: Mandibular reconstruction with the free fibula flap (FFF) has become a standardized procedure. The situation is different with oral rehabilitation, so the purpose of this study was to investigate the frequency of implant placement and prosthetic restoration. Additionally, the patients' situation, motivation, and treatment course were structurally assessed. Materials and methods: All cases between January 2013 and December 2018 that underwent mandibular reconstruction in our department with a free fibula flap and gave written informed consent to participate were interviewed with two structured questionnaires about their restoration and quality of life. Additionally, medical records, general information, status of implants and therapy, and metric analyses of the inserted implants were performed. Results: In total 59 patients were enrolled and analyzed in this monocentric study. Overall, oral rehabilitation was achieved in 23.7% at the time of investigation. In detail, implants were inserted in 37.3% of patients and showed an 83.3% survival of dental implants. Of these implanted patients, dental implants were successfully restored with a prosthetic restoration in 63.6. Within this subgroup, satisfaction with the postoperative aesthetic and functional result was 79.9% and with the oral rehabilitation process was 68.2%. Satisfaction with the implant-borne prosthesis was 87.5%, with non-oral-squamous-cell-carcinoma patients being statistically significantly more content with the handling (p=0.046) and care (p=0.031) of the prosthesis. Discussion: Despite the well-reconstructed bony structures, there is a need to increase the effort of achieving oral rehabilitation, especially looking at the patient's persistent motivation for the procedure.

Immune contexture of paediatric cancers.

BACKGROUND: The clinical development of immune checkpoint-targeted immunotherapies has been disappointing so far in paediatric solid tumours. However, as opposed to adults, very little is known about the immune contexture of paediatric malignancies. METHODS: We investigated by gene expression and immunohistochemistry (IHC) the immune microenvironment of five major paediatric cancers: Ewing sarcoma (ES), osteosarcoma (OS), rhabdomyosarcoma (RMS), medulloblastoma (MB) and neuroblastoma (NB; 20 cases each; n = 100 samples total), and correlated them with overall survival. RESULTS: NB and RMS tumours had high immune cell gene expression values and high T-cell counts but were low for antigen processing cell (APC) genes. OS and ES tumours showed low levels of T-cells but the highest levels of APC genes. OS had the highest levels of macrophages (CSF1R, CD163 and CD68), whereas ES had the lowest. MB appeared as immune deserts. Tregs (FOXP3 staining) were higher in both RMS and OS. Most tumours scored negative for PD-L1 in tumour and immune cells, with only 11 of 100 samples positive for PD-L1 staining. PD-L1 and OX40 levels were generally low across all five indications. Interestingly, NB had comparable levels of CD8 by IHC and by gene expression to adult tumours. However, by gene expression, these tumours were low for T-cell cytotoxic molecules GZMB, GZMA and PRF1. Surprisingly, the lower the level of tumour infiltrative CD8 T-cells, the better the prognosis was in NB, RMS and ES. Gene expression analyses showed that MYCN-amplified NB have higher amounts of immune suppressive cells such as macrophages, myeloid-derived suppressor cells and Tregs, whereas the non-MYCN-amplified tumours were more infiltrated and had higher expression levels of Teff. CONCLUSIONS: Our results describe the quality and quantity of immune cells across five major paediatric cancers and provide some key features differentiating these tumours from adult tumour types. These findings explain why anti-PD(L)1 might not have had single agent success in paediatric cancers. These results provides the rationale for the development of biologically stratified and personalised immunotherapy strategies in children with relapsing/refractory cancers.

Pseudoprogression of CNS metastatic disease of alveolar soft part sarcoma during anti-PDL1 treatment.

Immune checkpoint inhibitors are increasingly used in treatment of metastatic renal cell carcinoma, melanoma, and nonsmall cell lung cancer, as well as in clinical trials for novel targets. We present a pediatric patient with metastatic alveolar soft part sarcoma who was treated with MPDL3280 (Atezolizumab), a monoclonal anti-programmed death ligand-1 antibody. Imaging results for the patient suggested disease progression of multiple brain metastases with stable systemic disease. The patient met response evaluation criteria in solid tumors (RECIST) criteria of progression of disease and was removed from treatment with MPDL3280. Subsequent surgical resection of the brain lesions revealed nonviable tumor with extensive lymphocytic infiltrates consistent with pseudoprogression. This case report adds to a growing number of reports that question reliance on RECIST criteria and suggest need for further refinement of RECIST or irRECIST during immune checkpoint inhibitor treatment for central nervous system metastatic lesions.

An evolutionarily conserved transcriptional response to viral infection in Caenorhabditis nematodes.

BACKGROUND: Caenorhabditis elegans is a powerful model organism for probing many biological processes including host-pathogen interactions with bacteria and fungi. The recent identification of nematode viruses that naturally infect C. elegans and Caenorhabditis briggsae provides a unique opportunity to define host-virus interactions in these model hosts. RESULTS: We analyzed the transcriptional response of pathogen infected C. elegans and C. briggsae by RNA-seq. We identified a total of 320 differentially expressed genes (DEGs) in C. elegans following Orsay virus infection. The DEGs of known function were enriched for ubiquitin ligase related genes; however, the majority of the genes were of unknown function. Interestingly, many DEGs that responded to Orsay virus infection were similar to those induced by Nematocida parisii infection, which is a natural microsporidia pathogen of C. elegans that like Orsay virus infects intestinal cells. Furthermore, comparison of the Orsay virus DEGs in C. elegans to Santeuil virus DEGs in C. briggsae identified 58 C. elegans genes whose orthologs were likewise differentially expressed in C. briggsae, thereby defining an evolutionarily conserved response to viral infection. CONCLUSIONS: The two different species C. elegans and C. briggsae, which diverged ~18 million years ago, share a common set of transcriptionally responsive genes to viral infection. Furthermore, a subset of these genes were also differentially expressed following infection by a eukaryotic pathogen, N. parisii, suggesting that these genes may constitute a broader pan-microbial response to infection.

Engineering recombinant Orsay virus directly in the metazoan host Caenorhabditis elegans.

The recent identification of Orsay virus, the first virus that is capable of naturally infecting Caenorhabditis elegans, provides a unique opportunity to explore host-virus interaction studies in this invaluable model organism. A key feature of this system is the robust genetic tractability of the host, C. elegans, which would ideally be complemented by the ability to genetically manipulate Orsay virus in parallel. To this end, we developed a plasmid-based reverse genetics system for Orsay virus by creating transgenic C. elegans strains harboring Orsay virus cDNAs. Both wild-type and mutant Orsay viruses, including a FLAG epitope-tagged recombinant Orsay virus, were generated by use of the reverse genetics system. This is the first plasmid-based virus reverse genetics system in the metazoan C. elegans. The Orsay virus reverse genetics we established will serve as a fundamental tool in host-virus interaction studies in the model organism C. elegans. Importance: To date, Orsay virus is the first and the only identified virus capable of naturally infecting Caenorhabditis elegans. C. elegans is a simple multicellular model organism that mimics many fundamental features of human biology and has been used to define many biological properties conserved through evolution. Thus, the Orsay virus-C. elegans infection system provides a unique opportunity to study host-virus interactions. In order to take maximal advantage of this system, the ability to genetically engineer mutant forms of Orsay virus would be highly desirable. Most efforts to engineer viruses have been done with cultured cells. Here we describe the creation of mutant viruses directly in the multicellular organism C. elegans without the use of cell culture. We engineered a virus expressing a genetically tagged protein that could be detected in C. elegans. This provides proof of concept for modifying Orsay virus, which will greatly facilitate studies in this experimental system.

Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions.

Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated into Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha-delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses.

Orsay, Santeuil and Le Blanc viruses primarily infect intestinal cells in Caenorhabditis nematodes.

The discoveries of Orsay, Santeuil and Le Blanc viruses, three viruses infecting either Caenorhabditis elegans or its relative Caenorhabditis briggsae, enable the study of virus-host interactions using natural pathogens of these two well-established model organisms. We characterized the tissue tropism of infection in Caenorhabditis nematodes by these viruses. Using immunofluorescence assays targeting proteins from each of the viruses, and in situ hybridization, we demonstrate viral proteins and RNAs localize to intestinal cells in larval stage Caenorhabditis nematodes. Viral proteins were detected in one to six of the 20 intestinal cells present in Caenorhabditis nematodes. In Orsay virus-infected C. elegans, viral proteins were detected as early as 6h post-infection. The RNA-dependent RNA polymerase and capsid proteins of Orsay virus exhibited different subcellular localization patterns. Collectively, these observations provide the first experimental insights into viral protein expression in any nematode host, and broaden our understanding of viral infection in Caenorhabditis nematodes.

Comparison of novel MLB-clade, VA-clade and classic human astroviruses highlights constrained evolution of the classic human astrovirus nonstructural genes.

Eight serotypes of human astroviruses (the classic human astroviruses) are causative agents of diarrhea. Recently, five additional astroviruses belonging to two distinct clades have been described in human stool, including astroviruses MLB1, MLB2, VA1, VA2 and VA3. We report the discovery in human stool of two novel astroviruses, astroviruses MLB3 and VA4. The complete genomes of these two viruses and the previously described astroviruses VA2 and VA3 were sequenced, affording seven complete genomes from the MLB and VA clades for comparative analysis to the classic human astroviruses. Comparison of the genetic distance, number of synonymous mutations per synonymous site (dS), number of non-synonymous mutations per non-synonymous site (dN) and the dN/dS ratio in the protease, polymerase and capsid of the classic human, MLB and VA clades suggests that the protease and polymerase of the classic human astroviruses are under distinct selective pressure.

Complete genome sequence of Le Blanc virus, a third Caenorhabditis nematode-infecting virus.

Orsay virus and Santeuil virus, the first known viruses capable of naturally infecting the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, respectively, were recently identified by high-throughput sequencing of wild Caenorhabditis strains. By similar analysis of another wild C. briggsae isolate, we have now discovered and sequenced the complete genome of a third novel virus, Le Blanc virus, that is distantly related to Orsay and Santeuil viruses. All three viruses are positive-sense RNA viruses with bipartite genomes that are most closely related to nodaviruses. Identification of a third virus capable of infecting Caenorhabditis nematodes enables comparative analysis of this clade of viruses and strengthens this model for investigating virus-host interactions.

Astrovirus MLB2 viremia in febrile child.

Astroviruses cause diarrhea, but it is not known whether they circulate in human plasma. Astrovirus MLB2 was recently discovered in diarrhea samples from children. We detected MLB2 in the plasma of a febrile child, which suggests that MLB2 has broader tropism than expected and disease potential beyond the gastrointestinal tract.

Natural and experimental infection of Caenorhabditis nematodes by novel viruses related to nodaviruses.

An ideal model system to study antiviral immunity and host-pathogen co-evolution would combine a genetically tractable small animal with a virus capable of naturally infecting the host organism. The use of C. elegans as a model to define host-viral interactions has been limited by the lack of viruses known to infect nematodes. From wild isolates of C. elegans and C. briggsae with unusual morphological phenotypes in intestinal cells, we identified two novel RNA viruses distantly related to known nodaviruses, one infecting specifically C. elegans (Orsay virus), the other C. briggsae (Santeuil virus). Bleaching of embryos cured infected cultures demonstrating that the viruses are neither stably integrated in the host genome nor transmitted vertically. 0.2 µm filtrates of the infected cultures could infect cured animals. Infected animals continuously maintained viral infection for 6 mo (∼50 generations), demonstrating that natural cycles of horizontal virus transmission were faithfully recapitulated in laboratory culture. In addition to infecting the natural C. elegans isolate, Orsay virus readily infected laboratory C. elegans mutants defective in RNAi and yielded higher levels of viral RNA and infection symptoms as compared to infection of the corresponding wild-type N2 strain. These results demonstrated a clear role for RNAi in the defense against this virus. Furthermore, different wild C. elegans isolates displayed differential susceptibility to infection by Orsay virus, thereby affording genetic approaches to defining antiviral loci. This discovery establishes a bona fide viral infection system to explore the natural ecology of nematodes, host-pathogen co-evolution, the evolution of small RNA responses, and innate antiviral mechanisms.

Type IIB procollagen NH(2)-propeptide induces death of tumor cells via interaction with integrins alpha(V)beta(3) and alpha(V)beta(5).

Cartilage is resistant to tumor invasion. In the present study, we found that the NH(2)-propeptide of the cartilage-characteristic collagen, type IIB, PIIBNP, is capable of killing tumor cells. The NH(2)-propeptide is liberated into the extracellular matrix prior to deposition of the collagen fibrils. This peptide adheres to and kills cells from chondrosarcoma and cervical and breast cancer cell lines via the integrins alpha(v)beta(5) and alpha(v)beta(3). Adhesion is abrogated by blocking with anti alpha(v)beta(5) and alpha(v)beta(3) antibodies. When alpha(v) is suppressed by small intefering RNA, adhesion and cell killing are blocked. Normal chondrocytes from developing cartilage do not express alpha(v)beta(3) and alpha(v)beta(5) integrins and are thus protected from cell death. Morphological, DNA, and biochemical evidence indicates that the cell death is not by apoptosis but probably by necrosis. In an assay for invasion, PIIBNP reduced the number of cells crossing the membrane. In vivo, in a tumor model for breast cancer, PIIBNP was consistently able to reduce the size of the tumor.

Human stool contains a previously unrecognized diversity of novel astroviruses.

Human astroviruses are a leading cause of gastrointestinal disease. Since their discovery in 1975, 8 closely related serotypes have been described in humans, and more recently, two new astrovirus species, astrovirus MLB1 and astrovirus VA1, were identified in diarrhea patients. In this study, we used consensus astrovirus primers targeting the RNA polymerase to define the diversity of astroviruses present in pediatric patients with diarrhea on two continents. From 416 stool specimens comprising two different cohorts from Vellore, India, 35 samples were positive. These positive samples were analyzed further by either sequencing of the approximately 400 bp amplicon generated by the consensus PCR or by performing additional RT-PCR specific for individual astroviruses. 19 samples contained the classic human astrovirus serotypes 1-8 while 7 samples were positive for the recently described astrovirus MLB1. Strikingly, from samples that were positive in the consensus PCR screen but negative in the specific PCR assays, five samples contained sequences that were highly divergent from all previously described astroviruses. Sequence analysis suggested that three novel astroviruses, tentatively named astroviruses VA2, MLB2 and VA3, were present in these five patient specimens (AstV-VA2 in 2 patients, AstV-MLB2 in 2 patients and AstV-VA3 in one patient). Using the same RT-PCR screening strategy, 13 samples out of 466 tested stool specimens collected in St. Louis, USA were positive. Nine samples were positive for the classic human astroviruses. One sample was positive for AstV-VA2, and 3 samples were positive for AstV-MLB2 demonstrating that these two viruses are globally widespread. Collectively, these findings underscore the tremendous diversity of astroviruses present in fecal specimens from diarrhea patients. Given that a significant fraction of diarrhea etiologies is currently unknown, it is plausible that these or other yet unrecognized astroviruses may be responsible for at least part of the undiagnosed cases.

Single high-energy impact load causes posttraumatic OA in young rabbits via a decrease in cellular metabolism.

Articular cartilage deterioration commonly occurs following traumatic joint injury. Patients with posttraumatic osteoarthritis (PTA) experience pain and stiffness in the involved joint causing limited mobility and function. The mechanism by which PTA occurs has not been fully delineated. The goal of this study was to determine if a single high-energy impact load could cause the development of PTA in 3-month-old NZ White rabbits. Each rabbit underwent the application of a single, rapid, high-energy impact load to the posterior aspect of their right medial femoral condyle using a previously validated mechanism. At regular intervals (0, 1, 6 months) the injured cartilage was harvested and analyzed for the presence of PTA. Each specimen was assessed histologically for cell and tissue morphology and chondrocyte metabolism, including BMP-2 production and synthesis of extracellular matrix (type II procollagen mRNA). Cartilage from the contralateral sham limb, as well as uninjured cartilage from the experimental limb served as internal controls for each animal. Significant changes were found in the morphology of the cartilage including proteoglycan loss along with decreased BMP-2 and type II procollagen mRNA staining. These findings confirm that a single high-energy impact load can cause the development of PTA by disrupting the extracellular matrix and by causing a decrease in chondrocyte metabolism.

Ultrastructural changes in platelet aggregates of HIV patients: a scanning electron microscopy study.

Several hematological abnormalities associated with HIV have been documented, but the mechanisms responsible for the cytopenias in AIDS patients are complex and not always completely understood. Thrombocytopenia, which occurs in about 40% of patients with HIV infection, may be caused by increased peripheral platelet destruction, a defect in platelet production due to the impaired formation of platelets by HIV-infected magakaryocytes, or a combination of these. The aim of this study was to compare the morphology of the platelet aggregates in platelet-rich plasma (PRP) clots prepared from HIV patients with those of controls without HIV. These platelet aggregates were studied using the scanning electron microscope to determine the effect of the virus on platelet ultrastructure. The results showed that although the platelets do aggregate, the morphology was changed with membrane blebbing as well as torn cellular membranes. Membrane blebbing is typically associated with apoptosis. It is concluded that the altered morphology of platelet aggregates in HIV patients may be related to thrombocytopenia as a result of peripheral platelet destruction.

Site-1 protease is essential for endochondral bone formation in mice.

Site-1 protease (S1P) has an essential function in the conversion of latent, membrane-bound transcription factors to their free, active form. In mammals, abundant expression of S1P in chondrocytes suggests an involvement in chondrocyte function. To determine the requirement of S1P in cartilage and bone development, we have created cartilage-specific S1P knockout mice (S1P(cko)). S1P(cko) mice exhibit chondrodysplasia and a complete lack of endochondral ossification even though Runx2 expression, Indian hedgehog signaling, and osteoblastogenesis is intact. However, there is a substantial increase in chondrocyte apoptosis in the cartilage of S1P(cko) mice. Extraction of type II collagen is substantially lower from S1P(cko) cartilage. In S1P(cko) mice, the collagen network is disorganized and collagen becomes entrapped in chondrocytes. Ultrastructural analysis reveals that the endoplasmic reticulum (ER) in S1P(cko) chondrocytes is engorged and fragmented in a manner characteristic of severe ER stress. These data suggest that S1P activity is necessary for a specialized ER stress response required by chondrocytes for the genesis of normal cartilage and thus endochondral ossification.

Ultrastructural comparison of the morphology of three different platelet and fibrin fiber preparations.

The aim of the current study was to investigate the ultrastructural morphology of three different sources of fibrin networks and platelets, namely, lyophilized human platelet-rich plasma (LPRP), freshly prepared human platelet-rich plasma (FPRP), and human platelet concentrate (HPC). The ultrastructural morphology of the three different fibrin networks was studied using the scanning electron microscope (SEM). Turbidity curves were drawn at 405 nm at room temperature and fibrinogen concentrations were measured. Scanning electron micrographs showed that all clots produced thick major fibrin fibers as well as a well-defined fine fibrin network, which appeared to be a superimposed process that occurred after the major fibrin network was established. These features were decidedly more pronounced in the HPC specimens. Turbidity curves of the three types of plasma showed differences in LPRP and FPRP. Fibrinogen concentrations of all three preparations were in the normal ranges. Because of the great similarity between LPRP, HPC, and FPRP, we suggest that LPRP could be used successfully to study morphological changes in fibrin fibers and platelets, which may occur after exposure to certain therapeutic agents. However, functionality studies such as turbidity curves should concurrently be included. We therefore conclude that from a basic science point of view, LPRP is a valuable research tool and that such results may add information that could be valuable for clinical application.