Peter Dias

Strong Immunostaining for Myogenin in Rhabdomyosarcoma Is Significantly Associated with Tumors of the Alveolar Subclass

Rhabdomyosarcomas are a heterogeneous group of tumors with respect to their molecular basis, degree of differentiation, histology, and clinical behavior. Because of the wide variation of tumor morphology, it is often difficult to distinguish between the distinct subtypes of rhabdomyosarcomas. By using cryosections of tumor specimens and immunohistochemistry, in the present study we show that strong expression of myogenin in rhabdomyosarcoma is associated with alveolar histology (P = <0.0001, Fishers exact test). Although staining for myogenin was observed in 22 of 26 rhabdomyosarcomas, all alveolar rhabdomyosarcomas (nine of nine) showed high levels of staining for myogenin, as defined by the frequency and intensity of staining of the tumor cells. The staining pattern suggests that the tumor cells are clonally derived from myogenin-positive progenitor cells. In contrast, most embryonal rhabdomyosarcomas (13 of 15) were either negative or showed a low level of staining for myogenin. In these tumors a larger proportion of tumor cells were distinctly negative for myogenin. Six of seven alveolar rhabdomyosarcomas that strongly stained for myogenin were also positive for Pax3-7/Forkhead (FKHR) by polymerase chain reaction/reverse transcriptase-polymerase chain reaction. One of two embryonal rhabdomyosarcomas that strongly stained for myogenin was retrospectively found to be positive for Pax3/FKHR transcripts. Quantitative analysis for myogenin by Western blotting using a smaller subset of rhabdomyosarcomas revealed that in general there was a good correlation between immunohistochemical staining and Western blotting (P = 0.01, Pearson Correlation), although the former technique was more sensitive for detecting tumors with low levels of the protein. On average, alveolar rhabdomyosarcomas expressed at least threefold more myogenin than embryonal rhabdomyosarcomas. Our data show that staining for myogenin will be a simple, rapid, and accurate adjunct for distinguishing between alveolar and embryonal rhabdomyosarcomas. We propose that embryonal rhabdomyosarcomas result from an early block in myogenesis, before the expression of myogenin. In contrast, we propose that alveolar rhabdomyosarcomas either originate from a late block in myogenesis (after expression of myogenin) or that the pathological mechanisms involved in these neoplasms also induce strong expression of this protein.

MyoD1 protein expression in alveolar soft part sarcoma as confirmatory evidence of its skeletal muscle nature.

A typical case of alveolar soft part sarcoma was found to express in a strong and widespread fashion the marker MyoD1 protein. This nuclear phosphoprotein is the product of a regulatory gene that controls the commitment of a cell to myogenic lineage; it therefore provides strong and perhaps definitive evidence in support of the skeletal muscle nature of this enigmatic neoplasm.

Desmin Positivity in Primitive Neuroectodermal Tumors of Childhood

In this report, we describe two rosette-forming primitive neuroectodermal tumors that were found to contain desmin by both immunohistochemistry and Western blotting. Electron microscopy on both cases was consistent with primitive neuroectodermal tumors and revealed that the tumor cells contained cytoplasmic bundles of intermediate filaments. In both cases, studies for MyoDl protein using immunohistochemistry and Western blotting were negative. Thus, the detection of desmin in a pediatric neoplasm does not absolutely exclude the diagnosis of primitive neuroectodermal tumor and should not be considered as prima facie evidence that a small-cell tumor is a rhabdomyosarcoma.

A Mouse Model of Lethal Synergism Between Influenza Virus and Haemophilus influenzae

Secondary bacterial infections that follow infection with influenza virus result in considerable morbidity and mortality in young children, the elderly, and immunocompromised individuals and may also significantly increase mortality in normal healthy adults during influenza pandemics. We herein describe a mouse model for investigating the interaction between influenza virus and the bacterium Haemophilus influenzae. Sequential infection with sublethal doses of influenza and H. influenzae resulted in synergy between the two pathogens and caused mortality in immunocompetent adult wild-type mice. Lethality was dependent on the interval between administration of the bacteria and virus, and bacterial growth was prolonged in the lungs of dual-infected mice, although influenza virus titers were unaffected. Dual infection induced severe damage to the airway epithelium and confluent pneumonia, similar to that observed in victims of the 1918 global influenza pandemic. Increased bronchial epithelial cell death was observed as early as 1 day after bacterial inoculation in the dual-infected mice. Studies using knockout mice indicated that lethality occurs via a mechanism that is not dependent on Fas, CCR2, CXCR3, interleukin-6, tumor necrosis factor, or Toll-like receptor-4 and does not require T or B cells. This model suggests that infection with virulent strains of influenza may predispose even immunocompetent individuals to severe illness on secondary infection with H. influenzae by a mechanism that involves innate immunity, but does not require tumor necrosis factor, interleukin-6, or signaling via Toll-like receptor-4.

Insulinoma-Released Exosomes or Microparticles Are Immunostimulatory and Can Activate Autoreactive T Cells Spontaneously Developed in Nonobese Diabetic Mice

Exosomes (EXO) are secreted intracellular microparticles that can trigger inflammation and induce Ag-specific immune responses. To test possible roles of EXO in autoimmunity, we isolated small microparticles, mainly EXO, from mouse insulinoma and examined their activities to stimulate the autoimmune responses in NOD mice, a model for human type 1 diabetes. We demonstrate that the EXO contains strong innate stimuli and expresses candidate diabetes autoantigens. They can induce secretion of inflammatory cytokines through a MyD88-dependent pathway, and activate purified APC and result in T cell proliferation. To address whether EXO or the secreted microparticles are possible autoimmune targets causing islet-specific inflammation, we monitored the T cell responses spontaneously developed in prediabetic NOD mice for their reactivity to the EXO, and compared this reactivity between diabetes-susceptible and -resistant congenic mouse strains. We found that older NOD females, which have advanced islet destruction, accumulated more EXO-reactive, IFN-γ–producing lymphocytes than younger females or age-matched males, and that pancreatic lymph nodes from the prediabetic NOD, but not from the resistant mice, were also enriched with EXO-reactive Th1 cells. In vivo, immunization with the EXO accelerates insulitis development in nonobese diabetes-resistant mice. Thus, EXO or small microparticles can be recognized by the diabetes-associated autoreactive T cells, supporting that EXO might be a possible autoimmune target and/or insulitis trigger in NOD or congenic mouse strains.

Protein Kinase C θ Is Not Essential for T-Cell-Mediated Clearance of Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen with significant homology to human pathogens Epstein-Barr virus and Kaposis sarcoma-associated herpesvirus. T cells are essential for primary clearance of MHV-68 and survival of mice following intranasal infection. Previous reports have suggested that protein kinase C θ (PKCθ) is essential for T-cell activation and cytokine production in vitro. To determine the role of this molecule in vivo during the immune response to a viral infection, PKCθ−/− mice were infected with MHV-68. Despite the essential role of T cells in viral clearance, PKCθ−/− mice survived infection, cleared lytic virus, and maintained effective long-term control of latency. CD8 T-cell expansion, trafficking to the lung, and cytotoxic activity were similar in PKCθ+/+ and PKCθ−/− mice, whereas antiviral antibody and T-helper cell cytokine production were significantly lower in PKCθ−/− mice than in PKCθ+/+ mice. These studies demonstrate a differential requirement for PKCθ in the immune response to MHV-68 and show that PKCθ is not essential for the T-cell activation events leading to viral clearance.

Immunohistochemistry of MyoD1 in adult pleomorphic soft tissue sarcomas.

Adult pleomorphic rhabdomyosarcomas are difficult to distinguish from other pleomorphic soft tissue sarcomas. Immunostaining with the monoclonal antibody MyoD1 5.8A has recently shown promise as a sensitive and specific marker of rhabdomyosarcomas of childhood. To determine the usefulness of MyoD1 in distinguishing adult pleomorphic rhabdomyosarcoma from other pleomorphic sarcomas, we studied 21 cases of pleomorphic sarcoma, including six cases diagnosed as rhabdomyosarcoma. Immunostaining was accomplished using monoclonal antibodies against MyoD1, desmin, α-smooth-muscle actin, and muscle-specific actin on cryostat sections of frozen tumor. All cases of pleomorphic rhabdomyosarcoma stained positively with both desmin and MyoD1. Five cases diagnosed as nonrhabdomyosarcomatous pleomorphic sarcomas stained positively with desmin, but only one showed weak MyoD1 positivity. Three of these five cases were also positive with α-smooth-muscle actin. The results of this study suggest that the sensitivity and specificity of the MyoD1 antibody in the differential diagnosis of adult pleomorphic soft tissue sarcomas approaches that seen in pediatric rhabdomyosarcomas.

CD4 T-Cell Help Programs a Change in CD8 T-Cell Function Enabling Effective Long-Term Control of Murine Gammaherpesvirus 68: Role of PD-1-PD-L1 Interactions

ABSTRACT We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II−/− (CII−/−) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII−/− mice caused a significant reduction in lung viral titers, in contrast to those from control CII−/− mice. Anti-CD40 treatment also greatly prolonged survival of infected CII−/− mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII−/−, CD40−/−, or CD80/86−/− mice, compared with that in wild-type or CD28/CTLA4−/− mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.

M2SR, a novel live single replication influenza virus vaccine, provides effective heterosubtypic protection in mice.

Despite the annual public health burden of seasonal influenza and the continuing threat of a global pandemic posed by the emergence of highly pathogenic/pandemic strains, conventional influenza vaccines do not provide universal protection, and exhibit suboptimal efficacy rates, even when they are well matched to circulating strains. To address the need for a highly effective universal influenza vaccine, we have developed a novel M2-deficient single replication vaccine virus (M2SR) that induces strong cross-protective immunity against multiple influenza strains in mice. M2SR is able to infect cells and expresses all viral proteins except M2, but is unable to generate progeny virus. M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) protected mice against lethal challenge with influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic). The vaccine induced strong systemic and mucosal antibody responses of both IgA and IgG classes. Strong virus-specific T cell responses were also induced. Following heterologous challenge, significant numbers of IFN-γ-producing CD8 T cells, with effector or effector/memory phenotypes and specific for conserved viral epitopes, were observed in the lungs of vaccinated mice. A substantial proportion of the CD8 T cells expressed Granzyme B, suggesting that they were capable of killing virus-infected cells. Thus, our data suggest that M2-deficient influenza viruses represent a promising new approach for developing a universal influenza vaccine.

Autoimmune Responses to Exosomes and Candidate Antigens Contribute to Type 1 Diabetes in Non-Obese Diabetic Mice

Purpose of ReviewThe initial autoimmune trigger of type 1 diabetes (T1D) remains unclear. In non-obese diabetic (NOD) mice, islet inflammation starts early in life, suggesting the presence of an endogenous trigger for the spontaneous autoimmune response in this T1D mouse model. In this review, we argue that abnormal release of exosomes might be the trigger of the early inflammatory and autoimmune responses in the islets.Recent FindingsExosomes are nano-sized membrane complexes that are secreted by cells following fusion of late endosomes and/or multivesicular bodies with the plasma membrane. They are known extracellular messengers, communicating among neighboring cells via transporting large molecules from parent cells to recipient cells. Recent evidence demonstrates that these extracellular vesicles can modulate immune responses. It has been shown that insulinoma and islet mesenchymal stem cell-released exosomes are potent immune stimuli that can induce autoreactive B and T cells. Searching for candidate antigens in the exosomes identified endogenous retrovirus (ERV) Env and Gag antigens, which are homologous to an endogenous murine leukemia retrovirus. Autoantibodies and autoreactive T cells spontaneously developed in NOD mice can react to these retroviral antigens. More importantly, expression of the retroviral antigens in the islet mesenchymal stem cells is associated with disease susceptibility, and the expression is restricted to T1D-susceptible but not resistant mouse strains.SummaryExosomes are novel autoimmune targets, carrying autoantigens that can stimulate innate and adaptive immune responses. An abnormal or excess release of exosomes, particularly those ones containing endogenous retroviral antigens might be responsible for triggering tissue-specific inflammatory and autoimmune responses.