Pseudorabies virus usurps non-muscle myosin heavy chain IIA to dampen viral DNA recognition by cGAS for antagonism of host antiviral innate immunity.

Alphaherpesvirus pseudorabies virus (PRV) causes severe economic losses to the global pig industry and has garnered increasing attention due to its broad host range including humans. PRV has developed a variety of strategies to antagonize host antiviral innate immunity. However, the underlying mechanisms have not been fully elucidated. In our previous work, we demonstrated that non-muscle myosin heavy chain IIA (NMHC-IIA), a multifunctional cytoskeleton protein, attenuates innate immune responses triggered by RNA viruses. In the current study, we reported a previously unrecognized role of NMHC-IIA in counteracting PRV-induced cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) production. Mechanistically, PRV infection led to an elevation of NMHC-IIA, strengthening the interaction between poly (ADP-ribose) polymerase 1 (PARP1) and cGAS. This interaction impeded cGAS recognition of PRV DNA and hindered downstream signaling activation. Conversely, inhibition of NMHC-IIA by Blebbistatin triggered innate immune responses and enhanced resistance to PRV proliferation both in vitro and in vivo. Taken together, our findings unveil that PRV utilizes NMHC-IIA to antagonize host antiviral immune responses via impairing DNA sensing by cGAS. This in-depth understanding of PRV immunosuppression not only provides insights for potential PRV treatment strategies but also highlights NMHC-IIA as a versatile immunosuppressive regulator usurped by both DNA and RNA viruses. Consequently, NMHC-IIA holds promise as a target for the development of broad-spectrum antiviral drugs.IMPORTANCECyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis plays a vital role in counteracting alphaherpesvirus infections. Alphaherpesviruses exploit various strategies for antagonizing cGAS-STING-mediated antiviral immune responses. However, limited examples of pseudorabies virus (PRV)-caused immunosuppression have been documented. Our findings reveal a novel role of non-muscle myosin heavy chain IIA (NMHC-IIA) in suppressing PRV-triggered innate immune responses to facilitate viral propagation both in vitro and in vivo. In detail, NMHC-IIA recruits poly (ADP-ribose) polymerase 1 (PARP1) to augment its interaction with cGAS, which impairs cGAS recognition of PRV DNA. Building on our previous demonstration of NMHC-IIA's immunosuppressive role during RNA virus infections, these findings indicate that NMHC-IIA acts as a broad-spectrum suppressor of host antiviral innate immunity in response to both DNA and RNA viruses. Therefore, NMHC-IIA will be a promising target for the development of comprehensive antiviral strategies.

4H12, a Murine Monoclonal Antibody Directed against Myosin Heavy Chain-9 Expressed on Acinar Cell Carcinoma of Pancreas with Potential Therapeutic Application

Background: Pancreatic acinar cell carcinoma (PACC) is a rare type of pancreatic exocrine neoplasm that is frequently diagnosed at late stages with a high rate of metastasis. Identification of new biomarkers for PACC can improve our knowledge of its biology, early detection, or targeted therapy. In this study, hybridoma technology was used to generate mAbs against Faraz-ICR, a pancreatic acinar cell carcinoma cell line. Methods: Cell ELISA and flow cytometry were used for screening, and the 4H12 hybridoma clone was selected for further analysis. The 4H12 mAb was specific for myosin heavy chain-9 (MYH9) as determined by Immunoprecipitation, Western blot, and mass spectrometry. Results: This antibody reacted variably with other cancer cells, in comparison to Faraz-ICR cell. Besides, by immunohistochemical staining, the acinar cell tumor, which was the source of Faraz-ICR, showed high MYH9 expression. Among 21 pancreatic ductal adenocarcinoma cases, nine (42.8%) expressed MYH9 with low intensity, while 10 (47.8%) and 2 (9.5%) cases expressed MYH9 with moderate to strong intensities, respectively. The 4H12 mAb inhibited the proliferation of Faraz-ICR cells in a dose-dependent manner from 0.75 to 12.5 µg/ml concentrations (p < 0.0001 and p < 0.002). IC50 values were achieved at 12.09 ± 4.19 µg/ml and 7.74 ± 4.28 µg/ml after 24- and 48-h treatment, respectively. Conclusion: Our data suggest that the 4H12 mAb can serve as a tool for investigating the role of MYH9 pancreatic cancer biology and prognosis.

Helicobacter urease suppresses cytotoxic CD8+ T-cell responses through activating Myh9-dependent induction of PD-L1.

As a key virulence factor for persistent colonization, urease B subunit (UreB) is considered to be an ideal vaccine antigen against Helicobacter pylori infection. However, the role and molecular mechanisms of UreB involved in immune microenvironment dysregulation still remain largely unknown. In the present study, we evaluated the effects of UreB on macrophage activation and found that UreB induced PD-L1 accumulation on bone marrow-derived macrophages (BMDMs). Co-culture assays further revealed that UreB-induced PD-L1 expression on BMDMs significantly decreased the proliferation and secretion of cytolytic molecules (granzyme B and perforin) of splenic CD8+ T cells isolated from inactivated H. pylori-immunized mice. More importantly, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation techniques, it has been confirmed that myosin heavy chain 9 (Myh9) is a direct membrane receptor for UreB and is required for PD-L1 up-regulation on BMDMs. Molecular studies further demonstrated that the interaction between UreB and Myh9 decreased GCN2 autophosphorylation and enhanced the intracellular pool of amino acids, leading to the up-regulation of S6K phosphorylation, a commonly used marker for monitoring activation of mTORC1 signaling activity. Furthermore, blocking mTORC1 activation with its inhibitor Temsirolimus reversed the UreB-induced PD-L1 up-regulation and the subsequent inhibitory effects of BMDMs on activation of cytotoxic CD8+ T-cell responses. Overall, our data unveil a novel immunosuppressive mechanism of UreB during H. pylori infection, which may provide valuable clues for the optimization of H. pylori vaccine.

Influenza Infection has Fiber Type-Specific Effects on Cellular and Molecular Skeletal Muscle Function in Aged Mice.

Skeletal muscle myopathies represent a common non-pulmonary manifestation of influenza infection, leading to reduced physical function and hospitalization in older adults. However, underlying mechanisms remain poorly understood. Our study examined the effects of influenza virus A pulmonary infection on contractile function at the cellular (single fiber) and molecular (myosin-actin interactions and myofilament properties) levels in soleus and extensor digitorum longus muscles of aged (20 months) C57BL/6 male mice that were healthy or flu-infected for 7 (7-days post-infection; 7-DPI) or 12 days (12-DPI). Cross-sectional area (CSA) of myosin heavy chain (MHC) IIA and IIB fibers was reduced at 12-DPI relative to 7-DPI and healthy. Maximal isometric force in MHC IIA fibers was also reduced at 12-DPI relative to 7-DPI and healthy, resulting in no change in specific force (maximal isometric force divided by CSA). In contrast, MHC IIB fibers produced greater isometric force and specific force at 7-DPI compared to 12-DPI or healthy. The increased specific force in MHC IIB fibers was likely due to greater myofilament lattice stiffness and/or an increased number or stiffness of strongly bound myosin-actin cross-bridges. At the molecular level, cross-bridge kinetics were slower in MHC IIA fibers with infection, while changes in MHC IIB fibers were largely absent. In both fiber types, greater myofilament lattice stiffness was positively related to specific force. This study provides novel evidence that cellular and molecular contractile function is impacted by influenza infection in a fiber type-specific manner, suggesting potential molecular mechanisms to help explain the impact of flu-induced myopathies.

Antibodies in cerebral cavernous malformations react with cytoskeleton autoantigens in the lesional milieu.

Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms. Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.

Quantification of biomarkers for beef meat qualities using a combination of Parallel Reaction Monitoring- and antibody-based proteomics.

We and others have identified biomarker candidates of tenderness or marbling, two major attributes of bovine meat-eating qualities for consumers' satisfaction. In this study, Reverse Phase Protein Arrays (RPPA) and targeted mass spectrometry assays using Parallel Reaction Monitoring (PRM) were developed to test whether 10 proteins pass the sequential qualification and verification steps of the challenging biomarker discovery pipeline. At least MYH1, TPI1, ALDH1A1 and CRYAB were qualified by RPPA or PRM as being differentially abundant according to marbling values of longissimus thoracis and semimembranosus muscles. Significant mathematical relationships between the individual abundance of each of the four proteins and marbling values were verified by linear or logistic regressions. Four proteins, TNNT1, MDH1, PRDX6 and ENO3 were qualified and verified for tenderness, and the abundance of MDH1 explained 49% of the tenderness variability. The present PRM and RPPA results pave the way for development of useful meat industrial multiplex-proteins assays.

Monocytes prime autoreactive T cells after myocardial infarction.

In humans, loss of central tolerance for the cardiac self-antigen α-myosin heavy chain (α-MHC) leads to circulation of cardiac autoreactive T cells and renders the heart susceptible to autoimmune attack after acute myocardial infarction (MI). MI triggers profound tissue damage, releasing danger signals and self-antigen by necrotic cardiomyocytes, which lead to recruitment of inflammatory monocytes. We hypothesized that excessive inflammation by monocytes contributes to the initiation of adaptive immune responses to cardiac self-antigen. Using an experimental model of MI in α-MHC-mCherry reporter mice, which specifically express mCherry in cardiomyocytes, we detected α-MHC antigen in myeloid cells in the heart-draining mediastinal lymph node (MLN) 7 days after MI. To test whether monocytes were required for cardiac self-antigen trafficking to the MLN, we blocked monocyte recruitment with a C-C motif chemokine receptor type 2 (CCR2) antagonist or immune modifying nanoparticles (IMP). Blockade of monocyte recruitment reduced α-MHC antigen detection in the MLN after MI. Intramyocardial injection of the model antigen ovalbumin into OT-II transgenic mice demonstrated the requirement for monocytes in antigen trafficking and T-cell activation in the MLN. Finally, in nonobese diabetic mice, which are prone to postinfarction autoimmunity, blockade of monocyte recruitment reduced α-MHC-specific responses leading to improved tissue repair and ventricular function 28 days after MI. Taken together, these data support a role for monocytes in the onset of pathological cardiac autoimmunity following MI and suggest that therapeutic targeting of monocytes may mitigate postinfarction autoimmunity in humans.NEW & NOTEWORTHY Our study newly identifies a role for inflammatory monocytes in priming an autoimmune T-cell response after myocardial infarction. Select inhibition of monocyte recruitment to the infarct prevents trafficking of cardiac self-antigen and activation of cardiac myosin reactive T cells in the heart-draining lymph node. Therapeutic targeting of inflammatory monocytes may limit autoimmune responses to improve cardiac remodeling and preserve left ventricular function after myocardial infarction.

Microbiota-derived peptide mimics drive lethal inflammatory cardiomyopathy.

Myocarditis can develop into inflammatory cardiomyopathy through chronic stimulation of myosin heavy chain 6-specific T helper (TH)1 and TH17 cells. However, mechanisms governing the cardiotoxicity programming of heart-specific T cells have remained elusive. Using a mouse model of spontaneous autoimmune myocarditis, we show that progression of myocarditis to lethal heart disease depends on cardiac myosin-specific TH17 cells imprinted in the intestine by a commensal Bacteroides species peptide mimic. Both the successful prevention of lethal disease in mice by antibiotic therapy and the significantly elevated Bacteroides-specific CD4+ T cell and B cell responses observed in human myocarditis patients suggest that mimic peptides from commensal bacteria can promote inflammatory cardiomyopathy in genetically susceptible individuals. The ability to restrain cardiotoxic T cells through manipulation of the microbiome thereby transforms inflammatory cardiomyopathy into a targetable disease.

Myocardial infarction triggers cardioprotective antigen-specific T helper cell responses.

T cell autoreactivity is a hallmark of autoimmune diseases but can also benefit self-maintenance and foster tissue repair. Herein, we investigated whether heart-specific T cells exert salutary or detrimental effects in the context of myocardial infarction (MI), the leading cause of death worldwide. After screening more than 150 class-II-restricted epitopes, we found that myosin heavy chain alpha (MYHCA) was a dominant cardiac antigen triggering post-MI CD4+ T cell activation in mice. Transferred MYHCA614-629-specific CD4+ T (TCR-M) cells selectively accumulated in the myocardium and mediastinal lymph nodes (med-LN) of infarcted mice, acquired a Treg phenotype with a distinct pro-healing gene expression profile, and mediated cardioprotection. Myocardial Treg cells were also detected in autopsies from patients who suffered a MI. Noninvasive PET/CT imaging using a CXCR4 radioligand revealed enlarged med-LNs with increased cellularity in MI-patients. Notably, the med-LN alterations observed in MI patients correlated with the infarct size and cardiac function. Taken together, the results obtained in our study provide evidence showing that MI-context induces pro-healing T cell autoimmunity in mice and confirms the existence of an analogous heart/med-LN/T cell axis in MI patients.

CpG enhances the immunogenicity of heterologous DNA-prime/protein-boost vaccination with the heavy chain myosin of Brugia malayi in BALB/c mice.

The recombinant heavy chain myosin of Brugia malayi (Bm-Myo) has earlier been reported as a potent vaccine candidate in our lab. Subsequently, we further enhanced its efficacy employing heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) immunization approach that produced superior immune-protection than protein or DNA vaccination. In the present study, we evaluated the efficacy of heterologous prime boost vaccination in combination with CpG, synthetic oligodeoxynucleotides (ODN) adjuvant in BALB/c mice. The results showed that CpG/Myo-pcD+Bm-Myo conferred 84.5 ± 0.62% protection against B. malayi infective larval challenge which was considerably higher than Myo-pcD+Bm-Myo (75.6 ± 1.10%) following immunization. Although, both the formulations of immunization elicited robust production of specific IgG antibody and their isotypes (IgG1, IgG2a, IgG2b, and IgG3); however, CpG/Myo-pcD+Bm-Myo predominantly enhanced the level of IgG2a suggesting Th1 biased immune response in presence of CpG. Furthermore, spleen isolated from mice that immunized with CpG/Myo-pcD+Bm-Myo had greater accumulation of CD4+, CD8+, and CD19+ B cells and there was an augmented expression of co-stimulatory molecules CD40, CD86 on host dendritic cells (DCs). In contrast to Myo-pcD+Bm-Myo group, the splenocytes of CpG/Myo-pcD+Bm-Myo immunized mice developed comparatively higher pro-inflammatory cytokines IL-2 and IFN-γ leaving anti-inflammatory cytokine levels unchanged. Moreover, CpG formulation also upregulated the RNA expression of IL-12 and TNF-α in spleenocytes. The current findings suggest that the use of CpG would be more advantageous as an adjuvant predominantly in DNA/protein prime boost vaccine against Bm-Myo and presumably also for filarial infection.

Luminex-Coupled EliFACS: A Multiparametric Method to Enumerate and Functionally Characterize Antigen-Specific T cells in Human Peripheral Blood.

We describe a Luminex-coupled EliFACS assay that integrates multiplexing technology, enzyme-linked immunospot (ELISPOT), and intracellular cytokine FACS staining for the detection of multiple parameters of antigen-specific T-cell activation in human peripheral blood. Although our protocol is for measuring T-cell responses against cardiac myosin heavy chain and myelin basic protein, the major autoantigens in myocarditis and multiple sclerosis, respectively, these methods could be used for the detection of T-cell responses to other antigens, including foreign antigens.

MYH9 Key Amino Acid Residues Identified by the Anti-Idiotypic Antibody to Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 5 Involve in the Virus Internalization by Porcine Alveolar Macrophages.

MYH9 has been identified as an indispensable cellular protein for porcine reproductive and respiratory syndrome virus (PRRSV) entry into permissive cells using the monoclonal anti-idiotypic antibody (Mab2-5G2) recognizing an antibody that specifically interacts with PRRSV glycoprotein 5 (GP5). More recently, we found that Mab2-5G2 interacted with the MYH9 C-terminal domain, designated PRA, which is required for PRRSV internalization. In this study, we demonstrate that blocking of MYH9 with Mab2-5G2 significantly diminished PRRSV internalization by porcine alveolar macrophage (PAM) via interruption of direct interaction between GP5 and MYH9, and thus remarkably inhibited subsequent infection of PAMs by PRRSV-2 isolates. Moreover, the three-dimensional structure of the Mab2-5G2 Fab-PRA complex determined via homology modeling predicted potential docking sites required for PRRSV internalization. Further analysis of Mab2-5G2-binding sites within PRA highlighted that the amino acids E1670, K1673, E1679, and I1683 in PRA are the key Mab2-5G2-binding residues. Notably, recombinant PRA protein blocked the interaction between PRRSV GP5 and cellular MYH9 by preventing translocation of MYH9 from the cytoplasm to the cell membrane, an essential step for PRRSV virion internalization. Meanwhile, porcine cell line permissive for PRRSV bearing point mutation of E1670A in MYH9 demonstrated reduced susceptibility for PRRSV infection. In conclusion, this work increases understanding of both PRRSV pathogenesis and the mechanistic role played by MYH9 in PRRSV infection.

Ultrastructural and immunohistochemical study of phenotypic switch in gastrointestinal smooth muscle cells.

Dedifferentiation is a loss of phenotypic specialization that converts differentiated cells into adult stem cells in order to proliferate and differentiate into replacement tissue. This occurs in several tissues from various organs, such as smooth muscle cells (SMCs) of the mammalian gastrointestinal tract. The aim of this study was to describe ultrastructural and immunohistochemical changes in SMCs which could be compatible with a dedifferentiation process in human and rabbit intestinal muscles. Ultrastructural study and immunohistochemical staining (SMemb and MyoD) on human and rabbit duodenum tissue sections were performed. In both species, this dedifferentiation process is characterized by a loss of intercellular junctions, increased intercellular spaces, cytoskeletal disorganization, perinuclear accumulation of large vacuoles that tend to fuse, rupture of the vacuole membrane and release of cytoplasmic fragments. Dedifferentiated cells show the characteristic phenotype of a mesenchymal cell with scarce perinuclear cytoplasm, long cytoplasmic prolongations and finely distributed granular chromatin in the nucleus. These morphological changes are accompanied by a modulation to a less mature phenotype showing immunoreactivity for the embryonic form of the myosin heavy chain and for the myogenic regulatory factor MyoD. We suggest that SMC dedifferentiation includes the elimination of the contractile apparatus, the activation of the nucleus and the re-expression of embryonic markers. We described an ultrastructural dedifferentiation process possible in intestinal SMCs. This dedifferentiation process seems to play a key role in the homeostasis of the intestinal muscle.

Muscle fiber type diversity revealed by anti-myosin heavy chain antibodies.

Different forms of myosin heavy chains (MyHCs), coded by a large family of sarcomeric MYH genes, are expressed in striated muscles. The generation of specific anti-MyHC antibodies has provided a powerful tool to define the fiber types present in skeletal muscles, their functional properties, their response to conditions that affect muscle plasticity and their changes in muscle disorders. Cardiomyocyte heterogeneity has been revealed by the serendipitous observation that different MyHCs are present in atrial and ventricular myocardium and in heart conduction tissue. Developmental MyHCs present in embryonic and fetal/neonatal skeletal muscle are re-expressed during muscle regeneration and can be used to identify regenerating fibers in muscle diseases. MyHC isoforms provide cell type-specific markers to identify the signaling pathways that control muscle cell identity and are an essential reference to interpret the results of single-cell transcriptomics and proteomics.

A comprehensive comparison of four species of Onchidiidae provides insights on the morphological and molecular adaptations of invertebrates from shallow seas to wetlands.

The Onchidiidae family is ideal for studying the evolution of marine invertebrate species from sea to wetland environments. However, comparative studies of Onchidiidae species are rare. A total of 40 samples were collected from four species (10 specimens per onchidiid), and their histological and molecular differences were systematically evaluated to elucidate the morphological foundations underlying the adaptations of these species. A histological analysis was performed to compare the structures of respiratory organs (gill, lung sac, dorsal skin) among onchidiids, and transcriptome sequencing of four representative onchidiids was performed to investigate the molecular mechanisms associated with their respective habitats. Twenty-six SNP markers of Onchidium reevesii revealed some DNA polymorphisms determining visible traits. Non-muscle myosin heavy chain II (NMHC II) and myosin heavy chain (MyHC), which play essential roles in amphibian developmental processes, were found to be differentially expressed in different onchidiids and tissues. The species with higher terrestrial ability and increased integrated expression of Os-MHC (NMHC II gene) and the MyHC gene, illustrating that the expression levels of these genes were associated with the evolutionary degree. This study provides a comprehensive analysis of the adaptions of a diverse and widespread group of invertebrates, the Onchidiidae. Some onchidiids can breathe well through gills and skin when under seawater, and some can breathe well through lung sacs and skin when in wetlands. A histological comparison of respiratory organs and the relative expression levels of two genes provided insights into the adaptions of onchidiids that allowed their transition from shallow seas to wetlands. This work provides a valuable reference and might encourage further study.

HMGB1 silencing in macrophages prevented their functional skewing and ameliorated EAM development: Nuclear HMGB1 may be a checkpoint molecule of macrophage reprogramming.

High-mobility group box 1 (HMGB1), an important inflammatory factor, plays significant roles in CD4+T cell differentiation, cancer and autoimmune disease development. Our previous data have demonstrated that HMGB1 contributes to macrophage reprogramming and is involved in experimental autoimmune myocarditis (EAM) development. In contrast to the well-explored function of HMGB1, little is known about the nuclear function. Whether HMGB1 can serve as an architectural factor and control functional skewing of macrophages remains unclear. Therefore, the present work was performed to address the above speculation. The adenovirus-mediated shRNA (Ad-shRNA) was employed to knock down HMGB1 in RAW264.7 and monocytes/macrophages of EAM mice. Our data showed that in vitro HMGB1 silencing limited functional skewing of macrophages and down-regulated inflammatory factors secretion, which can't be reversed by the exogenous HMGB1. In M1 polarization system, the phosphorylations of NF-κB, p38 and Erk1/2 were inhibited following HMGB1 silencing. In vivo, HMGB1 silencing could effectively ameliorate EAM development. Our data suggest that HMGB1 may be a checkpoint nuclear factor of macrophage reprogramming. Our findings also provide an exciting therapeutic method for inflammatory disorders.

A One-Step Immunostaining Method to Visualize Rodent Muscle Fiber Type within a Single Specimen.

In this study, we present a quadruple immunostaining method for rapid muscle fiber typing of mice and rats using antibodies specific to the adult myosin heavy chain (MyHC) isoforms MyHC1, 2A, 2X, and 2B, which are common marker proteins of distinct muscle fiber types. We developed rat monoclonal antibodies specific to each MyHC isoform and conjugated these four antibodies to fluorophores with distinct excitation and emission wavelengths. By mixing the four types of conjugated antibodies, MyHC1, 2A, 2X, and 2B could be distinguished within a single specimen allowing for facile delineation of skeletal muscle fiber types. Furthermore, we could observe hybrid fibers expressing MyHC2X and MyHC2B together in single longitudinal muscle sections from mice and rats, that was not attained in previous techniques. This staining method is expected to be applied to study muscle fiber type transition in response to environmental factors, and to ultimately develop techniques to regulate animal muscle fiber types.

Immunization with Brugia malayi Myosin as Heterologous DNA Prime Protein Boost Induces Protective Immunity against B. malayi Infection in Mastomys coucha.

The current control strategies employing chemotherapy with diethylcarbamazine, ivermectin and albendazole have reduced transmission in some filaria-endemic areas, there is growing interest for complementary approaches, such as vaccines especially in light of threat of parasite developing resistance to mainstay drugs. We earlier demonstrated recombinant heavy chain myosin of B. malayi (Bm-Myo) as a potent vaccine candidate whose efficacy was enhanced by heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) vaccination in BALB/c mice. BALB/c mouse though does not support the full developmental cycle of B. malayi, however, the degree of protection may be studied in terms of transformation of challenged infective larvae (L3) to next stage (L4) with an ease of delineating the generated immunological response of host. In the current investigation, DNA vaccination with Bm-Myo was therefore undertaken in susceptible rodent host, Mastomys coucha (M. coucha) which sustains the challenged L3 and facilitates their further development to sexually mature adult parasites with patent microfilaraemia. Immunization schedule consisted of Myo-pcD and Myo-pcD+Bm-Myo followed by B. malayi L3 challenge and the degree of protection was evaluated by observing microfilaraemia as well as adult worm establishment. Myo-pcD+Bm-Myo immunized animals not only developed 78.5% reduced blood microfilarial density but also decreased adult worm establishment by 75.3%. In addition, 75.4% of the recovered live females revealed sterilization over those of respective control animals. Myo-pcD+Bm-Myo triggered higher production of specific IgG and its isotypes which induced marked cellular adhesion and cytotoxicity (ADCC) to microfilariae (mf) and L3 in vitro. Both Th1 and Th2 cytokines were significantly up-regulated displaying a mixed immune response conferring considerable protection against B. malayi establishment by engendering a long-lasting effective immune response and therefore emerges as a potential vaccination method against LF.

Phosphoinositide 3-kinase inhibitor LY294002 ameliorates the severity of myosin-induced myocarditis in mice.

BACKGROUND: Myocarditis, characterized by myocyte necrosis, fibrosis, and degeneration with mononuclear cell infiltration, always causes heart failure in patients. Phosphoinositide 3-kinase (PI3K) is a pivotal kinase known to regulate inflammatory responses in cardiac diseases. Although previous research has suggested that PI3K was involved in cardiac diseases such as myocardial infarction, it is still unclear whether the inhibition of PI3K is essential for the treatment of myosin-induced myocarditis. The aim of this study was to explore whether pharmacological blockade of PI3K is able to protect mice against experimental autoimmune myocarditis (EAM). MATERIALS AND METHODS: We used the cardiac myosin-induced murine EAM model to investigate the therapeutic effect of PI3K inhibitor LY294002 on autoimmune myocarditis in mice. RESULTS: LY294002 significantly alleviated EAM injury in mice, as indicated by the reduction of cardiac necrosis, inflammatory infiltrates, and CD3(+) T cells. LY294002 also decreased the expression of p-Akt upon cardiac myosin treatment in the cardiac tissue of the mice. In the present study, LY294002 resulted in a moderate reduction in absolute CD4(+) cell numbers and a significant decrease in the absolute numbers of CD8(+) cells. Consequently, LY294002 increased the CD4(+)/CD8(+) ratio compared with peptide treatment alone. CONCLUSION: This report provides evidence that PI3K inhibitor LY294002 has potent effects against cardiac injury caused by EAM, suggesting that it has therapeutic value for the treatment of myocarditis.