Formation Of Multinucleated Syncytia Research Articles

Large and Small Assembly: Combining Functional Macromolecules with Small Peptides to Control the Morphology of Skeletal Muscle Progenitor Cells.

The material properties of natural tissues, such as skeletal muscle, are highly sophisticated and are synthetically challenging to mimic. Using natural biomacromolecules to functionalize self-assembled peptide (SAP) hydrogels has the potential to increase the utility of these materials by more closely reproducing the natural cellular environment. Here, to demonstrate that a conserved co-assembly pathway can retain distinct function, the biocompatible peptide derivative Fmoc-FRGDF was co-assembled with either a sulfated polysaccharide, fucoidan, or the provisional matrix proteoglycan, versican. Our results demonstrate that thermodynamically driven co-assembly with biologically active macromolecules is facile, stable, and does not affect the final assembled nanostructure. Biologically, the incorporation of these functionally distinct molecules had no effect on C2C12 myoblast proliferation and viability but strongly altered their morphology. The surface area of myoblasts cultured on the fucoidan scaffold was reduced at 24 and 72 h post seeding, with a reduction in the formation of multinucleated syncytia. Myoblasts cultured on versican scaffolds were smaller compared to cells grown on the empty vector scaffolds at 24 h but not 72 h post seeding, with multinucleated syncytia formation being unaffected. This work allows programmed and distinct morphological effects of cell behavior, paving the way for further mechanistic studies.

A Microsporidian Infection in Phoronids (Phylum Phoronida): Microsporidium phoronidi n. sp. from a Phoronis embryolabi.

Microsporidia-like spores (2.0-3.0×1.3-1.5μm) were discovered upon examination of histological sections taken from Phoronis embryolabi Temereva, Chichvarkhin 2017 found inhabiting burrows of shrimps Nihonotrypeae japonica (Decapoda, Callianassidae) from the Sea of Japan, Russia. Ultrastructural examination of spores revealed one nucleus and a uniform polar filament of 7-11 coils. Representatives of the phylum Phoronida have never been recorded as hosts of microsporidia. Parasites developed in vasoperitoneal tissue and caused formation of multinucleate syncytia. Basing on unique host and fine morphology, we assign the novel finding to Microsporidium phoronidi n. sp. and place provisionally in the collective genus Microsporidium.

NT-17 * TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) WITH AN ONCOLYTIC MEASLES VIRUS IN COMBINATION WITH INHIBITORS OF THE PI3-KINASE PATHWAY

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a highly malignant pediatric brainstem tumor with less than 10% of patients surviving beyond 2 years. Surgical resection of this diffusely infiltrative tumor is not possible, radiation therapy is palliative not curative, and chemotherapy is ineffective. Clearly, more effective therapy for DIPG is necessary. The genomic analyses on tissue samples from biopsy and autopsy have frequently implicated amplification in the PI3K-mTOR pathway signaling in DIPG tumors. The vaccine strain of measles virus (MV) has been shown to be an effective oncolytic therapy in brain tumors in murine xenograft models. METHODS AND RESULTS: Flow cytometry showed that human DIPG cells abundantly express the CD46 receptor on the membrane that is preferentially utilized by MV for cellular entry. MV efficiently infects DIPG cells, replicates in these cells, and causes the formation of multinucleated syncytia. MV kills DIPG cells in dose and time dependent manner with more than 70% cell death observed at MOI of 0.5 after 96hrs. The mTORC1 inhibitor, rapamycin, and the Akt inhibitor, MK2206, individually attenuated the proliferation of DIPG cells (p < 0.05). Importantly, the killing of DIPG cells by MV was synergistically increased when used in combination with rapamycin or MK2206, as determined by the Chou-Talalay equation (synergism if combination index (CI) < 1). The CI values for MV-rapamycin and MV-MK2206 treatments were 0.4 and 0.8 respectively, at EC50. Cells treated with MV in combination with the inhibitor produced 10-times more virus than cells treated with the virus alone. The switch from apoptosis caused by MV alone to autophagy seen when inhibitors are used may be responsible for the increased virus titer. CONCLUSION: Measles virus has therapeutic potential in the treatment of DIPG tumors and the efficacy may be improved by simultaneously targeting the Akt-mTOR pathway.

Centrosomal proteins are reorganized in fused trophoblastic cells. (LB57)

Cell‐cell fusion is a cardinal event in the formation of multinucleate syncytia and is part of the normal biology of the syncytiotrophoblast (STB) layer of the human placenta. We have found numerous differences between non‐fused and fused trophoblastic cells in culture.We have now compared centrosomal proteins and microtubules in non‐fused and fused BeWo cells. In non‐fused BeWo cells, the centrosomal proteins, pericentrin and gamma‐tubulin, were present as one or two perinuclear dot‐like structures. Following forskolin‐induced cell fusion, the immunofluorescence labeling pattern for pericentrin and gamma‐tubulin was dramatically altered and appeared to increase in amount and had a “cloud‐like” distribution. Microtubule nucleation experiments in which microtubules regrow following depolymerization with nocodazole were conducted. In non‐fused cells, microtubules regrow from the single microtubule organization center (MTOC). However, in fused cells there were multiple sites of regrowth corresponding to pericentrin and gamma‐tubulin positive‐foci. Examination of human placental tissue revealed that centrosomal proteins in the STB were distributed in a manner similar to that found in fused BeWo cells while mononuclear cells within the placenta have MTOCs similar to those in non‐fused BeWo cells. Thus the cell culture model recapitulates the novel situation in the tissue.Grant Funding Source: Supported by NIH RO1HD058084

RAGE inhibits human respiratory syncytial virus syncytium formation by interfering with F-protein function

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.

Asymmetric Mbc, active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion inDrosophila

Myoblast fusion is an intricate process that is initiated by cell recognition and adhesion, and culminates in cell membrane breakdown and formation of multinucleate syncytia. In the Drosophila embryo, this process occurs asymmetrically between founder cells that pattern the musculature and fusion-competent myoblasts (FCMs) that account for the bulk of the myoblasts. The present studies clarify and amplify current models of myoblast fusion in several important ways. We demonstrate that the non-conventional guanine nucleotide exchange factor (GEF) Mbc plays a fundamental role in the FCMs, where it functions to activate Rac1, but is not required in the founder cells for fusion. Mbc, active Rac1 and F-actin foci are highly enriched in the FCMs, where they localize to the Sns:Kirre junction. Furthermore, Mbc is crucial for the integrity of the F-actin foci and the FCM cytoskeleton, presumably via its activation of Rac1 in these cells. Finally, the local asymmetric distribution of these proteins at adhesion sites is reminiscent of invasive podosomes and, consistent with this model, they are enriched at sites of membrane deformation, where the FCM protrudes into the founder cell/myotube. These data are consistent with models promoting actin polymerization as the driving force for myoblast fusion.

The Nipah Virus Fusion Protein Is Cleaved within the Endosomal Compartment

Nipah virus (NiV) is a recently emerged and highly pathogenic paramyxovirus that causes a systemic infection in animals and humans and can infect a wide range of cultured cells. Interestingly, the NiV fusion (F) protein has a single arginine at the cleavage site similar to paramyxoviruses that are activated by exogenous trypsin-like enzymes only present in specific cells and tissues and therefore only cause localized infections. We show here that NiV F activation is not mediated by an exogenous serum protease but by an endogenous ubiquitous cellular protease after endocytosis of the protein. In addition to endocytosis, acidification of the endosome is a prerequisite for F cleavage. These results show that activation of the NiV F protein depends on a type of proteolytic cleavage that is clearly different from what is known for other paramyxoviral and orthomyxoviral fusion proteins. To our knowledge, this is the first example of a viral class I fusion protein whose activation depends on clathrin-mediated constitutive endocytosis.

Systemic therapy of myeloma xenografts by an attenuated measles virus

Conditionally replicating viruses are promising agents for the treatment of malignancy. Here it is shown that the live attenuated Edmonston-B vaccine strain of measles virus (MV-Edm) replicates selectively in human myeloma cells and has potent antitumor activity. In vitro, replication of MV-Edm was restricted in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs) but proceeded efficiently in a panel of 6 myeloma cell lines-ARH-77, RPMI 8226, JJN-3, MM1, KAS-6/1, and KMS-11-and in primary myeloma cells isolated by CD138 sorting from the bone marrow aspirates of 6 patients. MV-Edm infection induced potent cytopathic effects in these myeloma cells, resulting in the formation of multinucleated syncytia that eventually became nonviable. In contrast, syncytial formation in PHA-stimulated PBLs was minimal after MV-Edm infection. In vivo, MV-Edm was antitumorigenic and inhibited the establishment of myeloma cells as xenografts in immunocompromised mice. When injected directly into ARH-77 myeloma xenografts in the mice, MV-Edm caused complete regression of these xenografts. MV-Edm administered intravenously into the tail veins of mice also showed significant antineoplastic activity against established RPMI 8226 and ARH-77 xenografts. In particular, the ARH-77 myeloma xenografts were exquisitely sensitive to MV-Edm therapy, and tumors in all mice regressed completely. In light of its selectivity for myeloma cells and its potent antineoplastic activity against myeloma xenografts in vivo, MV-Edm merits further development for the treatment of multiple myeloma.

CXCR4 on human endothelial cells can serve as both a mediator of biological responses and as a receptor for HIV-2

It has been shown that deletion of the chemokine receptor, CXCR4, causes disordered angiogenesis in mouse models. In the present studies, we examined the distribution and trafficking of CXCR4 in human endothelial cells, tested their responses to the CXCR4 ligand, SDF-1, and asked whether endothelial cell CXCR4 can serve as a cell surface receptor for the binding of viruses. The results show that CXCR4 is present on endothelial cells from coronary arteries, iliac arteries and umbilical veins (HUVEC), but expression was heterogeneous, with some cells expressing CXCR4 on their surface, while others did not. Addition of SDF-1 caused a rapid decrease in CXCR4 surface expression. It also caused CXCR4-mediated activation of MAPK, release of PGI 2, endothelial migration, and the formation of capillary-like structures by endothelial cells in culture. Co-culture of HUVEC with lymphoid cells that were chronically infected with a CD4-independent/CXCR4-tropic variant of HIV-2 resulted in the formation of multinucleated syncytia. Formation of the syncytia was inhibited by each of several different CXCR4 antibodies. Thus, our findings indicate: (1) that CXCR4 is widely expressed on human endothelial cells; (2) the CXCR4 ligand, SDF-1, can evoke a wide variety of responses from human endothelial cells; and (3) CXCR4 on endothelial cells can serve as a receptor for isolates of HIV that can utilize chemokine receptors in the absence of CD4.

Generation of lymphocyte cell lines coexpressing CD4 and wild-type or mutant HIV type 1 glycoproteins: implications for HIV type 1 Env-induced cell lysis.

To gain more insight into the processes leading to HIV-1 Env-induced cell death, we aim to coexpress stably wild-type and relevant mutant variants of both HIV-1 Env and human CD4 in lymphocyte cell lines. Here we report on the generation and characterization of several cell lines inducibly or constitutively expressing wild-type or cleavage-defective HIV-1 glycoproteins and human CD4 either singly or in combination. Coexpression of CD4 and wild-type Env led to the formation of multinucleated syncytia, to growth arrest and cell death, effects that all could be prevented by cultivation in the presence of monoclonal antibodies that inhibit cell surface membrane fusion. Cell lines coexpressing CD4 and mutated, noncleavable Env, detectable at the cell surface and still retaining CD4-binding capacity, were not retarded in their growth and cytolysis did not occur. These results indicate that cell lysis requires cell surface interaction of CD4 and gp120/41 and cleavage of gp160 to gp120 and gp41.

Restriction of Fusion Protein mRNA as a Mechanism of Measles Virus Persistence

Vero cells persistently infected with the hamster neurotropic strain (HNT-PI) of measles virus are deficient in the release of extracellular virus and syncytia formation, suggesting that mutations occur within the viral envelope proteins. Nucleotide sequence comparisons indicated that the coding regions of the matrix (M) and fusion (F) genes of HNT-PI were relatively conserved compared with those of their lytic progenitor virus Philadelphia 26 (Ph26), whereas the hemagglutinin (H) gene differed by 4.2% at the amino acid level. Northern blot analyses demonstrated the predominance of bicistronic M/F transcripts in HNT-PI at a 5:1 ratio over F monocistronic mRNA. Accordingly, no F protein could be detected in the HNT-PI cell line, although both the M and H proteins were produced in amounts comparable to those of Ph26. When the Semliki Forest virus replicon was used, coexpression of the HNT-PI F and Ph26 H genes resulted in the formation of multinucleated syncytia in transfected Veto cell cultures. Since the HNT-PI F protein was fusogenic, the restriction of its monocistronic mRNA is postulated to be a contributing factor in the reduction of cell fusion and ultimately in the maintenance of the persistent infection.

Pathologic and immunocytochemical studies of morbillivirus infection in striped dolphins (Stenella coeruleoalba).

Hundreds of striped dolphins (Stenella coeruleoalba) died along the Spanish Mediterranean coast during the second half of 1990. We necropsied 58 dolphins. Partial collapse of the lungs with patchy atelectasis, subcutaneous edema, icterus, and stomatitis were the most prominent gross morphologic changes. Histologically, a bronchiolo-interstitial pneumonia was the most frequent lesion (72% of the animals). It was characterized by hyperplasia of alveolar epithelial type II cells and formation of multinucleate syncytia in alveolar and bronchiolar lumina. Other prominent lesions were encephalitis (69%), lymphoid depletion, and formation of multinucleate syncytia in the cortex of lymph nodes. The distribution of morbillivirus antigen was investigated in 23 well-preserved dolphins using a monoclonal antibody against the hemagglutinin glycoprotein of phocine distemper virus. Positive immunostaining was found in brain (77%), in lung (70%), and in mesenteric (61%), mediastinal (47%), and prescapular (45%) lymph nodes. Phocine distemper virus antigen was demonstrated less frequently in trachea, stomach, biliary epithelium, intestine, kidney, and mammary gland. Necrotizing-hemorrhagic pneumonia and encephalitis due to Aspergillus fumigatus were seen in three dolphins, whereas two animals had lesions of toxoplasmosis. Changes in our dolphins were similar to those caused by distemper in seals and porpoises. The origin of the dolphin virus and the relationships among dolphin, seal, and porpoise morbilli viruses are unknown.

Role of CD4 in Normal Immunity and HIV Infection

In this report we have attempted to review our knowledge of the role(s) of CD4 in human T-cell function and the consequences of interactions between CD4 molecules and the human immunodeficiency virus (HIV). The observation in 1981 that antibodies to certain epitopes of CD4 inhibited the immune functions of CD4+ T cells led to the initial suggestion that CD4 molecules play a direct role in T-cell function. Although the precise functions of CD4 remain incompletely understood, a preponderance of evidence suggests that this molecule may in fact serve several critical roles. At least one such role is that of interacting directly with MHC class II molecules on antigen-presenting cells, presumably facilitating cell-to-cell interactions. On activated CD4+ T cells, CD4 molecules can also interact directly with the T-cell receptor complex to influence the immune response. Unfortunately, in addition to interacting with the T-cell receptor and class II MHC determinants, CD4 serves as a high affinity receptor for HIV, the causative agent of AIDS. Not only does interaction between the virus and CD4 initiate viral fusion to the cell membrane and HIV entry but, in addition, a similar molecular interaction initiates fusion between HIV-infected and uninfected CD4+ cells, resulting in the formation of multinucleated syncytia. Since uninfected CD4+ cells are, in effect, recruited into such syncytia, this mechanism may account in part for the depletion of CD4+ T cells in HIV-infected patients. Soluble forms of CD4 produced either by genetic engineering or solid phase peptide synthesis can completely block HIV infectivity and syncytia formation in vitro, remarkably without apparent effects on T-cell immunity. Such molecules are currently being explored for their possible therapeutic effects on HIV infection in vivo.

Isolation of Virus‐producing Transformants from Human Gastric Cancer Cell Line, HGC‐27, Infected with Human T‐cell Leukemia Virus Type I

A human anaplastic gastric cancer cell line, HGC‐27, showed marked degeneration with formation of multinucleated syncytia and cell detachment of nearly all cells which began 24 hr after and reached a maximum 2 to 3 days after co‐cultivation with X‐irradiated MT‐2 cells, HTLV‐I producing human cord leukocytes. Less severe degeneration without formation of syncytia was also observed in the cultures inoculated with cell‐free MT‐2 culture media. Morphologically altered cells began to proliferate and formed piled up colonies in some of the cultures co‐cultivated with X‐irradiated MT‐2 cells after a long culture period. The two clones designated HGC/MT2 (Cl‐1) and HGC/MT2 (Cl‐2) were separated by cell cloning. HGC/MT2 (Cl‐1) and HGC/MT2 (Cl‐2) cells were positive for HTLV‐I gag proteins (p19 and p24) and pX gene products, p40x, as demonstrated by immunohistochemistry and immunoblotting analysis, contained HTLV‐I provirus DNA, and consistently produced type C virus particles.

The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus.

Acquired immune deficiency syndrome (AIDS) is characterized by opportunistic infections and by 'opportunistic neoplasms' (for example, Kaposi's sarcoma). Persistent generalized lymphadenopathy (PGL) is epidemiologically associated with AIDS, especially in male homosexuals. A subset of T lymphocytes positive for the CD4 antigen (also termed T4 antigen), is depleted in AIDS and PGL patients. A retrovirus found in T-cell cultures from these patients is strongly implicated in the aetiology of AIDS because of the high frequency of isolation and the prevalence of specific antibodies in the patients. Here we have detected cell-surface receptors for the AIDS retrovirus (human T-cell leukaemia virus-III (HTLV-III) and lymphadenopathy-associated virus-1 (LAV-1) isolates) by testing the susceptibility of cells to infection with pseudotypes of vesicular stomatitis virus bearing retroviral envelope antigens, and by the formation of multinucleated syncytia on mixing virus-producing cells with receptor-bearing cells. Receptors were present only on cells expressing CD4 antigen; among 155 monoclonal antibodies tested, each of the 14 anti-CD4 antibodies inhibited formation of syncytia and blocked pseudotypes. Productive infection of CD4+ cells with HTLV-III or LAV-1 markedly reduced cell-surface expression of CD4. In contrast, receptors for HTLV-I and HTLV-II were not restricted to CD4+ cells, were not blocked by anti-CD4 antibodies; cells productively infected with HTLV-I and HTLV-II expressed surface CD4. Hence, we conclude that the CD4 antigen is an essential and specific component of the receptor for the causative agent of AIDS.

Recovery of a new herpesvirus from the ground squirrel (Citellus citellus).

A new herpesvirus recovered from the kidneys of mature ground squirrels (Citellus citellus) produced an effect in tissue culture cells resembling that of herpesviruses, including the formation of multinucleated syncytia and type A intranuclear inclusions. The isolate caused latent infection in the natural host, but it proved fatal for intracerebrally inoculated suckling mice and it produced pocks on the chorioallantoic membrane of embryonated eggs. Electron-microscopic examination of infected cells revealed intranuclear virus particles exhibiting a size and ultrastructure characteristic of herpesviruses. The isolate was ether-resistant and a DNA nucleic acid type was inferred from observations on inhibition by fluorinated pyrimidine. This ground squirrel herpesvirus was found to be antigenically distinct from Herpesvirus hominis and from ground squirrel cytomegalovirus, with no detectable cross-reactivity.