Fusion and regenerative therapies: is immortality really recessive?

Harnessing cellular fusion as a potential tool for regenerative therapy has been under tentative investigation for decades. A look back the history of fusion experiments in gerontology reveals that whereas some studies indicate that aging-related changes are conserved in fused cells, others have demonstrated that fusion can be used as a tool to revoke cellular senescence and induce tissue regeneration. Recent findings about the role of fusion processes in tissue homeostasis, replenishment, and repair link insights from fusion studies of previous decades with modern developments in stem cell biology and regenerative medicine. We suggest that age-associated loss of regenerative capacity is associated with a decline of effectiveness in stem cell fusion. We project how studies into the fusion of stem cells with tissue cells, or the fusion between activator stem cells and patient cells might help in the development of applications that "rejuvenate" certain target cells, thereby strategically reinstating a regeneration cascade. The outlook is concluded with a discussion of the next research milestones and the potential hazards of fusion therapies.     

Fusion of EGFP and porcine α 1,3GT genes decrease GFP expression

ObjectiveTo investigate the effect of fusion proteins expressed by the fused gene of porcine α 1, 3 galactosyltransferase (α1, 3 GT) and enhanced green fluorescent protein (EGFP) on the green fluorescence intensity of EGFP.MethodsThe fragment containing α 1, 3GT was firstly recovered after the pcDNA3.1- α 1, 3GT recombinant vector were digested with Bam HI and Eco RI, and then, the resultant fragment was ligated to the pEGFP-N1 vector which was also digested with the same enzymes. The new recombinant eukaryotic expression pEGFP/α 1, 3GT vector was obtained and sequenced. The pEGFP/α 1, 3GT was used to transfect human lung carcinoma cells A549 and HEKC 293FT, and the expression of EGFP was quantitatively analyzed by fluorescent microscope and flow cytometry.ResultsThe positive percentage of A549 was 80.5%, and that of 293 FT was 86.5% 48 hours after the two cell lines both were transfected by pEGFP-N1. The positive percentage of A549 was 75.8%, and that of 293 FT was 81.2% 48 hours after the two cell lines were transfected by pEGFP/α 1, 3GT. The mean fluorescence intensities of A549 transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 1.21 and 0.956, respectively when compared with that of A549 without transfection. Meanwhile, the those of the 293FT that were transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 7.66 and 1.00, respectively when compared with that of 293FT cells without transfection.ConclusionsThese results suggested that the expression of EGFP gene fused with porcine α 1, 3GT gene was partly inhibited.     

Fusion of imaging technologies: how,when, and for whom?

Over the past decade, electroanatomic mapping has emerged as a useful tool for complex ablation procedures. A more recent advancement is the development of image integration. Image integration refers to the process of registering a previously acquired MRI or CT scan of the heart with the mapping space during the ablation procedure. The technique of image integration is now relied on by many electrophysiology laboratories to guide complex ablation procedures, particularly atrial fibrillation ablation and ablation of patients with ventricular tachycardia in the setting of structural heart disease. An even more recent development is image fusion. This refers to taking information about the myocardial substrate, especially intramyocardial scar, and registering it with the active mapping space. This technique remains in its infancy but shows great promise in facilitating complex ablation procedures. The purpose of the article is to review the development, state of the art, and future of these image integration and fusion techniques.     

Early Events in Chikungunya Virus Infection—From Virus Cell Binding to Membrane Fusion

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research.     

Fusion of Na-ASP-2 with human immunoglobulin Fcγ abrogates histamine release from basophils sensitized with anti-Na-ASP-2 IgE

Na-ASP-2 is a major protein secreted by infective third-stage larvae (L3) of the human hookworm Necator americanus upon host entry. It was chosen as a lead vaccine candidate for its ability to elicit protective immune responses. However, clinical development of this antigen as a recombinant vaccine was halted because it caused allergic reactions among some of human volunteers previously infected with N.?americanus. To prevent IgE-mediated allergic reactions induced by Na-ASP-2 but keep its immunogenicity as a vaccine antigen, we designed and tested a genetically engineered fusion protein, Fcγ/Na-ASP-2, composed of full-length Na-ASP-2 and truncated human IgG Fcγ1 that targets the negative signalling receptor FcγRIIb expressed on pro-allergic cells. The chimeric recombinant Fcγ/Na-ASP-2 protein was expressed in Pichia pastoris and shared the similar antigenicity as native Na-ASP-2. Compared to Na-ASP-2, the chimeric fusion protein efficiently reduced the release of histamine in human basophils sensitized with anti-Na-ASP-2 IgE obtained from individuals living in a hookworm-endemic area. In dogs infected with canine hookworm, Fcγ/Na-ASP-2 resulted in significantly reduced immediate-type skin reactivity when injected intradermally compared with Na-ASP-2. Hamsters vaccinated with Fcγ/Na-ASP-2 formulated with Alhydrogel(?) produced specific IgG that recognized Na-ASP-2 and elicited similar protection level against N.?americanus L3 challenge as native Na-ASP-2.     

Localization of fossa ovalis and Brockenbrough needle prior to left atrial ablation using three-dimensional mapping with EnSite Fusion™

Background  

Transseptal catheterization of the interatrial septum has traditionally been performed under the guidance of fluoroscopy, echocardiography, and hemodynamic pressure monitoring. We hypothesized that the fossa ovalis could be identified on pre-ablation chest computerized tomography (CT) scan utilizing EnSite Verismo™ and Fusion™ software thereby permitting its real-time visualization during transseptal puncture.     

Exogenous PML/RARα Fusion Gene Responds to All-trans Retinoic Acid Results in Differentiation of the Human B Cell Line

The interaction of an exogenous PML/RARα fusion gene, associated with acute promyelocytic leukemia, with all-trans retinoic acid (ATRA) was examined in B-lymphoid cell lines. RPMI8866 cells were transfected with PML/RARα cDNA in the expression vector pGD and two stable transformants (RPMI8866Y-4 and RPMI8866Y-17) were established by selection with G418. ATRA inhibited the growth of those stable transformants, as assessed by [³H]-thymidine incorporation, but had no effect on the growth of control cells stably transformed with neomycin resistant gene alone. ATRA also increased expression of CD38 and immunoglobulin production in RPMI8866Y-4 cells but not in control cells. When these results are taken together, it can be observed that the exogenous PML/RARα fusion gene responds to ATRA, which results in cell differentiation of the human B cell line.     

Chronic Eosinophilic Leukemia with the FIP1L1-PDGFR± Fusion Gene in a Patient with a History of Combination Chemotherapy

Hypereosinophilic syndrome (HES) was diagnosed in December 2000 in a 43-year-old man on the basis of persistent eosinophilia (11.7 x 10(9)/L) and a normal karyotype of the bone marrow cells. He had developed intra-abdominal non-Hodgkin's lymphoma and in 1992 had received 3 courses of combination chemotherapy with doxorubicin (Adriamycin), cyclophosphamide, vincristine, methotrexate, bleomycin, and prednisolone. The patient was orally given prednisolone (10 mg/day) and cyclophosphamide (50 mg/day) as HES treatment without a subsequent improvement of the eosinophilia. In May 2003, anemia (hemoglobin, 7.9 g/dL) and thrombocytopenia (65 x 10(9)/L) manifested with progressive eosinophilia (21.0 x 10(9)/L) and a small number of blasts. The patient became febrile and was admitted in July 2003. Cytogenetic reexamination of the bone marrow cells disclosed the deletion of 4q12, indicating the presence of a fusion of the Fip1-like 1 (FIP1L1) gene to the plateletderived growth factor receptor alpha (PDGFRalpha) gene and consequently the clonal nature of his hematopoietic cells. DNA sequence analysis demonstrated that the breakpoints of the FIP1L1 and PDGFRalpha genes were present in exon 9 and exon 12, respectively. Treatment with imatinib mesylate (300 mg/day) promptly brought about complete remission. Although a number of similar eosinophilic cases have been reported, our patient may be the first such patient with a history of chemotherapy.     

Fusion of epithelial cells by Epstein–Barr virus proteins is triggered by binding of viral glycoproteins gHgL to integrins αvβ6 or αvβ8

Epstein–Barr^, virus (EBV) is a ubiquitous human herpesvirus that is causally implicated in the development of lymphoid and epithelial tumors. Entry of virus requires fusion of virus envelopes and cell membranes. Fusion with B lymphocytes requires virus glycoprotein gB and a 3-part complex of glycoproteins, gHgLgp42. It is triggered by interactions between glycoprotein 42 (gp42) and HLA class II. However, fusion with epithelial cells is impeded by gp42 and instead is triggered by interactions between an unknown epithelial protein and a 2-part complex of gHgL. We report here that gHgL binds with high affinity to epithelial cells and that affinity of binding is increased by 3 orders of magnitude in the presence of Mn²⁺. Binding and infection can be reduced by fibronectin and vitronectin, by down-regulation of integrin αv, or by a peptide corresponding to 13 aa of gH which include a KGDE motif. Fusion of cells expressing gB and gHgL can be blocked by vitronectin or triggered by addition of soluble truncated integrins αvβ6 and αvβ8. We conclude that the direct interaction between EBV gHgL and integrins αvβ6 and αvβ8 can provide the trigger for fusion of EBV with an epithelial cell.Epstein–Barr virus (EBV) is carried by >90% of the adult population worldwide. Many individuals are infected asymptomatically in childhood, but, although primary infection in adolescence or later often is accompanied by infectious mononucleosis, the major impact of the virus results from its role as a tumor initiator or tumor progressor. EBV is associated with both lymphoid and epithelial malignancies, reflecting its primary tropism for these 2 cell types (1).The proteins involved in EBV penetration of a B cell are more clearly defined than those required for epithelial cells (reviewed in ref. 2). Attachment is mediated by an interaction between envelope glycoprotein gp350 and the complement receptor type 2 (CR2) or CD21. Fusion, as for all herpesviruses, requires the core fusion machinery (3) of glycoproteins gB and gH and its partner gL, which is essential to the folding and transport of gH. Triggering of the core fusion machinery from a metastable to an active state requires an interaction with HLA class II, which functions as a coreceptor or entry mediator on the B-cell surface. The interaction is mediated by an additional glycoprotein, gp42, which, like gL, binds directly to gH to form a 3-part complex, gHgLgp42.Attachment of EBV to epithelial cells can be mediated by gp350, but on CR2-negative cells it also can be mediated by a 2-part complex, gHgL. A soluble truncated form of the complex, gHtgL, can bind specifically to epithelial cells but not to B cells (4), and a gH-null virus, which can still use gp350 to bind to CR2-positive cells, loses the ability to bind to a CR2-negative epithelial cell (5, 6). In addition, HLA class II, which is not constitutively expressed on epithelial cells, is unavailable to trigger fusion. Instead, fusion with an epithelial cell requires an unknown coreceptor, gB, and the gHgL complex that lacks gp42. Virus carries both 3-part gHgLgp42 complexes and 2-part gHgL complexes to accommodate infection of 2 cell types. Only 3-part complexes can mediate fusion with B cells, but only 2-part gHgL complexes can mediate entry of epithelial cells (7), and changes in the levels of gp42 by sequestration and degradation in an HLA class II-positive B cell, but not in an HLA class II-negative epithelial cell, switch virus tropism (8). The presence of gp42 blocks the ability of gHgL to mediate virus binding to an epithelial cell that lacks CR2 (4) and also blocks the ability of virus bound via gp350 to a CR2-positive cell to infect (7).These observations, together with the findings that a monoclonal antibody to gHgL not only blocked gHgL binding and virus binding to a CR2-negative cell but also blocked entry of bound virus into a CR2-positive epithelial cell (4, 5), suggested that the molecule that serves as an epithelial gHgL receptor might also serve as the missing coreceptor needed to trigger epithelial cell fusion by EBV glycoproteins. We previously have speculated that this triggering might in fact be the primary function of the gHgL receptor, because, although the binding of the virus to the molecule is relatively robust, the ability of the virus to enter the cell when using the molecule, rather than CR2, for attachment is not (4). We report here that 1 set of proteins that can function as gHgL receptors are αv-containing integrins. Downregulation of αv expression reduced binding and infection, as did a peptide corresponding to residues of 184–196 of the gH precursor, which include a putative integrin-binding motif, KGDXXXL. Further, soluble forms of human integrins αvβ6 and αvβ8 triggered epithelial cell fusion mediated by EBV gB and gHgL. We conclude that integrins are able to serve both for attachment and fusion of EBV with epithelial cells, although their most important role is likely to be in fusion.     

Clinical Features of Hypereosinophilic Syndrome: FIP1L1-PDGFRA Fusion Gene—Positive Disease is a Distinct Clinical Entity with Myeloproliferative Features and a Poor Response to Corticosteroid

We conducted a retrospective analysis of the clinical features of 20 patients with severe eosinophilia at our institution, including 10 cases of hypereosinophilic syndrome (HES) (5 definite and 5 probable cases) and 10 cases of other eosinophilic disorders. Of the 20 patients, 14 initially received prednisolone treatment, which resulted in rapid improvement and normalization of eosinophilia within 8 weeks; however, 2 patients with splenomegaly showed poor control of eosinophilia in response to corticosteroid treatment. In addition, the FIP1L1-PDGFRA fusion gene was detected only in these 2 cases. One of the FIP1L1-PDGFRA - positive HES cases featured bone marrow fibrosis. Treatment of this patient with imatinib mesylate resulted in a dramatic improvement of eosinophilia, organomegaly, and the bone marrow fibrosis. Taken together, our data and previous reports suggest that FIP1L1-PDGFRA - positive HES is a distinct clinical entity with myeloproliferative features and showing a poor response to corticosteroid treatment.     

Paradigmenwechsel in der universit?ren Medizin? Zur Fusion und Privatisierung von Universit?tskliniken am Beispiel von Marburg und Gie?en

1. Die Fusion der Universitätskliniken in Mittelhessen ist eine Zwangsheirat mit Risiko (Der Spiegel) [1]. Sie ist in der gegenwärtigen politischen und finanziellen Lage des landes aber wohl kaum vermeidbar. Aus Marburger Sicht ist es schmerzlich, die Gewinne und Rückstellungen eines wirtschaftlich bestens geführten Universitätsklinikums in die Defizite einer weniger effizient geführten Gießener Klinik und ggf. in deren zukünftige Baumaßnahmen geben zu müssen.2. Beide Fakultäten leiden unter einer extrem niedrigen Landeszuweisung für Forschung und Lehre, die bezogen auf die Studentenzahl und Professoren beträchtlich unter dem deutschen Mittelwert liegt. Alle 3 hessischen Medizinfachbereiche gehören zu den 5 am schlechtesten von den jeweiligen Bundesländern finanzierten Fakultäten. Beide Universitätskliniken, Gießen allerdings wesentlich mehr als Marburg, sehen sich für die kommenden 7–10 Jahre mit einem Investitionsstau in die Bausubstanz, beide in gleichem Umfang in die Großgeräte (HBFG) konfrontiert.