人源细胞对猪内源性逆转录病毒易感性的初步研究

目的 对6种人源细胞对猪内源性逆转录病毒(PERV)的易感性进行研究。方法 用PERV病毒上清感染培养中的6种人源细胞后，用PCR和RT—PCR法研究PERV是否感染这6种人源细胞。结果 张氏肝细胞和人胚软骨细胞对PERV不易感，其余4种人源细胞对PERV易感。结论 不同组织来源的人源细胞对PERV的易感性是不同的。     

Activation of microglia by retroviral infection correlates with transient clearance of prions from the brain but does not change incubation time

Prion diseases are fatal transmissible diseases, where conversion of the endogenous prion protein (PrP^C) into a misfolded isoform (PrP^Sc) leads to neurodegeneration. Microglia, the immune cells of the brain, are activated in neurodegenerative disorders including prion diseases; however, their impact on prion disease pathophysiology is unclear with both beneficial PrP^Sc‐clearing and detrimental potentially neurotoxic effects. Moreover, monocytes entering the brain from the periphery during disease course might add to disease pathophysiology. Here, the degree of microglia activation in the brain of prion infected mice with and without an additional intraperitoneal retrovirus infection was studied. Peripheral murine retrovirus infection leads to activation of parenchymal microglia without recruitment of monocytes. This activation correlated with transient clearance or delay in accumulation of infectious prions specifically from the brain at early time points in the diseases course. Microglia expression profiling showed upregulation of genes involved in protein degradation coinciding with prion clearance. This enforces a concept where microglia act beneficial in prion disease if adequately activated. Once microglia activation has ceased, prion disease reemerges leading to disease kinetics undistinguishable from the situation in prion‐only infected mice. This might be caused by the loss of microglial homeostatic function at clinical prion disease.     

Tunable assembly of amyloid-forming peptides into nanosheets as a retrovirus carrier

Using and engineering amyloid as nanomaterials are blossoming trends in bionanotechnology. Here, we show our discovery of an amyloid structure, termed “amyloid-like nanosheet,” formed by a key amyloid-forming segment of Alzheimer’s Aβ. Combining multiple biophysical and computational approaches, we proposed a structural model for the nanosheet that is formed by stacking the amyloid fibril spines perpendicular to the fibril axis. We further used the nanosheet for laboratorial retroviral transduction enhancement and directly visualized the presence of virus on the nanosheet surface by electron microscopy. Furthermore, based on our structural model, we designed nanosheet-forming peptides with different functionalities, elucidating the potential of rational design for amyloid-based materials with novel architecture and function.Numerous proteins and polypeptides have been found to self-assemble into amyloid fibrils under certain conditions (1). They are associated not only with dozens of devastating diseases including Alzheimer’s and Parkinson’s diseases (2) but are integral to many biological processes such as hormone storage, signal transduction, and cell surface adhesion (3–5). Separate from the context of their parent proteins, synthesized peptide segments can self-assemble into amyloid-like fibrils in vitro as well (6, 7). Fibrils formed by diverse proteins and peptides all share a common cross-β structure, composed of interdigitated β-sheets termed “the zipper-like fibril spine” (8, 9). The self-assembly process is a consequence of backbone hydrogen bonding for the single β-sheet layer formation and side-chain interaction (e.g., hydrophobic interaction, π-stacking, and van der Waals) for pairing β-sheet layers together (10). Their highly repetitive and ordered architecture, in particular for the short peptide fibrils, exhibits favorable properties including high thermal stability and stiffness, biocompatibility, controllable self-assembly, surface patterning and integration of functionality, and inexpensive production by chemical synthesis (11–13). These exceptional properties promote the exploitation of amyloid fibril as an emerging class of bionanomaterials (14).Several studies have demonstrated that natural amyloidogenic and designed amphiphilic peptides are capable of self-assembling into nanostructures with topographies including fibril, film, nanotube, hydrogel, and liquid crystals (15–21), and these nanostructures have been used for nanowires, biosensors, 3D culturing, environmental carbon capture, retroviral gene transfer, light harvesting, and catalysis (22–26). Amyloid fibrils were also hybridized with other nanomaterials such as graphene and DNA origami in hopes of creating new properties and functions (27–29). In this study, we expand the amyloid material field’s scope by the finding, structure characterization, and functionalization of a previously unidentified architecture—the amyloid-like nanosheet. We showed that KLVFFAK, a key amyloid-forming heptapeptide of the Italian familial form of Alzheimer’s Aβ (30), self-assembles into a 2D nanosheet with a width of over 200 nm, far larger than typical fibrils (10–20 nm) (Fig. 1). We characterized the molecular structure of the nanosheet with both biophysical and computational approaches. The unique structural architecture of the nanosheet enables us to use it as a highly effective enhancer for retroviral transduction and to directly observe virus condensation on the nanosheet. Based on our structural model, we further designed two series of artificial peptides. One highlights the potential of the nanosheet as a robust platform for integrating different functionalities, and the other sheds light on the de novo design of amyloid-like nanosheets.Open in a separate windowFig. 1.Characterization of the KLVFFAK nanosheet. (A) KLVFFAK is the key amyloid-forming segment of the Italian familial form of Aβ. Its chemical formation is shown in β-strand, and its length is ∼2.2 nm estimated from its structural model (SI Appendix, Materials and Methods). (B) AFM image (Lower) and section analysis (Upper) of the KLVFFAK nanosheet on mica. The thickness of the KLVFFAK nanosheet is about the length of the KLVFFAK β-strand, indicating an upright alignment of the peptide strands forming the nanosheet. (C) TEM images of the KLVFFAK nanosheet and a typical amyloid fibril of peptide VQIVYK (Inset). (D) Optical image (Upper) of the Congo red-stained nanosheet. It shows green birefringence when viewed between crossed polarizers (Lower). (E) X-ray diffraction image of nanosheet showing a cross-β diffraction pattern characteristically seen in fibrils (2). (F) FTIR profile indicating that the nanosheet is composed of antiparallel β-sheets.     

Transfer of p16inka/CDKN2 gene in leukaemic cell lines inhibits cell proliferation

The gene encoding for p16^ink4a, a negative regulator of transition between G1 and S phase, is homozygously deleted in a large proportion of acute lymphoblastic leukaemias (ALL). Transfer of p16^ink4a gene in several solid tumour cell lines with functional pRb and lacking both p16^ink4a alleles has resulted in a dramatic reduction of cell proliferation, and the aim of this work was to confirm this effect in leukaemic (especially ALL) cell lines. We tested the proliferation in liquid medium and in soft agar after transfer of p16^ink4a gene by a retroviral vector in leukaemic cell lines with homozygous p16^ink4a gene deletion (K562, CEM, Jurkat cell lines) or with p16^ink4a gene hemizygous deletion and a point mutation inactivating the remaining allele (HL60 cell line). The viral titre obtained after transfection of PA317 amphotropic packaging cell line, which has a p16^ink4a gene homozygous deletion, was low, suggesting that p16^ink4a gene expression could impair viral production of retroviral packaging cell lines derived from the NIH3T3 cell line. After retroviral transfer of p16^ink4a in cell lines and G418 selection in liquid medium, a strong cell proliferation inhibition was observed for K562, CEM and Jurkat, but no inhibition was seen for HL60. A strong growth reduction in soft agar was also observed with p16^ink4a-transduced CEM, Jurkat and K562 cells, with a moderate growth reduction in the HL60 cell line. The growth inhibition in liquid culture, of K562 and Jurkat cell lines, was confirmed by electroporation transfer of the p16^ink4a gene. Our findings show that p16^ink4a gene transfer has a growth-inhibitory effect in leukaemic cell lines with p16^ink4a gene homozygous deletion. These data suggest that p16 could be a suitable gene for gene therapy in ALL.     

A polyclonal CD4+ and CD8+ lymphocytosis in a patient doubly infected with HTLV-I and HIV-1: a clinical and molecular analysis

HTLV-I is associated with adult T-cell leukemia/lymphoma (ATL) characterized by monoclonal expansions of CD4+ T-lymphocytes. In this report we describe a histologically benign, polyclonal HTLV-I infection in a patient exhibiting both an absolute CD4+ and CD8+ lymphocytosis. Three T-cell lines containing integrated HTLV-I proviral copies established from this patient were initially polyclonal, but with time all grew out the same two clones as determined by analysis of their T-cell antigen receptor beta chain gene rearrangements. The patient subsequently developed pulmonary and nasopharyngeal nodules containing HTLV-I infected cells. Restriction analysis of the patient's HTLV-I provirus revealed no differences from prototype HTLV-I and the tax gene was normally expressed in vivo and in vitro. The patient's T-lymphocytosis and HTLV-I+ pulmonary tract nodules were put into a complete clinical remission by treatment with alkylating agents and steroids. Subsequently, the patient developed a severe immunodeficiency state and expired. Retrospective serologic and gene amplification assays for HIV-1 demonstrated that he had been doubly infected from the time of presentation. Postmortem analysis by polymerase chain reaction revealed the presence of both HTLV-I and HIV-1 in lymphatic tissues and the testes; HIV-1 was also detected in brain tissue.     

Gene therapy of monogenic and cardiovascular disorders

The concept of gene therapy involves the introduction of genetic material into patient cells to cure or alleviate the symptoms of a disease by complementing a damaged gene or by giving the cell a new function. The belief that gene therapy would soon reach the clinic has been widely spread, frequently resulting in controversies when these expectations were not met. Nevertheless, over the last 10-year period, the experience from a number of clinical trials has taught us that gene transfer is technically feasible, but that the gene delivery vehicles, or vectors, for the transfer of genetic material are still suboptimal and that treatment may have severe side effects. This review will provide examples of different genetic disorders for which gene therapy is an option and has been attempted. It will also briefly discuss the existing vector systems and mention their advantages and drawbacks.     

Avian endogenous provirus (ev-3) env gene sequencing: Implication for pathogenic retrovirus origination

The avian endogenous env gene product blocks the surface receptor and, as a result, cells become immune to related exogenous retroviruses. On the other hand, the same sequence can be included in the pathogenic retrovirus genome, as shown by oligonucleotide mapping. However, since the complete env gene sequence was not known, the comparison of genomic nucleotide sequences was not possible. Therefore an avian endogenous provirus with an intact env gene was cloned from a chicken gene bank and the regions coding for the C terminus of the gp85 and gp37 proteins were sequenced. Comparison of this sequence with those of other retroviruses proved that one of the pathogenic viruses associated with osteopetrosis is a cross between avian endogenous virus and Rous sarcoma virus.