Intra-tissue Characteristics of Chloroplasts in the Lamina and Petiole of Mature Winter Leaf of Arum italicum Miller

Abstract: Laminae and petioles from mature winter leaves of Arum italicum were studied in order to obtain information on the sun—shade intra-tissue properties of chloroplasts. This inference was based on the: (1) micro- and submicroscopic characteristics of the chloroplasts, (2) cytochemical localizations of functional PS I and PS II, (3) pigment patterns and compositions, (4) immunolocalization of Rubisco, and (5) net photosynthesis. It was inferred that all the chloroplasts across the lamina had adaptations to intermediate shade conditions, without a sun-shade dimorphism between the palisade and the spongy tissues. In the petiole, where normally-structured chloroplasts were surprisingly present in the entire thickness of the organ, a structural and chemical dimorphism was found between the outer chlorenchyma and the inner aerenchyma where intermediate shade-type and extreme shade-type chloroplasts were present, respectively. However, some anomalies in the pigment composition were noted chiefly in the inner aerenchyma (low concentrations of β-carotene and lutein, absence of zeaxanthin, presence of unusual pigments, for instance lutein epoxide, lutein cis-isomer, and chlorophyllide a). The Rubisco immunolabelling in the outer chlorenchyma of the petiole was similar to that in the lamina, while it was very scant in the inner aerenchyma. Net photosynthesis in the petiole was about 75% of that recorded in the lamina. These data suggest that the petiole of the mature winter leaf of A. italicum closely co-operates with the lamina for enhancing light capture and utilization.     

The interferon-inducible IFI16 gene inhibits tube morphogenesis and proliferation of primary, but not HPV16 E6/E7-immortalized human endothelial cells

Immunohistochemical analysis has demonstrated that the human IFI16 gene, in addition to the hematopoietic tissues, is highly expressed in endothelial cells and squamous stratified epithelia. In this study, we have developed a reliable HSV-derived replication-defective vector (TO-IFI16) to efficiently transduce IFI16 into primary human umbilical vein endothelial cells (HUVEC), which are usually poorly transfectable. HUVEC infection with TO-IFI16 virus suppressed endothelial migration, invasion and formation of capillary-like structures in vitro. In parallel, sustained IFI16 expression inhibited HUVEC cell cycle progression, accompanied by significant induction of p53, p21, and hypophosphorylated pRb. Further support for the involvement of these pathways in IFI16 activity came from the finding that infection with TO-IFI16 virus does not impair the in vitro angiogenic activity and cell cycle progression of HUVEC immortalized by HPV16 E6/E7 oncogenes, which are known to inactivate both p53 and pRb systems. This use of a reliable viral system for gene delivery into primary human endothelial cells assigns a potent angiostatic activity to an IFN-inducible gene, namely IFI16, and thus throws further light on antiangiogenic therapy employing IFNs.     

Protection from bacterial infection by a single vaccination with replication-deficient mutant herpes simplex virus type 1

Adaptive immune responses in which CD8(+) T cells recognize pathogen-derived peptides in the context of major histocompatibility complex class I molecules play a major role in the host defense against infection with intracellular pathogens. Cells infected with intracellular bacteria such as Listeria monocytogenes, Salmonella enterica serovar Typhimurium, or Mycobacterium tuberculosis are directly lysed by cytotoxic CD8(+) T cells. For this reason, current vaccines for intracellular pathogens, such as subunit vaccines or viable bacterial vaccines, aim to generate robust cytotoxic T-cell responses. In order to investigate the capacity of a herpes simplex virus type 1 (HSV-1) vector to induce strong cytotoxic effector cell responses and protection from infection with intracellular pathogens, we developed a replication-deficient, recombinant HSV-1 (rHSV-1) vaccine. We demonstrate in side-by-side comparison with DNA vaccination that rHSV-1 vaccination induces very strong CD8(+) effector T-cell responses. While both vaccines provided protection from infection with L. monocytogenes at low, but lethal doses, only rHSV-1 vaccines could protect from higher infectious doses; HSV-1 induced potent memory cytotoxic T lymphocytes that, upon challenge by pathogens, efficiently protected the animals. Despite the stimulation of relatively low humoral and CD4-T-cell responses, rHSV-1 vectors are strong candidates for future vaccine strategies that confer efficient protection from subsequent infection with intracellular bacteria.     

Heparan Sulfate Proteoglycan Binding by Herpes Simplex Virus Type 1 Glycoproteins B and C, Which Differ in Their Contributions to Virus Attachment, Penetration, and Cell-to-Cell Spread

Herpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB’s role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK⁻ also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK⁻ gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK⁻. Compared with wild-type virus, KgBpK⁻ showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the lacZ gene (KCZ) was substantially more impaired (ca. 65%-reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK⁻gC⁻) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK⁻ mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity.     

Protection from herpes simplex virus type 1 lethal and latent infections by secreted recombinant glycoprotein B constitutively expressed in human cells with a BK virus episomal vector.

The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.     

Identification of replication-competent HSV-1 Cgal strain targets in a mouse model of human hepatocarcinoma xenograft

Recent studies based on animal models have shown the advantages and potential of oncolytic viral therapy using HSV-1 -based replication-competent vectors in the treatment of liver tumors, but little is known about the cellular targets that are modulated during viral infection. In the present work, we have studied the effects of intratumoral injections of HSV-1 Cgal⁺ strain in a murine model of human hepatoma xenografts. Viral replication was assessed for more than 1 month, leading to a significant reduction of tumor growth rate mediated, in part, by a cyclin B dependent cell proliferation arrest. Early events resulting in this effect were analyzed using a proteomic approach. Protein extracts from xenografted human hepatomas treated with saline or HSV-1 Cgal⁺ strain during 24 h were compared by 2-D DIGE and differential spots were identified by nanoLC-ESI-MS/MS. Alterations on glutathione S transferase 1 Omega, and ERp29 suggest novel HSV-1 Cgal⁺ targets in solid liver tumors. Additionally, ERp29 showed a complex differential isoform pattern upon HSV-1 Cgal⁺ infection, suggesting regulatory mechanisms based on post-translational modification events.     

A lentiviral vector-based, herpes simplex virus 1 (HSV-1) glycoprotein B vaccine affords cross-protection against HSV-1 and HSV-2 genital infections

Genital herpes is caused by herpes simplex virus 1 (HSV-1) and HSV-2, and its incidence is constantly increasing in the human population. Regardless of the clinical manifestation, HSV-1 and HSV-2 infections are highly transmissible to sexual partners and enhance susceptibility to other sexually transmitted infections. An effective vaccine is not yet available. Here, HSV-1 glycoprotein B (gB1) was delivered by a feline immunodeficiency virus (FIV) vector and tested against HSV-1 and HSV-2 vaginal challenges in C57BL/6 mice. The gB1 vaccine elicited cross-neutralizing antibodies and cell-mediated responses that protected 100 and 75% animals from HSV-1- and HSV-2-associated severe disease, respectively. Two of the eight fully protected vaccinees underwent subclinical HSV-2 infection, as demonstrated by deep immunosuppression and other analyses. Finally, vaccination prevented death in 83% of the animals challenged with a HSV-2 dose that killed 78 and 100% naive and mock-vaccinated controls, respectively. Since this FIV vector can accommodate two or more HSV immunogens, this vaccine has ample potential for improvement and may become a candidate for the development of a truly effective vaccine against genital herpes.     

An S/MAR-based infectious episomal genomic DNA expression vector provides long-term regulated functional complementation of LDLR deficiency

Episomal gene expression vectors offer a safe and attractive alternative to integrating vectors. Here we describe the development of a high capacity episomal vector system exploiting human episomal retention sequences to provide efficient vector maintenance and regulated gene expression through the delivery of a genomic DNA locus. The iBAC-S/MAR vector is capable of the infectious delivery and retention of large genomic DNA transgenes by exploiting the high transgene capacity of herpes simplex virus type 1 (HSV-1) and the episomal retention properties of the scaffold/matrix attachment region (S/MAR). The iBAC-S/MAR vector was used to deliver and maintain a 135kb genomic DNA insert carrying the human low density lipoprotein receptor (LDLR) genomic DNA locus at high efficiency in CHO ldlr^−/− a7 cells. Long-term studies on CHO ldlr^−/− a7 clonal cell lines carrying iBAC-S/MAR-LDLR demonstrated low copy episomal stability of the vector for >100 cell generations without selection. Expression studies demonstrated that iBAC-S/MAR-LDLR completely restored LDLR function in CHO ldlr^−/− a7 cells to physiological levels and that this expression can be repressed by ~70% by high sterol levels, recapitulating the same feedback regulation seen at the endogenous LDLR locus. This vector overcomes the major problems of vector integration and unregulated transgene expression.