In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence.

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides.

Prevention of measles virus infection by intranasal delivery of fusion inhibitor peptides.

UNLABELLED: Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV H and the fusion (F) envelope glycoprotein; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad-repeat (HR) regions of F can potently inhibit MV infection at the entry stage. We show here that specific features of H's interaction with its receptors modulate the susceptibility of MV F to peptide fusion inhibitors. A higher concentration of inhibitory peptides is required to inhibit F-mediated fusion when H is engaged to its nectin-4 receptor than when H is engaged to its CD150 receptor. Peptide inhibition of F may be subverted by continued engagement of receptor by H, a finding that highlights the ongoing role of H-receptor interaction after F has been activated and that helps guide the design of more potent inhibitory peptides. Intranasal administration of these peptides results in peptide accumulation in the airway epithelium with minimal systemic levels of peptide and efficiently prevents MV infection in vivo in animal models. The results suggest an antiviral strategy for prophylaxis in vulnerable and/or immunocompromised hosts. IMPORTANCE: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that parenterally delivered fusion-inhibitory peptides protect mice from lethal CNS MV disease. Here we show, using established small-animal models of MV infection, that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. Since the fusion inhibitors are stable at room temperature, this intranasal strategy is feasible even outside health care settings, could be used to protect individuals and communities in case of MV outbreaks, and could complement global efforts to control measles.

Current animal models: transgenic animal models for the study of measles pathogenesis.

Animal models are highly important to understand the pathologic mechanisms of viral diseases. Therefore, the lack of a suitable animal model has greatly hindered the research into the pathogenesis of measles. Identification of two human receptors for measles virus, CD46 and CD150 (SLAM) has opened new perspectives in this field. During the last decade, numerous transgenic animal models have been developed in order to humanize mice and use them to study measles infection and virus-host interactions. Despite their limitations, these models have provided remarkable insights in different aspects of measles infection, providing a better understanding of virus-induced neuropathology, immunosuppression, mechanisms of virus virulence, and contribution of innate and adaptive immune response in viral clearance. They should certainly continue to help in studies of the host and viral factors that are important in measles infection and in developing of new antiviral agents and measles virus-based vaccines. In addition, as CD46 serves as a receptor for two other human viruses, some of these models may also find an important application in the study of adenovirus and herpesvirus 6 infection. In this review, we describe different CD46 and CD150 transgenic models and detail their utilization in the study of various aspects of measles pathogenesis.

[Nipah and Hendra viruses : emerging zoonotic pathogens]. / Les virus Nipah et Hendra : des agents pathogènes zoonotiques émergents.

Emerging new viruses present an enormous challenge in understanding their aetiology, pathogenesis and epidemiology. In the last decade two new viruses : Nipah virus in Malaysia and Hendra virus in Australia crossed species barrier from flying foxes to infect humans. While Hendra virus mainly induced pulmonary disease, Nipah virus provoked encephalitis with 40-70 % of mortality, causing important health and economic problems. Based on the similar genome structure, these 2 viruses are classified in a new genus, Henipaviruses, within the family of Paramyxoviridae and both are ranked internationally as biosecurity level 4 agents. Recent studies on the virulence, host range and cell tropism of these human pathogens provide more insight into unique biological properties of the emergent zoonotic viruses.

Suppression of Smad-1 mRNA expression level by Smad-2 likely control dichotomy of NF-κB and Smads mediated activation.

The aim of this study was to find out how NF-κB and Smad-mediated signaling influenced the expression of astrogliogenic versus neurogenic markers of brain development in U4C cells which were either enriched (Tg Jak-1) or deprived in Jak-1 molecule (Jak-1 KO). Genetically modified U4C cells were transfected with NF-kB reporter plasmid in order to follow its activation when cells were cotransfected with different combinations of Smads constructs. In wild type cells no significant activation of NF-κB was observed while genetically modified cells exhibited somewhat different pattern of NF-κB activation depending on the Smad constructs combination used. The absence of NF-κB activation in Jak-1 transgenic cells transfected with Smad-1 plus Smad-3 was accompanied by the appearance of apoptotic cells as revealed by DAPI staining. Smad-1 expression was undetectable in Jak-1 transgenic cells and was downregulated in wild type cells upon transfection with Smad-2. The absence of p65 nuclear translocation in Smad-2 transfected cells and the presence of Smad-4 in nucleus of the same cells indicates dichotomy in NF-κB and Smads mediated signaling pathways. The significance of this study is that helps to elucidate the point of collaboration among three different signaling pathways - Jak-1 mediated cytokine signaling, NF-κB and Smads mediated pathways.

Tumour cell proliferation is abolished by inhibitors of Na+/H+ and HCO3-/Cl- exchange.

Cell membrane-associated ion transporters, Na+/H+ exchanger and Na(+)-dependent HCO3-/Cl- antiport, were shown to be important in the regulation of acidic intracellular pH in different cell types. This study investigated the role of the ion exchangers and their inhibitors in the serum-induced proliferation of two murine tumour cell lines, P815 and L929. The presence of Na+/H+ exchanger [inhibited by amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA)] and Na(+)-dependent HCO3-/Cl- antiport [inhibited by 4,4'-diisothiocyanostilbene-2,2-disulphonic acid (DIDS)] was shown on the tumour cell line tested. EIPA suppressed tumour cell proliferation more strongly than amiloride, and its effect was further increased after intracellular acidification by nigericin. DIDS slightly inhibited proliferation of L929 cell line and did not influence proliferation of P815 cells. However, in nigericin acidified cells DIDS had a dose dependent antiproliferative effect. Furthermore, DIDS significantly increased antiproliferative effects of amiloride and EIPA, suggesting the activity of Na(+)-dependent HCO3-/Cl- antiport in tumour cell proliferation. These results demonstrate the importance of Na(+)-dependent HCO3-/Cl- exchange in addition to Na+/H+ antiport, in tumour cell proliferation and indicate the possibility that ion exchange inhibitors could act as antitumour reagents.

Lectin histochemistry of mouse vagina during the estrous cycle.

Estrous cycle-related histochemical changes in the vaginal epithelium of sexually mature female mice were studied with 30 fluorescein isothiocyanate (FITC)-labeled lectins. On the basis of the staining pattern the lectins were divided into five groups: I, seventeen lectins that reacted with mucinous surface layer of proestrus. This group comprised two subgroups: Ia, seven lectins that reacted exclusively with the mucinous layer, and Ib, ten lectins that reacted with mucinous cells and the underlying squamous epithelium of proestrus; II, two lectins that reacted with squamous epithelium of proestrus only but were unreactive with mucinous cells; III, three lectins that reacted in a phase-specific manner with squamous epithelium; IV, six lectins that showed increased luminal surface reactivity in diestrus and/or metestrus; and V, eleven lectins that were unreactive with vaginal epithelium. These data indicate that the cyclic changes in the morphology of the vaginal epithelium are accompanied by distinct lectin reactivity patterns.

Glycoproteins of mouse vaginal epithelium: differential expression related to estrous cyclicity.

We used lectin overlay blotting and SDS-PAGE to analyze the estrous cycle-specific expression of mouse vaginal epithelial glycoproteins. Seven lectins chosen for their differential carbohydrate-binding specificity revealed 15 glycoproteins that showed cycle-related expression. Each lectin had a unique binding pattern different from the patterns revealed by other lectins. However, several estrous cycle phase-specific glycoproteins reacted with more than one lectin. The most prominent of these glycoproteins (M(r) 92-95 KD) was weakly expressed in late diestrus and fully expressed only in proestrus, coincident with the transformation of two superficial layers of vaginal squamous epithelium into mucinous cuboidal cells. Electron microscopic lectin histochemistry revealed the glycoproteins in the mucinous granules of surface cuboidal cells and in the lumen of the vagina. Our results illustrate the complexity of glycoconjugate synthesis in mouse vagina and reveal the distinct cycle-specific patterns of individual glycoprotein expression. These cyclic glycoproteins could serve as vaginal biochemical markers for the specific phases of the estrous cycle.

Contrasuppression and tumor rejection.

The growth of a highly progressive MCA-induced tumor 3152-PRO is dependent on the activity of suppressor T cells (Ts). Injection of syngeneic mice with antibodies specific for Ts leads to enhanced tumor transplantation resistance of the 3152-PRO tumor. In addition, injection of recipient mice with highly immunogenic regressor tumors conjugated with trinitrophenyl (TNP) activates a T cell population which also mediates protection to transplantation of TNP-conjugated 3152-PRO tumor cells. One such tumor, 1591-RE, was investigated in detail to determine the phenotype and biologic activity of this T cell population in overcoming Ts cell activity. Induction of transplantation resistance requires the presence of TNP hapten on both the highly regressive immunizing tumor (and not its progressor variant 1591-PRO4), and on the challenge tumor 3152-PRO. The cell population from TNP-1591-RE immunized animals which mediates protection against the transplantation of TNP-3152-PRO is Thy-1+, CD4+, 8-, Lyt1+, I-J+, and Vicia villosa lectin adherent, the identical phenotype to antigen-specific contrasuppressor T cells in the contact sensitivity (CS) response to TNP in vivo. A T cell population of identical phenotype from TNP-1591-RE immunized mice can overcome the effects of antigen-specific Ts cells on PCl-immune cells in the adoptive transfer of CS in vivo. These results suggest that immunoregulatory cells that mediate protection against progressive tumors may be identical in function to antigen-specific contrasuppressor T cells.

Successful therapy to a progressive tumor using monoclonal-antibodies to T-cell regulatory molecules and interleukin-2.

Weekly injections of monoclonal antibodies (mAb) to T-cell regulatory molecules, into animals challenged with a progressively growing epithelial cell tumor 3152-PRO, decreased tumor incidence and caused significantly slower tumor growth, compared with animals given identical treatments of control rat mAb. Some of the mAb showed significant synergy with rIL-2 in the successful therapeutic treatment of 3152-PRO. Furthermore, spleen cells from treated mice generated strong tumor-specific cytotoxic activity. The successful elimination of progressively growing 3152-PRO tumor rendered animals specifically resistant to a secondary challenge of 3152-PRO tumor cells, but not to 2237-PRO or 3256-PRO, two other, noncrossreactive tumors.

Chorioangiomas.

Six cases of chorioangioma of the placenta and a review of relevant leterature are presented. The incidence of chorioangiomas is higher than generally realized because many small chorioangiomas are missed in routine examination of placentas. A case of chorioangioma with foci resembling hemangiopericytoma is presented. Cases of large chorioangiomas seem to occur when the placenta is large. Hydramnios is commonly found with large chiorangiomas, and both of these conditions may be conducive to premature births. There is an increased incidence of toxemia in pregnant patients with chorioangiomas, and the condition is unrelated to the size of the tumor. Diligent examination of infants associated with chorioangioma of the placenta is suggested, as these infants may have unsuspected congenital anomalies.

Aqueous mixture of pyrrolidone-5-hydroxamic acid-iron (III) complexes: isolation and characterization of tris (pyrrolidone-5-hydroxamato) iron (III).

The published procedure for the synthesis of pyrrolidone-5-hydroxamic acid was improved. The acidity constant of the pyrrolidone-5-hydroxamic acid was determined as pKa = 8.65. In an aqueous solution of iron (III) ions, pyrrolidone-5-hydroxamic acid binds ferric ion, forming a mixture of mono-, bis-, and tris(pyrrolidone-5-hydroxamato)iron (III) complexes. These complexes were studied by potentiometric and spectrophotometric methods. The tris compound was isolated as dark orange-red crystals and identified according to elemental analysis and IR spectral data as C15H21FeN6O9.6H2O, having the magnetic moment of 5.67 B.M.

Cyclic changes in the expression of endometrial proteins of the mouse during the normal estrus cycle.

Cellular proteins extracted from the endometrial tissue of normal sexually mature outbred mice were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to define the changes in protein expression that occur during the normal estrus cycle. Using one dimensional gradient SDS-PAGE it was possible to resolve approximately 80 protein bands, 8 of which showed cyclic changes. Five protein bands (Mr 28, 40, 135, 150 and 220 kD) were most prominent in the estrus phase and were barely detectable or not visible at all in the diestrus phase. Since the estrus phase is under the influence of estrogen, these five proteins were considered to be estrogen-induced. Two protein bands (Mr 35, and 37 kD) were more prominent in the diestrus phase. The 116 kD protein band was found only in the diestrus phase. These three proteins were considered to be induced by progesterone, the hormone that governs the diestrus phase of the cycle. None of the protein bands was found to be typical of the proestrus and metestrus phases of the cycle, although the 28 kD band appeared more prominent in proestrus than in other phases. These data show that the mouse estrus cycle is associated with phase-specific changes which can be reproducibly and reliably detected by SDS-PAGE.

Oviductal glycoproteins in sexually immature mice.

The expression of oviductal glycoproteins was studied in prepubertal mice by lectin overlay of electrophoretically separated cellular extracts. Two N-acetylgalactosamine specific lectins [Maclura pomifera (MPA), and Ricinus communis (RCA-I)] were used to study samples obtained from 7, 14, 21 and 28 day old virgin mice. A total of 9 developmentally regulated glycoprotein bands were identified. Several patterns of glycoprotein expression were noticed: glycoproteins expressed equally strongly in all stages of development; glycoproteins that were weakly expressed or not found in 7 day old or 14 and 21 day old mice but became more prominent and reached maximal expression in mature mice; glycoproteins expressed under the influence of maternal hormones at 7 days that became barely detectable in 14 and 21 day old mice but were strongly expressed in mature mice. These data show that the pattern of expression of oviductal glycoproteins changes during the prepubertal maturation of the oviduct.