Assessment of the biological performance of the needle-free injector INJEX using the isolated porcine forelimb.

BACKGROUND: The development and utilization of novel needle-free injection devices in order to minimize needle stick injuries make increasing demands for suitable assay systems, which reflect the physiological situation in humans as close as possible. OBJECTIVES: It was therefore the goal of the present study to test the biological performance of a needle-free injector (INJEX) by the use of porcine skin as a model with a high predictive value for the feasibility in humans because of its close similarity to human skin. METHODS: In order to use porcine skin in the context of the underlying tissues, the isolated porcine forelimb was chosen as an assay model for use with the INJEX injector. Ink or the fluorescent dye fluorescein-isothiocyanate was injected and the penetration depth was determined metrically and dye distribution histologically. To assess the resorption of heparin, needle injection was compared with needle-free injection in a perfused limb model. RESULTS: Increasing amounts of ink increasingly penetrated into subcutaneous tissue layers in a cone-shaped manner mainly following lead structures. Penetration was hampered by skin thickness and by the deep muscle fascia, which served as a penetration barrier. Resorption of heparin was similar irrespective of injection by the use of a needle or the INJEX device. CONCLUSIONS: The isolated porcine forelimb serves as a versatile tool for the assessment of the biological performance of needle-free injection devices such as INJEX. Further studies are necessary to correlate the model for drug delivery in humans.

Effect of the quassinoids glaucarubolone and simalikalactone D on growth of cells permanently infected with feline and human immunodeficiency viruses and on viral infections.

Growth of Crandall feline kidney cells permanently infected with feline immunodeficiency virus was inhibited by the anti-cancer quassinoid glaucarubolone whereas growth of uninfected cells was not inhibited. Similar results were obtained for human MOLT-4 cells infected with HIV-1. The results suggest that cell lines permanently infected with either the feline or the human lentivirus exhibit growth response characteristics to the quassinoids in common with other cell lines malignantly transformed. In addition the quassinoids may delay viral infection suggesting some commonality between the mechanism responsible for inhibition of the growth of the transformed phenotype and viral infection.

Internalization of human rhinovirus 14 into HeLa and ICAM-1-transfected BHK cells.

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection.

Transmission, species specificity, and pathogenicity of Aujeszky's disease virus.

Aujeszky's disease virus (ADV), also known as pseudorabies virus (PrV), is an alphaherpesvirus that causes fatal infections in a wide range of animal species. The virus shares a variety of biological properties with human pathogenic herpesviruses like herpes simplex virus or varicella-zoster virus. Although only limited data are available, it seems unlikely that PrV causes disease in immunocompetent humans, but may pose a risk for immunocompromised patients.

Glycoprotein B (gB) of pseudorabies virus interacts specifically with the glycosaminoglycan heparin.

We have previously shown that the pseudorabies virus (PrV) glycoproteins gB and gC (former PrV-gII and PrV-gIII) exhibit heparin-binding properties. While PrV-gC functions as the major adsorption protein, the biological role of the heparin-binding properties of PrV-gB are not understood. We used a gC-deleted PrV-mutant, PrV (dlg92/dltk), to analyse the heparin-binding properties of PrV-gB and the biological role of the PrV-gB-protein in adsorption. PrV-gB was the only glycoprotein of this vaccine strain binding to immobilised heparin in in vitro assays. Presence of the gC-protein was not necessary for the interaction of gB with heparin. Soluble heparin also interfered with adsorption of this mutant virus to a similar extent as it blocked adsorption of wild-type PrV (Ka), but it had only a minor inhibitory effect on infectivity of the mutant strain. These results show that PrV-gB interacts specifically with immobilized heparin and heparin-like structures on the cell surface, but this interaction is not required for a productive infection.

Protein-glycosaminoglycan interactions: infectiological aspects.

Glycosaminoglycans (GAGs) are linear heteropolysaccharides consisting of repeated disaccharide units that are variably N- and O-sulfated. Due to this heterogeneity, GAGs possess a high amount of structural information. Linked to a protein core to form a proteoglycan, GAGs are present on the surface of probably all mammalian tissues. During the recent years, a number of pathogens ranging from viruses to protozoans were found to interact specifically with cell surface GAGs to recognize and bind to their target cells. This review is intended to give a short overview over protein-GAG interaction under the aspects of infection.

Detection of specific human immunodeficiency virus IgM antibodies.

This study was done to demonstrate whether the use of the antigen-sandwich human immunodeficiency virus (HIV) antibody-screening assays (3rd generation assays), which detect all classes of anti-HIV immunoglobulins, leads to an earlier detection of HIV IgM compared to the 2nd generation HIV antibody-screening assays. We tested sequential bleeds of three donors obtained from commercially available seroconversion panels. Anti-HIV testing was done before and after high-performance liquid chromatography separation of IgG and IgM fractions. The positive result of the first bleedings from all three panels was linked to the IgM fraction, while at that time the IgG fraction was still negative. For subsequent samples drawn 5-9 days later, a positive signal was obtained with the IgG fraction in addition to a stronger positive signal obtained with the IgM fraction. We conclude that an assay capable of simultaneously detecting different immunoglobulin classes, including IgM, will help to narrow the "window period" for serological detection of seroconversion to HIV by detecting anti-HIV IgM-containing samples earlier than conventional assays using only anti-human IgG enzyme conjugates (indirect anti-HIV-screening assay, 2nd generation assays).

Cellular receptor structures for pseudorabies virus are blocked by antithrombin III.

Pseudorabies virus (PrV), an alphaherpesvirus of swine, uses cellular heparan sulfate residues as a receptor for attachment. Interaction of the virus with its receptor is mediated by the envelope glycoprotein C (PrV-gC), a protein with heparin-binding properties. We have previously shown that a region of this protein shows structural similarities to the high-affinity heparin-binding site of the serum protease-inhibitor antithrombin III (ATII). In this publication, we describe the effect of ATIII on interaction of PrV with its cellular receptor. ATIII bound specifically to heparan sulfate residues on the surface of herpesvirus-permissive RK13 cells. Binding of ATIII to RK13 cells interfered with adsorption of radioactively labelled PrV to these cells. Enzymatic treatment using heparinase I (E.C. 4.2.2.7) removed the receptor for PrV as well as the receptor for ATIII. Since amino acids 130-137 of the high affinity heparin-binding site of ATIII show structural similarities to amino acids 134-141 of PrV-gC, both sequences were synthesized as synthetic peptides. Although interaction of the peptide derived from ATIII with heparin was significantly stronger, both peptides interacted specifically with heparin in assays in vitro. These results suggest that PrV and ATIII interact with the same structure on the cellular surface.

A peptide-model for the heparin-binding property of pseudorabies virus glycoprotein III.

The pseudorabies virus glycoprotein III (PrV-gIII) has been identified previously as the major viral component binding to a heparin-like receptor on the surface of target cells. The amino acid sequence of gIII contains three regions corresponding to consensus sequences for heparin binding. A synthetic peptide corresponding to amino acids 134 to 141 of PrV-gIII bound heparin in a dot blot assay. In contrast, a synthetic peptide derived from amino acids 290-299 of PrV-gIII did not bind heparin. We therefore conclude that the region containing amino acid 134-141 is involved in binding to the heparin-like cellular receptor.

Processing of pseudorabies virus glycoprotein gII.

The glycoprotein complex gII of pseudorabies virus was isolated by immunoprecipitation with the monoclonal antibody M5, which was covalently linked to protein A-Sepharose. After sodium dodecyl sulfate-polyarylamide gel electrophoresis under reducing conditions and blotting onto poly(vinylidene difluoride) membrane, its subunits, gIIa, gIIb, and gIIc, were subjected to N-terminal sequencing. gIIa and gIIb start at position 59 and gIIc starts at position 503 according to the amino acid sequence deduced from the gene, indicating that there is one major protein (gIIa) which is cleaved into the two protein fragments gIIb and gIIc. Protein labeling with 14C-amino acids gave no indication that the three proteins (gIIa, gIIb, and gIIc) of the complex are present in equimolar ratios. It seems that gIIa is only a minor component of the complex, whereas gIIb and gIIc are contained in equimolar amounts.

Comparison of heparin-sensitive attachment of pseudorabies virus (PRV) and herpes simplex virus type 1 and identification of heparin-binding PRV glycoproteins.

To determine whether heparan sulphate residues on the cellular surface could serve as an attachment receptor for pseudorabies virus (PRV), the effect of heparin on PRV in plaque reduction and adsorption tests was investigated. PRV was significantly less sensitive to heparin than was herpes simplex virus type 1 (HSV-1). At concentrations of 500 micrograms/ml heparin the number of plaques formed by PRV was reduced to 7% of the untreated control whereas the number of plaques formed by HSV-1 was reduced to below 0.1%. Adsorption of PRV to host cells was also less sensitive to heparin treatment than was adsorption of HSV-1. Experiments concerning the binding sites of PRV showed that heparin binds to the disulphide-linked glycoprotein complex gII (PRV gB), gIII (PRV gC) and probably gV.

Entry of pseudorabies virus into CHO cells is blocked at the level of penetration.

Replication of pseudorabies virus (PrV) in Chinese hamster ovary (CHO) cells, a cell line naturally resistant to infection by herpesviruses, is blocked at the level of penetration. Virions bound to the surface of CHO cells are taken up into cytoplasmic vesicles and degraded.