Inactivation of viruses by beta-propiolactone in human cryo poor plasma and IgG concentrates.

Virus inactivation by cold treatment with beta-propiolactone (BPL) was investigated in human cryo poor plasma and purified IgG concentrates spiked with relevant human viruses or appropriate animal model viruses. The samples were treated with 0.1 or 0.25% BPL for 300 or 480 min, respectively. Residual infectivity was determined by standard microtitration assays on tissue culture cells. The inactivation of all viruses tested was more effective in IgG than in plasma. IgG: R1=4-5.5 log10 for vesicular stomatitis virus (VSV). Semliki Forest virus (SFV), bovine virus diarrhoea virus (BVDV), murine encephalomyelitis virus (MEV), feline calicivirus (FVC), suid parvovirus (PPV), simian virus 40 (SV40); R1=2-4 log10 for suid herpesvirus type 1 (SHV-1), bovine herpesvirus type 1 (BHV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIVagm3). Plasma: R1=3-5 log10 for VSV, SFV, BVDV, SHV-1, MEV:R1=0-3 log10 for HIV-1, SIVagm3 BHV-1, FCV, PPV, SV40. After addition of SIVagm3, HIV-2, and PPV to plasma or IgG, spontaneous inactivation without further addition of BPL was observed. These results demonstrate that treatment with BPL has a limited capacity to inactivate viruses. Different inactivation kinetics were observed in plasma and IgG concentrates. Therefore, virus inactivation by BPL must be tested for individual blood products independently and should not be extrapolated from other model systems.

Transport of viruses through fetal membranes: an in vitro model of perinatal transmission.

A model system for perinatal transmission of viral infections was developed and transport of infectious virus particles through fetal membranes was investigated. Viruses of different families known to cause serious intrauterine infections were selected, including relevant and model viruses: the DNA-viruses HSV-1 and -2 as well as the animal herpes viruses BHV-1 and SHV-1, the RNA-virus BVDV as a model for hepatitis C virus, HIV-1 and -2, and PPV as a model for parvovirus B19. Migration of infectious virus from the maternal to the fetal side of the membrane could be detected as early as 20 min after the start of incubation. A peak of virus migration was observed after 1-2 hr. 0.02-1% of HSV-1 and 0.03-0.2% of HSV-2 were transported from the maternal side of the membrane to the fetal side. Only 0.01% of PPV migrated to the fetal side, whereas no transport of BVDV was observed. HIV-1 (1.4%) and HIV-2 (0.8%) seemed to be transported at higher rates. The concept of an active transport of infectious virus is compatible with the kinetics of penetration of the fetal membrane. The question of whether different receptors for the individual viruses on the cellular surface account for differences in virus transport will require further investigation. The fetal membrane acts as a protective barrier for the fetus, reducing greatly infectious titers or even preventing completely penetration of virus.

Epithelial uptake and transport of cell-free human immunodeficiency virus type 1 and gp120-coated microparticles.

Cell-free human immunodeficiency virus type 1 (HIV-1) can be taken up and released by a monolayer of primary human gingival cells and remain infectious for CD4+ cells. Virus-sized latex particles covalently coated with purified native HIV-1 envelope glycoprotein gp120 are also transported through the primary epithelial cells. This process is significantly stimulated by increasing the intracellular cyclic AMP (cAMP) concentration. Inhibition experiments with mannan and alpha-methyl-mannopyranoside indicated that mannosyl groups are involved in the interaction between gp120 and gingival cells. An increase of cellular oligomannosyl receptors by incubation with the mannosidase inhibitor deoxymannojirimycin augmented transcellular transport of the gp120-coated particles. The results suggest that infectious HIV can penetrate gingival epithelia by a cAMP-dependent transport mechanism involving interaction of the lectin-like domain of gp120 and mannosyl residues on glycoproteins on the mucosal surface. Penetration of HIV could be inhibited by soluble glycoconjugates present in oral mucins.

Selective endocytosis of fluorothymidine and azidothymidine coupled to LDL into HIV infected mononuclear cells.

Drug targeting via lipoproteins may be of benefit for use of cytotoxic drugs like fluorothymidine (FLT) or azidothymidine (AZT). Both drugs are potent inhibitors of the human immunodeficiency virus (HIV) reverse transcriptase and are used in the therapy of HIV infection. With regard to this project, the selective endocytosis in HIV infected human macrophages was studied after covalent coupling of AZT and LDL to low density lipoproteins (LDL). Cultured human macrophages and the lymphocytic Molt 4/8 cell line were infected with HIV-1 in vitro and subsequently treated with FLT-LDL or AZT-LDL. Viral replication was followed by determination of cell-released capsid antigen p24. Internalisation into HIV-1 infected human macrophages by the scavenger receptor pathway leads to a dose dependent inhibition of HIV replication. Otherwise, in HIV infected, but scavenger receptor missing lymphocytes (Molt 4/8 cells), neither endocytosis nor inhibition of HIV replication results. Thus, covalent coupling of drugs to LDL leads to a macrophage specific transport. This strategy could possibly avoid toxic side effects in the therapeutic use of antiretroviral drugs and thus may open a way for an earlier chemotherapy in HIV infection.

Replication pattern of human immunodeficiency virus type 1 in mature Langerhans cells.

Langerhans cells (LC), the dendritic antigen presenting cells of the skin, mature into potent immunostimulatory cells during migration to regional lymph nodes, where they are identified as interdigitating cells (IDC). Since mature Langerhans cells (mLC) resemble IDC in phenotype and immunostimulatory capacity, we examined whether these cells were susceptible to infection with macrophagetropic and lymphotropic strains of human immunodeficiency virus type 1 (HIV-1). Highly purified cell preparations of mLC migrating from human epidermis expressed high amounts of major histocompatibility complex (MHC) class I and II antigens and of the accessory molecules CD40, CD80 and CD86, indicative of the phenotype of potent immunostimulatory cells. CD4 expression was upregulated on mLC during cultivation, independent of the presence of tumour necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the culture medium. The macrophagetropic HIV-1 strain SF162 replicated to higher titres in mLC than the lymphotropic strain IIIB. Both strains induced syncytia, with SF162 showing a more rapid cytopathic effect. Addition of TNF-alpha enhanced virus production, due to better cell viability under TNF-alpha treatment, whereas GM-CSF did not significantly influence viability of cells and replication pattern of the virus. These findings suggest that in the infected individual IDC in lymph nodes may function as target cells for HIV-1.

Cell specific uptake of antiretroviral drugs: AZT coupled to LDL inhibits HIV replication in human macrophages.

Macrophages, besides helper T-lymphocytes, are target cells for the human immunodeficiency virus (HIV). We report on a mechanism to deliver selectively antiretroviral drugs to cells of the monocyte/macrophage lineage. These cells and cells of the endothelium express scavenger receptors which mediate the transport of modified low density lipoprotein (LDL). LDL modified by covalently bound azidothymidine (AZT), a potent inhibitor of HIV replication, is internalized via this pathway into human macrophages. Treatment of HIV-1 infected human macrophages with AZT-LDL showed in vitro efficient inhibition of viral replication. In contrast, HIV replication in T-lymphocytes (Molt 4/8), which do not express scavenger receptors, is not inhibited by AZT-LDL but by free AZT.

Expression of nef, vpu, CA and CD4 during the infection of lymphoid and monocytic cell lines with HIV-1.

The expression of the capsid antigen (CA) and the two regulatory proteins nef and vpu as well as the CD4 cell surface receptor was followed in HIV-infected lymphoid and promonocytic cells. In the lytic phase of infection all three viral proteins were expressed; production of these proteins coincided with the increase of CA antigen and infectious virus in culture supernatants and with prominent cytopathic effects. After selection of persistently infected cells, the number of lymphoid cells expressing detectable levels of nef decreased to zero; the number of cells positive for CA ranged between 40 to 70%. In chronically infected promonocytic cells nef and vpu expression was reduced to undetectable levels, whereas most of the cells accumulated CA intracellularly. Infectious cell free virus and CA in the supernatant of promonocytic cells had low titers. CD4 surface expression declined in all cell lines investigated before cell free virus was detectable.

Interferon induction by cell-cell interaction of HIV-infected monocytes/macrophages with lymphocytes.

An in vitro model of interferon (IFN) induction in HIV infection was established. IFN are induced by a cooperative mechanism, involving monocytes/macrophages (M/M) as well as lymphocytes. M/M non-lytically infected with HTLV-IIIB did not produce IFN, but were able to induce high titres of IFN activity when cocultured with peripheral blood lymphocytes, cell-cell contact being required. IFN alpha as well as IFN gamma were induced, as shown by neutralization with specific antisera. The antiviral activity of HIV-induced IFN was shown by infectivity reduction assays, and the immune-modulating capacity was demonstrated by activation of M/M leading to neopterin release.

Altered urinary excretion of pteridines in neoplastic disease. Determination of biopterin, neopterin, xanthopterin, and pterin.

Urinary pteridine concentrations in healthy control subjects and patients with cancer and non-malignant diseases were determined by HPLC and TLC after partial purification by ion exchange and Sephadex chromatography. Elevated concentrations of neopterin were found in 70% of the 50 cancer patients investigated. In patients with non-Hodgkin's lymphoma (seven cases) or with liver metastases (12 cases) neopterin concentrations were significantly higher than in control subjects (p < 0.01). Biopterin was less frequently increased (22%). Xanthopterin was generally raised when neopterin and/or biopterin excretion was high. Neopterin/biopterin ratios were higher in some patients with cancer or with severe renal insufficiency than in controls. These findings suggest that alterations in pteridine metabolism are common in malignant disease. The pathogenic, diagnostic and therapeutic significance of these changes remains to be established.