IIIB Cells Research Articles

Evidence of epigenetic landscape shifts in mucopolysaccharidosis IIIB and IVA

Lysosomal storage diseases (LSDs) are a group of monogenic diseases characterized by mutations in genes coding for proteins associated with the lysosomal function. Despite the monogenic nature, LSDs patients exhibit variable and heterogeneous clinical manifestations, prompting investigations into epigenetic factors underlying this phenotypic diversity. In this study, we focused on the potential role of epigenetic mechanisms in the pathogenesis of mucopolysaccharidosis IIIB (MPS IIIB) and mucopolysaccharidosis IVA (MPS IVA). We analyzed DNA methylation (5mC) and histone modifications (H3K14 acetylation and H3K9 trimethylation) in MPS IIIB and MPS IVA patients’ fibroblasts and healthy controls. The findings revealed that global DNA hypomethylation is present in cell lines for both diseases. At the same time, histone acetylation was increased in MPS IIIB and MPS IVA cells in a donor-dependent way, further indicating a shift towards relaxed open chromatin in these MPS. Finally, the constitutive heterochromatin marker, histone H3K9 trimethylation, only showed reduced clustering in MPS IIIB cells, suggesting limited alterations in heterochromatin organization. These findings collectively emphasize the significance of epigenetic mechanisms in modulating the phenotypic variations observed in LSDs. While global DNA hypomethylation could contribute to the MPS pathogenesis, the study also highlights individual-specific epigenetic responses that might contribute to phenotypic heterogeneity. Further research into the specific genes and pathways affected by these epigenetic changes could provide insights into potential therapeutic interventions for these MPS and other LSDs.

Electrophysiological properties of maxillary trigeminal Aβ-afferent neurons of rats.

Aβ-afferents in maxillary or V2 trigeminal ganglion (TG) neurons are somatosensory neurons that may be involved in both non-nociceptive and nociceptive functions in orofacial regions. However, electrophysiological properties of these V2 trigeminal Aβ-afferent neurons have not been well characterized so far. Here, we used rat ex vivo trigeminal nerve preparations and applied patch-clamp recordings to large-sized V2 TG neurons to characterize their electrophysiological properties. All the cells recorded had afferent conduction velocities in the range of Aβ-afferent conduction speeds. However, these V2 trigeminal Aβ-afferent neurons displayed different action potential (AP) properties. APs showed fast kinetics in some cells but slow kinetics with shoulders in repolarization phases in other cells. Based on the derivatives of voltages in AP repolarization with time (dV/dt), we classified V2 trigeminal Aβ-afferent neurons into four types: type I, type II, type IIIa and type IIIb. Type I V2 trigeminal Aβ-afferent neurons had the largest dV/dt of repolarization, the fastest AP conduction velocities, the shortest AP and afterhyperpolarization (AHP) durations, and the highest AP success rates. In contrast, type IIIb V2 trigeminal Aβ-afferent neurons had the smallest dV/dt of AP repolarization, the slowest AP conduction velocities, the longest AP and AHP durations, and the lowest AP success rates. The type IIIb cells also had significantly lower voltage-activated K+ currents. For type II and type IIIa V2 trigeminal Aβ-afferent neurons, AP parameters were in the range between those of type I and type IIIb V2 trigeminal Aβ-afferent neurons. Our electrophysiological classification of V2 trigeminal Aβ-afferent neurons may be useful in future to study their non-nociceptive and nociceptive functions in orofacial regions.

Daphnane Diterpenoids from Trigonostemon lii and Inhibition Activities Against HIV-1

Natural products are the important source for the discovery of more potent anti-HIV agents. In this study, six daphnane diterpenoids including three unreported structures were isolated from Trigonostemon lii, which showed significant activities against HIV-1 strains replication in the nanomolar/picomolar range. Meanwhile, these diterpenoids significantly inhibited the fusion of H9/HIV-1 IIIB cells with uninfected C8166 cells, with the EC50s from 1.06 to 8.73 ng/mL, and did not show any inhibition activities against HIV-1 reverse transcriptase. Moreover, all of the diterpenoids shows significant inhibitions against T20-resistan HIV-1 strains, PNL4-3gp41(36G)V38E, N42S and pNL4-3gp41(36G)V38A, N42T. The results revealed that the six diterpenoids could be a new type of potential lead candidate as an HIV entry inhibitor, particularly for those infected by T20-resistant variants.Graphic

Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts

Enzyme replacement therapy for MPS IIIB (mucopolysaccharidosis type IIIB; also known as Sanfilippo B syndrome) has been hindered by inadequate mannose 6 phosphorylation and cellular uptake of rhNAGLU (recombinant human α-N-acetylglucosaminidase). We expressed and characterized a modified rhNAGLU fused to the receptor-binding motif of IGF-II (insulin-like growth factor 2) (rhNAGLU-IGF-II) to enhance its ability to enter cells using the cation-independent mannose 6-phosphate receptor, which is also the receptor for IGF-II (at a different binding site). RhNAGLU-IGF-II was stably expressed in CHO (Chinese-hamster ovary) cells, secreted and purified to apparent homogeneity. The Km and pH optimum of the fusion enzyme was similar to those reported for rhNAGLU. Both intracellular uptake and confocal microscopy suggested that MPS IIIB fibroblasts readily take up the fusion enzyme via receptor-mediated endocytosis that was inhibited significantly (P<0.001) by the monomeric IGF-II peptide. Glycosaminoglycan storage was reduced by 60% (P<0.001) to near background levels in MPS IIIB cells after treatment with rhNAGLU-IGF-II, with half-maximal correction at concentrations of 3-12 pM. A similar cellular uptake mechanism via the IGF-II receptor was also demonstrated in two different brain tumour-derived cell lines. Fusion of rhNAGLU to IGF-II enhanced its cellular uptake while maintaining enzymatic activity, supporting its potential as a therapeutic candidate for treating MPS IIIB.

Microvirin, a Novel α(1,2)-Mannose-specific Lectin Isolated from Microcystis aeruginosa, Has Anti-HIV-1 Activity Comparable with That of Cyanovirin-N but a Much Higher Safety Profile

Microvirin (MVN), a recently isolated lectin from the cyanobacterium Microcystis aeruginosa PCC7806, shares 33% identity with the potent anti-human immunodeficiency virus (HIV) protein cyanovirin-N (CV-N) isolated from Nostoc ellipsosporum, and both lectins bind to similar carbohydrate structures. MVN is able to inhibit infection by a wide variety of HIV-1 laboratory-adapted strains and clinical isolates of different tropisms and subtypes in peripheral blood mononuclear cells. MVN also inhibits syncytium formation between persistently HIV-1-infected T cells and uninfected CD4(+) T cells and inhibits DC-SIGN-mediated HIV-1 binding and transmission to CD4(+) T cells. Long term passaging of HIV-1 exposed to dose-escalating concentrations of MVN resulted in the selection of a mutant virus with four deleted high mannose-type glycans in the envelope gp120. The MVN-resistant virus was still highly sensitive to various other carbohydrate binding lectins (e.g. CV-N, HHA, GNA, and UDA) but not anymore to the carbohydrate-specific 2G12 monoclonal antibody. Importantly, MVN is more than 50-fold less cytotoxic than CV-N. Also in sharp contrast to CV-N, MVN did not increase the level of the activation markers CD25, CD69, and HLA-DR in CD4(+) T lymphocytes, and subsequently, MVN did not enhance viral replication in pretreated peripheral blood mononuclear cells. Therefore, MVN may qualify as a useful lectin for potential microbicidal use based on its broad and potent antiviral activity and virtual lack of any stimulatory properties and cellular toxicity.

Novel Recombinant Engineered gp41 N-terminal Heptad Repeat Trimers and Their Potential as Anti-HIV-1 Therapeutics or Microbicides

Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved alpha-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.

Selective inhibitory effects of hybrid liposomes on the growth of HIV type 1-infected cells in vitro

A good correlation between fifty-percent inhibitory concentration (IC 50) of hybrid liposomes (HL) composed of dimyristoylphosphatidylcholine and polyoxyethylene( n) dodecyl ether on the growth of MOLT-4/IIIB cells (MOLT-4 cells chronically infected with human immunodeficiency virus (HIV)) and the membrane fluidity of HL was obtained. Furthermore, the huge enhancement of virus production was observed in the latently HIV-infected (J 22-HL-60) cells after the treatment with HL.

Potent and Selective Inhibition of Tat-Dependent HIV-1 Replication in Chronically Infected Cells by a Novel Naphthalene Derivative JTK-101

In search for effective human immunodeficiency virus type 1 (HIV-1) transcription inhibitors, we have evaluated more than 100,000 compounds for their inhibitory effects on HIV-1 long terminal repeat (LTR)-driven reporter gene expression, and identified a novel naphthalene derivative, JTK-101. This compound could suppress tumour necrosis factor (TNF)-alpha-induced HIV-1 production in latently infected OM-10.1 cells at nanomolar concentrations. JTK-101 could also potently inhibit constitutive HIV-1 production in MOTL-4/IIIB. However, the antiviral activity of JTK-101 was found to be much weaker in acutely infected cells and the chronically infected cells U937/IIIB cells than in OM-10.1 and MOLT-4/IIIB cells. JTK-101 selectively suppressed TNF-alpha-induced HIV-1 mRNA synthesis in OM-10.1 cells in a dose-dependent fashion. JTK-101 modestly inhibited TNF-alpha-induced HIV-1 LTR-driven reporter gene expression, but potently inhibited Tat-induced gene expression. Immunoblot analysis revealed that low-level expression of the Tat cofactors CDK9 and cyclin T1 might contribute to the diminished antiviral activity in U937/IIIB cells. Furthermore, JTK-101 could not inhibit HIV-1 replication in chronically infected monocytes/macrophages, in which CDK9 and cyclin T1 were undetectable. These results suggest that JTK-101 exerts its anti-HIV-1 activity through the inhibition of known or unknown Tat cofactors, presumably CDK9/cyclin T1.

Neurological Correction of Lysosomal Storage in a Mucopolysaccharidosis IIIB Mouse Model by Adeno-associated Virus-Mediated Gene Delivery

Mucopolysaccharidosis (MPS) IIIB is characterized by mild somatic features and severe neurological diseases leading to premature death. No definite treatment is available for MPS IIIB patients. We constructed two recombinant adeno-associated virus (rAAV) vectors containing the human α-N-acetylglucosaminidase (NaGlu) cDNA driven by either a CMV or a neuron-specific enolase (NSE) promoter. In vitro, these rAAV vectors mediated efficient expression of recombinant NaGlu in human MPS IIIB fibroblasts and mouse MPS IIIB somatic and brain primary cell cultures. The secreted rNaGlu was taken up by both human and mouse MPS IIIB cells in culture and degraded the accumulated glycosaminoglycans (GAG). A direct microinjection (107 viral particles, 1 μl/10 minutes per injection) of vectors containing the NSE promoter resulted in long-term (6 months, the duration of the experiments) expression of rNaGlu in multiple brain structures/areas of adult MPS IIIB mice. Consistent with previous studies, the main target cells were neurons. However, while vector typically transduced an area of 400–500 μm surrounding the infusion sites, the correction of GAG storage involved neurons of a much broader area (1.5 mm) in a 6-month duration of experiments. These results provide a basis for the development of a treatment for neurological disease in MPS IIIB patients using AAV vectors.

Antiviral activity of UIC-PI, a novel inhibitor of the human immunodeficiency virus type 1 protease

The human immunodeficiency virus type 1 (HIV-1) protease inhibitor UIC-PI ( 1) was developed via structure-based design and incorporated a novel bis-tetrahydrofuran (bis-THF) ligand in the (R)-(hydroxyethyl)sulfonamide based isostere. The EC 50 and EC 90 of the compound in acutely-infected H9 cells were <1 and ∼1 nM, respectively. In chronically infected H9/HIV-1 IIIB cells, the EC 50 and EC 90 were 20 and 50 nM, respectively. In parallel studies comparing UIC-PI and saquinavir in H9/HIV-1 IIIB cells, viral p24 levels in culture supernatants were an order of magnitude lower with UIC-PI than with saquinavir.

Anti-HIV effects of chloroquine: mechanisms of inhibition and spectrum of activity.

To investigate the mechanisms and spectrum of the anti-HIV activity of chloroquine. MT-4 cells or peripheral blood mononuclear cells were infected with X4, R5 or R5/X4 HIV-1 strains from clades A-E and HIV-2. The cells were then treated with clinically relevant and achievable chloroquine concentrations (i.e. 0-12.5 microM), so as to determine the EC50. The effects of chloroquine on reverse transcription and integration were tested using a replication-defective reporter HIV-1 construct (pRRL.sin.hPGK.GFP). The effects of the drug on the viral envelope were assessed by syncytium assays and immunoprecipitation, using antibodies to different epitopes of gp120. In de-novo infected MT-4 cells, chloroquine selectively inhibited HIV-1 IIIB replication but not pRRL.sin.hPGK.GFP. In chronically HIV-1-infected H9 IIIB cells, chloroquine decreased the infectivity of the newly produced virus and the ability of these cells to form syncytia in co-culture with MT-2 cells. These effects were associated with structural changes in the gp120 glycoprotein, such as a reduction of reactivity with antibodies directed against the glycosylated 2G12 epitope. Although affecting a variable target such as gp120, chloroquine was capable of inhibiting X4, R5 and R5/X4 primary HIV-1 isolates from subtypes A, B, C, D, E and HIV-2. At clinically achievable concentrations chloroquine inhibits HIV-1 post-integrationally by affecting newly produced viral envelope glycoproteins, and the drug has broad-spectrum anti-HIV-1 and HIV-2 activity.

Antiviral properties of a mangrove plant, Rhizophora apiculata Blume, against human immunodeficiency virus

A polysaccharide extracted from the leaf of Rhizophora apiculata (RAP) was assessed in cell culture systems, for its activity against human and simian immunodeficiency viruses. RAP inhibited HIV-1 or HIV-2 or SIV strains in various cell cultures and assay systems. It blocked the expression of HIV-1 antigen in MT-4 cells and abolished the production of HIV-1 p24 antigen in peripheral blood mononuclear cells (PBMC); the 50% effective concentration (EC 50) of RAP in HIV-1 infected MT-4 cells and in PBMC was 10.7 and 25.9 μg/ml, respectively. RAP (100 μg/ml) completely blocked the binding of HIV-1 virions to MT-4 cells. RAP also reduced the production of viral mRNA when added before virus adsorption. RAP inhibited syncytium formation in cocultures of MOLT-4 cells and MOLT-4/HIV-1 IIIB cells. RAP did not prolong activated partial thromboplastin time (APTT) up to 500 μg/ml. These properties may be advantageous should RAP be considered for further development.

Synthesis and anti-HIV-1 activities of new pyrimido[5,4- b]indoles

A set of new pyrimido[5,4- b]indole derivatives that are structurally related to some non-nucleoside HIV-1 reverse transcriptase inhibitors were synthesized and biologically evaluated for their activity as inhibitors of wild and mutant HIV-1 RT types in an ‘in vitro’ recombinant HIV-1 RT screening assay, as well as anti-infectives in HLT4lacZ-1 IIIB cells. Preliminary structure–activity relationships suggest that activity is promoted by simultaneous substitution in positions 2 and 4, especially when chains of alkyldiamine type are present, and by electron-releasing substituents (methoxy) in positions 7 and 8. The inactivity or the very low activity of title derivatives does not suggest interest in AIDS therapy.

Delivery of an anti-HIV-1 ribozyme into HIV-infected cells via cationic liposomes

Cationic liposome-mediated intracellular delivery of a fluorescein-labeled chimeric DNA–RNA ribozyme targeted to the HIV-1 5′ LTR was investigated, using THP-1, THP-1/HIV-1 IIIB or HeLa/LAV cells. Different fluorescence patterns were observed when the cells were exposed to Lipofectamine, Lipofectin or DMRIE:DOPE (1:1) complexed to the ribozyme. With Lipofectamine intense cell-associated fluorescence was found. Incubation with Lipofectin resulted in less intense diffuse fluorescence, while with DMRIE an intense but sporadic fluorescence was observed. Differentiated THP-1/HIV-1 IIIB cells were more susceptible to killing by liposome–ribozyme complexes than THP-1 cells. Under non-cytotoxic conditions (a 4-h treatment) complexes of 5, 10 or 15 μM Lipofectin or DOTAP:DOPE (1:1) and ribozyme, at lipid:ribozyme ratios of 8:1 or 4:1, did not affect p24 production in THP-1/HIV-1 IIIB cells in spite of the intracellular accumulation of the ribozyme. A 24-h exposure of THP-1/HIV-1 IIIB cells to 5 μM Lipofectin or DOTAP:DOPE (1:1) complexed with either the functional or a modified control ribozyme reduced virus production by approximately 30%. Thus, the antiviral effect of the liposome-complexed ribozyme was not sequence-specific. In contrast, the free ribozyme at a relatively high concentration inhibited virus production by 30%, while the control ribozyme was ineffective, indicating a sequence-specific effect. Both Lipofectin and DOTAP complexed with ribozyme were toxic at 10 and 15 μM after a 24-h treatment. A 4-h treatment of HeLa/LAV cells with Lipofectin at 5, 10 or 15 μM was not toxic to the cells, but also did not inhibit p24 production. In contrast, treatment of HeLa CD4 + cells immediately after infection with HIV-1 IIIB at the same lipid concentrations and lipid:ribozyme ratios was cytotoxic. Our results indicate that the delivery of functional ribozyme into cells by cationic liposomes is an inefficient process and needs extensive improvement before it can be used in ex vivo and in vivo applications.

Synergistic inhibition of HIV-1 by an antisense oligonucleotide and nucleoside analog reverse transcriptase inhibitors

We have studied the effects of the gag antisense phosphorothioate oligonucleotide GEM 91® and mismatch antisense controls on the antiviral activities of ddC and other nucleoside analogs in HIV-infected MT-4 cells using a cytoprotection based assay. Under standard assay conditions, i.e. simultaneous incubation of drugs, HIV-1 IIIB and MT-4 cells, both GEM 91® and mismatch controls interacted synergistically with ddC resulting in an approximate 40-fold decrease in the IC 50 value of ddC; this suggests a potent but sequence non-specific effect of GEM 91®. Under post-adsorption assay conditions, i.e. pre-incubation of virus and cells and removal of excess HIV before drug addition, GEM 91® exhibited synergism with ddC, with an approximate 5-fold decrease in ddC IC 50 value. This favorable interaction was not seen with any of the mismatch oligonucleotides, suggesting the involvement of a sequence-specific mechanism of action. Similar results were seen with the thymidine analogs AZT and d4T in combination with GEM 91®. These data suggest a potential role for GEM 91® and future sequence-specific antisense drugs in combination with nucleoside analogs for the treatment of HIV infection. It is essential that potential interactions between new and existing classes of anti-HIV drugs are studied extensively as antiretroviral drug combinations become increasingly more complex.

Antiviral and Biological Properties of Dimethoxytrityl-Linked Guanine-Rich Oligodeoxynucleotides that Inhibit Replication by Primary Clinical Human Immunodeficiency virus Type 1 Isolates

We have investigated the biological and antiviral properties of a dimethoxytrityl (DmTr)-linked guanine (G)-rich oligodeoxynucleotide (ODN) with a phosphodiester linkage, SA-1042 (5' DmTr-TGGGAGGTGGGTCTG 3'), that has been shown to exhibit potent inhibition of human immunodeficiency virus type 1 (HIV-1)-induced cytopathic effect. In a study of the modification of the sequences of SA-1042, it was revealed that the inhibitory effect is exhibited in a highly sequence-specific manner, at the G-rich core sequence (5' TGGG 3'), especially near the 5' end. In a study of the gel mobility-antiviral activity relationship, DmTr modification and the G-rich core sequence of DmTr-ODNs were indispensable for the formation of a hyperstructure and for their antiviral activity. HIV-induced syncytium formation in co-cultures of MOLT-4 cells and chronically infected MOLT-4/HIV-1 IIIB cells was blocked by SA-1042 at a 50% inhibitory concentration (IC 50 ) of 6.5 μg mL -1 . Time-of-addition studies revealed that SA-1042 blocked virus attachment to cells. The susceptibility of 10 fresh HIV-1 clinical isolates [nine with syncytium-inducing (SI) phenotypes and one with a non-SI phenotype], and five laboratory strains with both phenotypes to SA-1042 was assessed by a focus reduction assay using CD4 + HeLa cells or by p24 antigen quantitative assay using human peripheral blood mononuclear cells. SA1042 was active in both assays. These results suggest that SA-1042, which inhibits the adsorption and fusion steps of HIV-1 replication through its hyperstructure formation and the G-rich core sequence, exhibits potent activity against primary clinical isolates.

Stavudine selectively induces apoptosis in HIV type 1-infected cells.

We evaluated the cytotoxic effects of various human immunodeficiency virus (HIV-1) reverse transcriptase inhibitors (zidovudine, didanosine, zalcitabine, stavudine, and nevirapine) on HIV-1-infected and uninfected T cell lines. Among the compounds, only stavudine (not the others) proved to be more cytotoxic to MOLT-4/IIIB cells (MOLT-4 cells chronically infected with HIV-1) than to uninfected MOLT-4 cells. Its 50% cytotoxic concentrations were 59.8 and 2.2 microM for MOLT-4 and MOLT-4/IIIB cells, respectively. Stavudine was also more cytotoxic to CEM/ROD (CEM cells chronically infected with HIV type 2) than to uninfected CEM cells. Microscopic analysis revealed that stavudine induced apoptosis in MOLT-4/IIIB cells. Apparent chromatin condensation in the nucleus was observed by electron microscopy. Furthermore, a DNA fragmentation ladder was detected by agarose gel electrophoresis. Addition of thymidine to the culture medium could rescue the cells from stavudine-induced apoptosis. The expression of anti-apoptotic protein Bcl-2 was partially downregulated in MOLT-4/IIIB cells after treatment with stavudine. This downregulation was not identified in MOLT-4 cells. These results indicate that stavudine selectively induces apoptosis in HIV-1-infected T cells and may have potential as a novel strategy for effective chemotherapy of the acquired immune deficiency syndrome (AIDS).

Antiviral Activity of a Sulphated Polysaccharide Extracted from the Marine Pseudomonas and Marine Plant Dinoflagellata against Human Immunodeficiency Viruses and other Enveloped Viruses

A natural sulphated mucopolysaccharide (OKU40), extracted from a marine plant Dinoflagellata, and an artificial sulphated polysaccharide (OKU41), prepared from a marine Pseudomonas, displayed antiviral activities against several enveloped viruses. OKU40 and OKU41 were found to be homogenous in electrophoresis and sedimation velocity and had a molecular weight of 8.0 × 1065.0 × 105respectively. The sulphation rate of OKU40 and OKU41 was 8.9% and 5.4%, respectively. Each OKU40 and OKU41 inhibited the cytopathic effect of human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and zidovudineresistant HIV-1 in MT-4 cells at similar concentrations to those of dextran sulphate (molecular weight: 5000) (50% inhibitory concentrations: 0.86-1.95 μg mL−1), whereas these compounds did not affect the growth and viability of mock-infected MT-4 cells at concentrations up to 500 μg mL−1. These compounds proved inhibitory not only to HIV-1 and HIV-2 but also to other enveloped viruses, i.e. herpes simplex virus type 1, influenza virus A and B, respiratory syncytial virus and measles virus. OKU40 and OKU41 suppressed syncytium formation induced by cocultivation of MOLT-4/IIIb and MOLT-4 cells at concentrations higher than 20 μg mL−1. Although OKU41 inhibited the binding of HIV-1 to the host cells and the binding of anti-gp120 monoclonal antibody to HIV-1 gp120, OKU40 did not inhibit these bindings, suggesting that the mechanism of anti-HIV activity of OKU40 and OKU41 may be primarily due to the inhibition of virus-cell fusion and viral adsorption to the host cells, respectively. Furthermore, these compounds did not inhibit to the blood coagulation process at a concentration that was significantly inhibitory to HIV replication. The compounds appear to have an interesting potential as virucidal agents.

In vitro inhibitory activity of citreoviridin against HIV-1 and an HIV-associated opportunist: Candida albicans.

Citreoviridin, a mycotoxin produced by some molds of the genera Penicillium and Aspergillus, inhibits the growth of bacteria of the Bacillus genus. Since significant information was not available on the effects of citreoviridin on eukaryotic cells and viruses, this molecule was tested on CD4+ T-lymphoid cell lines, on HIV-1 and on Candida albicans, which sometimes complicates HIV-infection. Antiviral activity was detected in H9 HTLV IIIB cells, a clone chronically infected by HIV-1. Citreoviridin reduced p24 in the supernatant of H9 HTLV IIIB in a dose-dependent manner with a pharmacological selectivity index of 2.6. In C. albicans, the effects of the mold-derivative were evaluated on some parameters associated with pathogenicity and virulence: cellular proliferation, germ tube production, expression of heat shock mannoproteins, release of proteases and phospholipases. At a 12.5 microM dose, citreoviridin showed a marked inhibitory effect on all parameters analyzed. As regards the mechanism of action, it is possible to hypothesize that the effects of citreoviridin may be due to a reduction of protein synthesis, since it inhibited the replication of HIV-1 at post-integrational stages and reduced the intracellular RNA and protein content in C. albicans.

Antibody to HIV-1 Tat protein inhibits the replication of virus in culture.

The HIV-1 transactivator protein Tat is essential for viral replication. Tat is released from infected cells and can be taken up and transactivate HIV-LTR in LTR-CAT transfected cell lines. The present study shows that the addition of monoclonal antibody to Tat in IIIB and MN-infected cultures reduces the HIV antigen production in a concentration dependent manner. These data suggest that external Tat might be important in the replication of HIV, exerting the effect in a paracrine fashion. Using 1 microgram/ml of anti-Tat antibody resulted in a decline of HIV antigen production to 33% and 45% of controls in IIIB and MN infected H9 cells, respectively. A time course experiment showed progressively increased inhibition of replication during 7 days of exposure to anti-Tat antibody, which could be due to increasing Tat concentration. The inhibitory effect of anti-Tat antibodies on the replication of HIV could play an important regulatory role during infection in vivo.