Cationic porphyrin-mediated photodynamic inactivation of Candida biofilms and the effect of miconazole.

The formation of biofilms by Candida and the increasing resistance of Candida species to antifungals contribute to the high recurrence rates of denture stomatitis. This increase has stimulated an interest in antimicrobial photodynamic therapy (aPDT) as an alternative treatment. We examined the photoactivity of the porphyrin-based photosensitizer, TMP-1363, against biofilms of C. albicans, C. glabrata, C. tropicalis and C. parapsilosis, and the effect of the combined use of miconazole and aPDT. Biofilms of three American Type Culture Collection (ATCC) strains and four clinical isolates developed on poly(methyl methacrylate) (PMMA) disks, were incubated with miconazole, followed by treatment with TMP-1363 for 30 min at 37°C. The plates were exposed to broadband visible light at a distance of 10 cm to the plate, for 30 min (irradiance at the surface of the plate: 32.5 mW/cm2). The metabolic activity of the biofilms was measured by the XTT assay. ATCC strains and C. glabrata 7531/06 were not sensitive to TMP-aPDT, whereas the metabolic activities of the remaining three clinical isolates were reduced to 64.2 ± 5.5% of controls. Miconazole at 25 µg/ml decreased the viability of all strains except the ATTCC strain C. albicans MYA274; however its combination with aPDT was effective against this strain, suggesting a synergistic interaction. Effects of miconazole and aPDT on C. albicans MYA 2732, C. albicans 6122/06 were additive. With C. tropicalis and C. parapsilosis, the combined treatment had a higher, but not entirely additive, cytotoxic effect. The combined use of miconazole and TMP-aPDT is advantageous in the treatment of biofilms of a number of Candida species and strains, but not all. The molecular basis of this differential response is not known.

Bone marrow transplantation alters lung antigen-presenting cells to promote TH17 response and the development of pneumonitis and fibrosis following gammaherpesvirus infection.

Hematopoietic stem cell transplantation (HSCT) efficacy is limited by numerous pulmonary complications. We developed a model of syngeneic bone marrow transplantion (BMT) followed by infection with murine gamma herpesvirus-68 that results in pneumonitis and fibrosis and mimics human "noninfectious" HSCT complications. BMT mice experience increased early lytic replication, but establish viral latency by 21 days post infection. CD4 T cells in BMT mice are skewed toward interleukin (IL)-17A rather than interferon (IFN)-γ production. Transplantation of bone marrow from Il-17a(-/-) donors or treatment with anti-IL-17A neutralization antibodies at late stages attenuates pneumonitis and fibrosis in infected BMT mice, suggesting that hematopoietic-derived IL-17A is essential for development of pathology. IL-17A directly influences activation and extracellular matrix production by lung mesenchymal cells. Lung CD11c+ cells of BMT mice secrete more transforming growth factor beta-ß1, and pro-TH17 mRNAs for IL-23 and IL-6, and less TH1-promoting cytokine mRNA for IFN-γ but slightly more IL-12 mRNA in response to viral infection. Adoptive transfer of non-BMT lung CD11c-enriched cells restores robust TH1 response and suppresses aberrant TH17 response in BMT mice to improve lung pathology. Our data suggest that "noninfectious" HSCT lung complications may reflect preceding viral infections and demonstrate that IL-17A neutralization may offer therapeutic advantage even after disease onset.

Miconazole activity against Candida biofilms developed on acrylic discs.

Oral candidiasis in the form of Candida-associated denture stomatitis (CaDS) is associated with Candida adhesion and biofilm formation on the fitting surface of poly (methyl methacrylate) (PMMA) dentures. Candida biofilms show considerable resistance to most conventional antifungal agents, a phenomenon that is considered a developmental-phase-specific event that may help explain the high recurrence rates associated with CaDS. The aim of this study was to examine the activity of miconazole towards in vitro-grown mature Candida biofilms formed on heat-cured PMMA discs as a standardized model. The effect of miconazole nitrate on Candida biofilms developed on acrylic discs was determined for C. albicans MYA-2732 (ATCC), C. glabrata MYA-275 (ATCC), and clinical isolates, C. albicans 6122/06, C. glabrata 7531/06, C. tropicalis 8122/06, and C. parapsilosis 11375/07. Candida biofilms were developed on heat-cured poly(methyl methacrylate) discs and treated with miconazole (0.5 - 96 µg/ml). The metabolic activity of the biofilms was measured by the XTT reduction assay. The minimum inhibitory concentrations (MICs) of miconazole against Candida species were determined by the microdilution method. The MICs for miconazole for the investigated strains ranged from 0.016-32 µg/ml. Treatment with miconazole resulted in a significant reduction of biofilm metabolic activity for all strains. The highest inhibition was observed at 96 µg/ml miconazole. In the case of C. glabrata MYA-275 and C. tropicalis 8122/06 this corresponded to 83.7% and 75.4% inhibition, respectively. The lowest reduction was observed for C. parapsilosis 11375/07-46.1%. For all Candida strains there was a strong correlation between MIC values and miconazole concentrations corresponding to a reduction of metabolic activity of the biofilm by 50%. Miconazole exhibits high antifungal activity against Candida biofilms developed on the surface of PMMA discs. The study provides support for the use of miconazole as an effective agent for the treatment of CaDS.

Do patients with chronic patellar tendinopathy have an altered somatosensory profile? A Quantitative Sensory Testing (QST) study.

The prevalence of tendinopathies in sports is high. The etiology and pain mechanisms of tendinopathies are not completely understood. Currently, little is known whether, or to which degree, somatosensory changes within the nervous system may contribute to the pain in tendinopathies. We conducted a patient controlled study in which we used the standardized QST protocol developed by the German Research Network on Neuropathic Pain. This protocol consists of seven different tests that measures 13 somatosensory parameters and can be seen as the gold standard to measure somatosensory function. Twelve athletes with clinically diagnosed chronic patellar tendinopathy (PT) mean duration 30 months (range 6-120) and 20 controls were included in the study. In two of the 13 QST parameters namely Mechanical Pain Threshold (P < 0.05) and Vibration Disappearance Threshold (P < 0.5) injured athletes were significantly more sensitive for the applied stimuli. None of the athletes had signs of Dynamic Mechanical Allodynia. Reduced mechanical pain thresholds or pinprick allodynia reflects the involvement of central sensitization upon the myelinated (Aδ-fibre) nociceptive input. From this explorative study, we conclude that sensitization may play a prominent role in the pain during and after sports activity in patella tendinopathy patients.

Transferrin lipoplex-mediated suicide gene therapy of oral squamous cell carcinoma in an immunocompetent murine model and mechanisms involved in the antitumoral response.

Suicide gene therapy has been used for the treatment of a variety of cancers. We reported previously the in vitro efficacy of the Herpes Simplex Virus Thymidine kinase (HSV-tk)/ganciclovir (GCV) system to mediate cytotoxicity in oral squamous cancer cells, using transferrin (Tf)-lipoplexes, prepared from cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and cholesterol. In the present study, we evaluated the antitumoral efficacy mediated by this lipoplex formulation in two suicide gene therapy strategies, HSV-tk/GCV and cytosine deaminase (CD)/5-fluorocytosine (5-FC), using a syngeneic, orthotopic murine model for head and neck squamous cell carcinoma. The cellular and molecular events associated with the antitumoral response elicited by both the therapeutic approaches were investigated by analyzing tumor cell death, tumor-infiltrating immune cells and tumor cytokine microenvironment. Significant tumor reduction was achieved upon intratumoral delivery of HSV-tk or CD genes mediated by Tf-lipoplexes, followed by intraperitoneal injection of GCV or 5-FC, respectively. Enhanced apoptosis, the recruitment of NK cells, CD4 and CD8 T-lymphocytes and an increase in the levels of several cytokines/chemokines were observed within the tumors. These observations suggest that suicide gene therapy with lipoplexes modifies the tumor microenvironment, and leads to the recruitment of immune effector cells that can act as adjuvants in reducing the tumor size.

The association between gingival crevicular fluid TGF-beta1 levels and periodontal status in HIV-1(+) patients.

BACKGROUND: The purpose of this study was to investigate the relationship between transforming growth factor-beta 1 (TGF-beta1) in gingival crevicular fluid (GCF) and the periodontal status of subjects who were positive for the human immunodeficiency virus (HIV)-1. METHODS: Medical and demographic variables, including age, cigarette smoking, CD4 cell count, and viral load values, were recorded. At the baseline and 6-month visits, gingival index (GI), plaque index, bleeding on probing, probing depth (PD), and attachment loss (AL) were recorded, and GCF samples were taken with paper strips from three periodontitis sites (GI >0; PD > or =5 mm; AL > or =3 mm), three gingivitis sites (GI >0; PD < or =3 mm; AL = 0), and two healthy sites (GI = 0; PD < or =3 mm; AL < or =2 mm) in 25 subjects who were HIV-1(+). GCF TGF-beta1 levels were determined by enzyme-linked immunosorbent assays. A statistical software package was used to analyze the data. RESULTS: The mean amounts of GCF TGF-beta1 were greater in gingivitis and periodontitis sites than in healthy sites (P <0.0001). GCF levels of TGF-beta1 correlated with PD, AL, age, smoking pack-years, CD4 cell count, and viral load at the baseline and 6-month visits (0.0001 < P <0.05). An active site was defined as a site that had > or =2 mm new AL during the 6-month study period. An active patient was defined as a patient who had one or more active site(s) during the study period. Repeated-measures analysis of 18 active sites versus 182 inactive sites indicated that GCF TGF-beta1 levels were higher in active sites than in inactive sites (P <0.0001). Eleven of the 25 study subjects had active sites at the end of the 6-month study period. The mean GCF TGF-beta1 level and the mean AL and PD for these 11 active subjects were higher than for the 14 inactive subjects (P <0.0001). CONCLUSION: In subjects who are HIV-1(+), sites with high GCF levels of TGF-beta1 are at significantly greater risk for the progression of established periodontitis.

Photodynamic therapy in dentistry.

Photodynamic therapy (PDT), also known as photoradiation therapy, phototherapy, or photochemotherapy, involves the use of a photoactive dye (photosensitizer) that is activated by exposure to light of a specific wavelength in the presence of oxygen. The transfer of energy from the activated photosensitizer to available oxygen results in the formation of toxic oxygen species, such as singlet oxygen and free radicals. These very reactive chemical species can damage proteins, lipids, nucleic acids, and other cellular components. Applications of PDT in dentistry are growing rapidly: the treatment of oral cancer, bacterial and fungal infection therapies, and the photodynamic diagnosis (PDD) of the malignant transformation of oral lesions. PDT has shown potential in the treatment of oral leukoplakia, oral lichen planus, and head and neck cancer. Photodynamic antimicrobial chemotherapy (PACT) has been efficacious in the treatment of bacterial, fungal, parasitic, and viral infections. The absence of genotoxic and mutagenic effects of PDT is an important factor for long-term safety during treatment. PDT also represents a novel therapeutic approach in the management of oral biofilms. Disruption of plaque structure has important consequences for homeostasis within the biofilm. Studies are now leading toward selective photosensitizers, since killing the entire flora leaves patients open to opportunistic infections. Dentists deal with oral infections on a regular basis. The oral cavity is especially suitable for PACT, because it is relatively accessible to illumination.

Metal particles in a ceramic matrix--scanning electron microscopy and transmission electron microscopy characterization.

This paper is concerned with ceramic matrix (Al(2)O(3)) composites with introduced metal particles (Ni, Fe). The composites were obtained via sintering of powders under very high pressure (2.5 GPa). Scanning electron microscopy and transmission electron microscopy were chosen as the tools for the identification and description of the shape, size and distribution of the metal particles. The Al(2)O(3)-Ni composite contained agglomerates of the Ni particles surrounded by ceramic grains and nanometre-size Ni particles located inside the ceramic grains and at the ceramic grain boundaries. In the Al(2)O(3)-Fe composite, the Fe particles were mostly surrounded by ceramic grains. Moreover, holes left by the Fe particles were found. The high pressure used in the fabrication of the composites changed the shape of the metal and ceramic powder grains via plastic deformation.

Influence of antifungal polyenes on the adhesion of Candida albicans and Candida glabrata to human epithelial cells in vitro.

Candidal adherence to mucosal surfaces is considered as the first step in the pathogenesis of oral candidiasis. We examined the effect of antifungal polyenes, amphotericin B, nystatin and natamycin, at sublethal and minimum inhibitory concentrations (MICs) on the adherence of Candida albicans and Candida glabrata to HeLa cervical carcinoma and HSC-3 oral squamous cell carcinoma cells. A total of six oral Candida isolates were used throughout the study. Two Candida strains, C. albicans (44990) and C. glabrata (MYA-275) were obtained from ATCC. Four Candida strains, C. albicans 19 and 24 and C. glabrata 15 and 21, were isolated from patients with documented Candida-associated denture stomatitis. Cells were either incubated with Candida in the presence of the drug, or pre-incubated with yeasts and exposed subsequently to the drug. In the drug-free controls, the mean number of C. albicans yeasts associated with HeLa cells obtained from all experiments (130.1+/-10.1 yeasts/mm(2)) was significantly greater than that for HSC-3 cells (114.7+/-10.1 yeasts/mm(2); P<0.025). For C. glabrata, the mean adherence to HeLa and HSC-3 cells was 84.4+/-5.5 and 84.4+/-3.3 yeasts/mm(2), respectively, and these values were not statistically different (P>0.4). Candidal adherence was significantly reduced when the tested polyenes were present during the "adherence phase". The obtained values were significantly different from the controls, except for the effect of nystatin at the MIC on the adherence of C. glabrata strain MYA-275 to HeLa cells (P<0.375). Amphotericin B had the highest effect against both Candida species, reducing adherence by approximately 50 and approximately 60%, at the MIC and sublethal concentrations, respectively. The susceptibility of cell-associated Candida to polyenes was decreased markedly and the treatment did not result in significant detachment of adherent yeasts. The reduction in adherence was between 2 and 10%, when compared to the drug-free controls. These findings suggest that sub-therapeutic levels of polyenes that are likely to persist in the oral cavity following topical treatment may modulate candidal colonization when present during the "adherence phase".

Delivery of novel macromolecular drugs against HIV-1.

The development of new low molecular weight drugs against human immunodeficiency virus Type 1 (HIV-1) targets other than reverse transcriptase (RT) and protease, such as the integrase and the envelope glycoprotein, is likely to take many years. Macromolecular drugs, including antisense oligonucleotides, ribozymes, RNA decoys and transdominant mutant proteins, may be able to interfere with a relatively large number of viral targets, thereby decreasing the likelihood of the emergence of drug-resistant strains. It may also be relatively easy to alter the sequence of some of the macromolecular drugs to counter emerging drug-resistant viruses. The delivery of antisense oligonucleotides and ribozymes to HIV-1 infected or potentially infectable cells by antibody-targeted liposomes, certain cationic lipid formulations and pH-sensitive liposomes results in significant anti-HIV-1 activity. These carriers not only facilitate cytoplasmic delivery but also protect the drugs from nuclease digestion. Delivery of therapeutic genes (another form of macromolecular drug) to target cells is an important challenge of gene therapy. Following delivery by a viral vector, sufficient levels of gene expression must be maintained over an extended period of time to have therapeutic activity. Robust expression of therapeutically useful ribozymes, antisense, decoys and aptamers can be achieved by the use of Pol III expression systems. Moloney murine leukaemia virus- (MoMuLV), adeno-associated virus (AAV)-, or HIV-derived vectors expressing a variety of therapeutic genes have been used successfully to inhibit HIV-1 replication in cultured cells.

Rev-binding aptamer and CMV promoter act as decoys to inhibit HIV replication.

We examined whether the antiviral effect of an HIV-1 Rev-binding aptamer [RBE(apt)] could be enhanced by a ribozyme directed against the HIV-1 env gene, and whether the antiviral activity was affected by different promoters. The efficacy of the aptamer and ribozyme DNAs was tested in HeLa cells co-transfected with the HIV-1 proviral clones, HXBDeltaBgl or pNL4-3, using transferrin-lipoplexes. The RBE(apt) and anti-env ribozyme genes were inserted into the pTZU6+27 plasmid, or constructed under the control of the human cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters. The parental vector plasmids were used as controls. Co-transfection of the pTZU6+27 RBE(apt) plasmid with HXBDeltaBgl, or pNL4-3, at a weight ratio of 5:1, inhibited p24 production by 70 and 45%, respectively. The RSV RBE(apt) plasmid co-transfected with either HIV clone, at the same weight ratio, reduced viral production by 88%. The addition of the anti-env ribozyme to the RSV RBE(apt) did not enhance its antiviral activity. When the constructs were under the control of the CMV promoter, the expression of the HIV plasmids was very low and was independent of the presence of the RBE(apt). Thus, the effect of the RBE(apt) was strongly dependent on the promoter of the tested construct. The anti-HIV activity of the CMV RBE(apt) construct was non-specific, because co-transfection with either pCMV. SPORT-betagal or pCMVlacZ significantly suppressed HIV production from the HIV proviral clones. The reduction in p24 could not be attributed to the non-specific toxicity of the transfection procedure. Transfection of acutely HIV-infected HeLa-CD4 cells with pCMV.SPORT-betagal reduced the p24 level by 35%, while the expression of the U6 RBE(apt) did not affect p24 production. The suppression of HIV production from the HIV proviral clones by the CMV promoter constructs in the co-transfection assays may be explained by competition for transcription factors (TFs) between HIV and CMV promoters. This observation points to the potential for misleading results in co-transfections involving CMV constructs and HIV.

Secretory leukocyte protease inhibitor (SLPI): oxidation of SLPI does not explain its variable anti-HIV activity.

Secretory leukocyte protease inhibitor (SLPI) has been proposed as a potential inhibitor of HIV-1 infection in human saliva. Although the ability of recombinant (r) SLPI to inhibit HIV-1 infection of macrophages and primary T-cells has been demonstrated by two independent laboratories, evidence to the contrary has also been reported. This study re-examines the anti-HIV effect of rSLPI and investigates the effects of repeated freeze-thawing and oxidation on the anti-HIV activity of rSLPI. rSLPI inhibited HIV-1BaL infection of human macrophages in a highly variable manner. HPLC and electrospray ionization mass spectrometry (ESI) analyses indicated that variability in our inhibition data could not be attributed to the degradation or oxidation of rSLPI. These results suggest that the variable anti-HIV effect of rSLPI may be due to differential expression of the cell-surface molecule(s) to which SLPI binds rather than to changes in the rSLPI molecule.

Anti-HIV activity of amphotericin B-cholesteryl sulfate colloidal dispersion in vitro.

We examined whether the anti-HIV-1 activity of the polyene antibiotic Amphotericin B (AMB) is retained following incorporation into sterically stabilized 'Stealth' liposomes (L-AMB) with prolonged circulation in vivo, or cholesteryl sulfate colloidal dispersions (CD-AMB). The effects of the different preparations on acute infection of H9 cells with HIV-1IIIB, spreading of the virus from chronically infected H9/HTLV-IIIB cells to SupT1 cells, and HIV-1-induced syncytium formation were evaluated. Infection was monitored by p24 levels in culture supernatants. L-AMB did not affect HIV-1 infection. When present only during initial infection, AMB (3-20 microg/ml) reduced p24 levels by 70-80% after 7 and 10 days post-infection, while CD-AMB inhibited p24 production by approximately 30-40% at day 7 and 50-60% at day 10. The inhibitory effect of CD-AMB and AMB was enhanced by continuous treatment of acutely infected cells. The reduction of p24 production during continuous treatment was not due to cytotoxicity. During spreading of infection from infected to uninfected cells, AMB almost completely inhibited virus production while CD-AMB reduced both p24 production and the cytopathic effect in a dose-dependent manner. HIV-1 induced syncytium formation was slightly inhibited by AMB but not by CD-AMB. Because CD-AMB is considerably less cytotoxic than AMB, its ability to inhibit HIV infection in vivo needs to be evaluated further.

Liposome-mediated delivery of antiviral agents to human immunodeficiency virus-infected cells.

Intracellular delivery of novel macromolecular drugs against human immunodeficiency virus type-1 (HIV-1), including antisense oligodeoxynucleotides, ribozymes and therapeutic genes, may be achieved by encapsulation in or association with certain types of liposomes. Liposomes may also protect these drugs against nucleases. Low-molecular-weight, charged antiviral drugs may also be delivered more efficiently via liposomes. Liposomes were targeted to HIV-1-infected cells via covalently coupled soluble CD4. An HIV-1 protease inhibitor encapsulated in conventional negatively charged multilamellar liposomes was about 10-fold more effective and had a lower EC90 than the free drug in inhibiting HIV-1 production in human monocyte-derived macrophages. The drug encapsulated in sterically stabilized liposomes was as effective as the free drug. The EC50 of the reverse transcriptase inhibitor 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was reduced by an order of magnitude when delivered to HIV-1-infected macrophages in pH-sensitive liposomes. A 15-mer antisense oligodeoxynucleotide against the Rev response element was ineffective in free form against HIV-1 replication in macrophages, while delivery of the oligonucleotide in pH-sensitive liposomes inhibited virus replication. The oligodeoxynucleotide encapsulated in sterically stabilized pH-sensitive liposomes with prolonged circulation in vivo, which were recently developed in the laboratories of the authors, was also highly effective. A ribozyme complementary to HIV-1 5'-LTR delivered in pH-sensitive liposomes inhibited virus production by 90%, while the free ribozyme caused only a slight inhibition. Cationic liposome-mediated co-transfection of the HIV-regulated diphtheria toxin A fragment gene and a proviral HIV clone into HeLa cells completely inhibited virus production, while the frame-shifted mutant gene was ineffective. Co-transfection of the proviral genome and a gene encoding a Rev-binding aptamer into HeLa cells via transferrin-associated cationic liposomes inhibited virus production. These studies indicate that liposomes can be used to facilitate the intracellular delivery of certain anti-HIV agents and to enhance their therapeutic effects. These properties may be particularly advantageous in the development of novel macromolecular drugs, which may be necessary because of the emergence of virus strains resistant to the currently available drugs.

Receptor ligand-facilitated cationic liposome delivery of anti-HIV-1 Rev-binding aptamer and ribozyme DNAs.

We examined whether HIV-1 gene expression could be inhibited by the anti-HIV Rev-binding aptamer [RBE(apt)], and whether the antiviral effect of the aptamer could be enhanced by a ribozyme directed against the HIV-1 env gene. Since cationic liposomes are relatively safe and non-immunogenic for in vivo gene delivery, we tested the effectiveness of the aptamer and ribozyme DNAs in HeLa cells, using Lipofectin reagent in a transient transfection assay. To increase the transfection efficiency, lipofectin was mixed with transferrin before subsequent addition of DNA. Co-transfection of HeLa cells with the RBE(apt) and the proviral HIV clone, HXBdeltaBgl, resulted in inhibition of virus production. Specific inhibition of viral p24 production following co-transfection of the RBE(apt) and HIV proviral DNAs was observed. These data provide strong support for the use of in vitro evolved ligands as potential anti-HIV agents. The addition of the anti-env ribozyme to the aptamer construct did not further enhance the antiviral activity, suggesting either that we had reached the limits of inhibition in this assay, or that the ribozyme was not able to access its target site with Rev bound to the RBE aptamer. The observed inhibition of p24 production could not be attributed to the non-specific toxicity of the transfection procedure, because no difference in viability was observed between the RBE(apt)- and the vector control-treated cells. All of the aptamer-ribozyme constructs as well as the RBE(apt) were similarly effective.

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-1IIIB 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-1IIIB 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 microM 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-1IIIB cells in spite of the intracellular accumulation of the ribozyme. A 24-h exposure of THP-1/HIV-1IIIB cells to 5 microM 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 microM after a 24-h treatment. A 4-h treatment of HeLa/LAV cells with Lipofectin at 5, 10 or 15 microM 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-1IIIB 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.

Platelet-activating factor mediates angiotensin II-induced proteinuria in isolated perfused rat kidney.

Isolated kidney preparations (IPK) from male Sprague Dawley rats perfused at constant pressure were used to evaluate the effect of angiotensin II (AII) and platelet-activating factor (PAF) on renal function and urinary protein excretion. Compared with basal, intrarenal infusion of AII at 8 ng/min caused a progressive increase in protein excretion (11 +/- 6 versus 73 +/- 21 micrograms/min) in parallel with a decline in renal perfusate flow (RPF) (29 +/- 3 versus 18 +/- 3 ml/min). Addition to the perfusate of PAF at 50 nM final concentration also induced proteinuria (9 +/- 4 versus 55 +/- 14 micrograms/min) but did not change RPF (29 +/- 3 versus 30 +/- 3 ml/min). Preexposure of isolated kidneys to the PAF receptor antagonist WEB 2086 prevented the increase in urinary protein excretion induced by AII infusion (basal: 13 +/- 6; post-AII: 12 +/- 7 micrograms/min) but failed to prevent the vasoactive effect of AII (RPF, basal: 30 +/- 2; post-AII: 21 +/- 3 ml/min). In additional experiments, dexamethasone reduced the proteinuric effect of PAF remarkably. These results indicate that in isolated kidney preparation: (1) AII infusion induced proteinuria and decreased RPF; and (2) the effect of AII in enhancing urinary protein excretion was completely prevented by a specific PAF receptor antagonist, which, however, did not influence the AII-induced fall in RPF. It is suggested that PAF plays a major role in AII-induced changes in the permselective function of the glomerular capillary barrier.

Cationic liposome-mediated expression of HIV-regulated luciferase and diphtheria toxin a genes in HeLa cells infected with or expressing HIV.

HIV-regulated expression of the diphtheria toxin A fragment gene (HIV-DT-A) is a potential gene therapy approach to AIDS. Since cationic liposomes are safe and non-immunogenic for in vivo gene delivery, we examined whether LipofectAMINE or DMRIE reagent could mediate the transfection of HIV-DT-A (pTHA43) or the HIV-regulated luciferase gene (pLUCA43) into HIV-infected or uninfected HeLa cells. pLUCA43 was expressed at a 10(3)-fold higher level in HeLa/LAV cells than in uninfected HeLa cells, while the extent of expression of RSV-regulated luciferase was the same in both cell lines. Co-transfection of HeLa cells with pTHA43 and the proviral HIV clone, HXB deltaBgl, resulted in complete inhibition of virus production. In contrast, the delivery of HIV-DT-A to chronically infected HeLa/LAV or HeLa/IIIB cells, or to HeLa CD4+ cells before infection, did not have a specific effect on virus production, since treatment of cells with control plasmids also reduced virus production. This reduction could be ascribed to cytotoxicity of the reagents. The efficiency of transfection, as measured by the percentage of cells expressing beta-gal, was approximately 5%. Thus, cationic liposome-mediated transfection was too inefficient to inhibit virus production when the DT-A was delivered by cationic liposomes to chronically- or de novo- infected cells. However, when both the virus and DT-A genes were delivered into the same cells by cationic liposomes, DT-A was very effective at inhibiting virus production. Our results indicate that the successful use of cationic liposomes for gene therapy will require the improvement of their transfection efficiency.

The anti-HIV-1 activity associated with saliva.

This review summarizes the data on the anti-human immunodeficiency virus (HIV) activity associated with saliva and the possible routes of oral transmission of HIV. Saliva can be passed from an HIV-infected individual to an uninfected person via sexual or non-sexual activities. The relative risk of HIV transmission through saliva is a subject of continuing concern for dental practitioners. HIV-infected individuals frequently have oral lesions that can cause bleeding and release of the virus into the oral cavity. In addition, viral p24 and HIV-1 RNA were detected in tonsils and adenoids even in asymptomatic seropositive individuals. Nevertheless, the potential HIV-infectivity of saliva is low, although both infectious HIV-1 and HIV DNA have been detected in saliva. This observation has led to the suggestion that saliva may contain factors that inhibit HIV-1 infectivity. At least two anti-HIV activities have been partially characterized: (i) physical entrapment of HIV by high-molecular-weight molecules (e.g., mucins), and (ii) inhibition of viral infection by soluble proteins. Several studies have indicated that, of the salivary proteins evaluated, recombinant secretory leukocyte protease inhibitor (rSLPI) could inhibit HIV-1 infection in macrophages at physiological concentrations. The anti-HIV activity of the serine protease inhibitor rSLPI is most likely due to its interaction with a cell-surface molecule(s) other than the primary HIV-1 receptor, CD4, and may involve (i) inhibition of cell-surface serine protease(s), and/or (ii) interaction with other human-specific co-factors essential for viral entry.