Effective and safe gene-based delivery of GLP-1 using chitosan/plasmid-DNA therapeutic nanocomplexes in an animal model of type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates blood glucose level post-prandially. It has been proposed that GLP-1 can be used in type 2 diabetes (T2D) mellitus treatment because of its insulinotropic action. Despite its remarkable advantages, GLP-1 suffers the disadvantage of an extremely short half-life owing to its degradation by the dipeptidyl peptidase IV protease. One way of overcoming this drawback is GLP-1 gene delivery. Here we show effective and safe gene-based delivery of GLP-1 using chitosan/plasmid-DNA therapeutic nanocomplexes (TNCs) in Zucker diabetic fatty (ZDF) animal model of T2D. The expression plasmid fused the GLP-1 gene to a Furin cleavage site was driven by a cytomegalovirus promoter/enhancer. TNCs were prepared by mixing this plasmid with chitosans of specific molecular weight (MW), degree of deacetylation (DDA) and ratio of chitosan amine to DNA phosphate (N:P ratio). Animals injected with the TNC chitosan 92-10-5 (DDA-MW-N:P) showed GLP-1 plasma levels of about fivefold higher than that in non-treated animals and the insulinotropic effect of recombinant GLP-1 was shown by a threefold increase in plasma insulin concentration when compared with untreated animals. Intraperitoneal glucose tolerance tests revealed an efficacious decrease of blood glucose compared with controls for up to 24 days after treatment, where injections of this formulation allowed near-normalization of blood glucose level. TNCs composed of specific chitosans and GLP-1-expressing plasmid constructs showed an impressive ability to harness the profound therapeutic potential of GLP-1 for the treatment of T2D mellitus.

Chitosan-plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies.

Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.

HIV-1 gp120/160 expressing transfected cell clones to evaluate the antibody-dependent cellular cytotoxicity (ADCC) during the course of HIV-1 infection.

Transfection by electroporation of the CEM.NKR cell line with a plasmid containing the HIVNL43 gp160 gene resulted in the establishment of HIV-1 gp120/160 expressing CEM.NKR cell clones. The cell-surface expression of the recombinant viral envelope is stable and does not lead to syncytium formation among a substantial number of cells expressing envelope glycoprotein, suggesting that these cells are suitable as targets to monitor HIV-specific ADCC activities with the stage or the progression of HIV-1 disease. By using these cells, the ability of sera from HIV-1 seropositive individuals to mediate ADCC against HIV-1 gp120/160 transfected cells in an antigen-specific manner was established. Approximately 63% of the HIV-infected individuals representing all stages of infection have virus-specific ADCC antibodies. Moreover, sera from healthy seropositive (CDC class II) subjects mediated substantially higher levels of ADCC activity (25.7 +/- 11.5%) than did sera from individuals in CDC class III (10.3 +/- 8.9%) or IVC1 (6.4 +/- 8.1%). The mean ADCC activity for the sera from the seronegative volunteers was 2 +/- 1.3%. The correlation between the ADCC level and CD4 cell depletion remains uncertain. Therefore, the established transfected cell line may be used to probe the role of gp120/160-specific ADCC activity in the development of AIDS and may also prove useful in screening human and murine monoclonal antibodies with potential ADCC activity. Such monoclonals may be useful for future immunotherapeutic agents in conjunction with antiviral chemotherapy.

Accurate and differential quantitation of HIV-1 tat, rev and nef mRNAs by competitive PCR.

An accurate method is described for measuring the relative abundance of HIV-1 regulatory mRNAs directly in clinical specimens. Specimen RNA is reverse transcribed and coamplified with a common competitor containing tat, rev and nef internal standards using fluorescent primers and a competitive polymerase chain reaction. After amplification, individual products are separated and analyzed on a fluorescent DNA sequencer. Using this approach, it was possible to measure reproducibly two-fold differences in the relative abundance of mRNAs with coding potential for tat, rev and nef from as little as 0.2 ng of total RNA extracted from peripheral blood mononuclear cells of HIV-1 infected persons. The ratio method eliminates the need to account for variability in RNA recovery during sample processing and provides a powerful tool for measuring the differential expression of HIV-1 regulatory genes in vivo.

Cannabis improves night vision: a case study of dark adaptometry and scotopic sensitivity in kif smokers of the Rif mountains of northern Morocco.

Previous reports have documented an improvement in night vision among Jamaican fishermen after ingestion of a crude tincture of herbal cannabis, while two members of this group noted that Moroccan fishermen and mountain dwellers observe an analogous improvement after smoking kif, sifted Cannabis sativa mixed with tobacco (Nicotiana rustica). Field-testing of night vision has become possible with a portable device, the LKC Technologies Scotopic Sensitivity Tester-1 (SST-1). This study examines the results of double-blinded graduated THC administration 0-20 mg (as Marinol) versus placebo in one subject on measures of dark adaptometry and scotopic sensitivity. Analogous field studies were performed in Morocco with the SST-1 in three subjects before and after smoking kif. In both test situations, improvements in night vision measures were noted after THC or cannabis. It is believed that this effect is dose-dependent and cannabinoid-mediated at the retinal level. Further testing may assess possible clinical application of these results in retinitis pigmentosa or other conditions.

Contribution to the knowledge of Rifian traditional medicine. II: Folk medicine in Ksar Lakbir district (NW Morocco).

An ethnobotanical survey of the medicinal plants used by the local population of the Ksar Lakbir district (NW Morocco) was conducted. One hundred and eighty-six species from 61 botanical families were recorded as well as their uses and modes of administration. Quantitative ethnopharmacological data (medicinal plant knowledge and use indices) were also evaluated and discussed.

Relationship between ammonia sensing properties of polyaniline nanostructures and their deposition and synthesis methods.

The ammonia absorption properties of polyaniline nanostructures are studied in terms of sensitivity, response and recovery times and stability. These characteristics are obtained by measuring, at room temperature, the absorbance variations at 632 nm. The nanostructures are synthesized either by interfacial or rapid or dropwise polymerizations with the oxidant-to-monomer mole ratio equals to 0.5 or 1. The influence of the deposition method (in-situ or drop-coating technique) as well as the nature of the dopant (HCl, CSA or I(2)) on the gas detection properties are also studied. The results show a strong dependence of the morphology on the deposition method, the in-situ technique leads to the best sensitivity and response time. For this deposition method, the nanostructures sensitivity, response time and regeneration rate depend on the synthesis method, the dopant and the mole ratio. The ageing effect after 8 months under ambient conditions and the mechanism of interaction between the polyaniline nanostructures and ammonia molecules are also presented.

Assessing changes in pupillary size in Rifian smokers of kif (Cannabis sativa L.).

Although the measurement of eye pupil variations is a common method in the only few cannabis effect research, there are no studies on short term effects of kif (Moroccan traditional preparation of cannabis) on eye pupil. The aim of the present paper is to present results about effect of a smoked kif preparation (Cannabis sativa L.) on pupil diameter variations after 30 mn. Two examiners measured the pupil diameter variations before and after kif smoking in 34 eyes of 17 volunteer-consumers in a dark closed room. Pupil diameter was estimated by Colvard pupillometer. Results reveal a significantly increase in pupil size post kif.

T-cell receptor-mediated anergy of a human immunodeficiency virus (HIV) gp120-specific CD4(+) cytotoxic T-cell clone, induced by a natural HIV type 1 variant peptide.

Human immunodeficiency virus type 1 (HIV-1) infection triggers a cytotoxic T-lymphocyte (CTL) response mediated by CD8(+) and perhaps CD4(+) CTLs. The mechanisms by which HIV-1 escapes from this CTL response are only beginning to be understood. However, it is already clear that the extreme genetic variability of the virus is a major contributing factor. Because of the well-known ability of altered peptide ligands (APL) to induce a T-cell receptor (TCR)-mediated anergic state in CD4(+) helper T cells, we investigated the effects of HIV-1 sequence variations on the proliferation and cytotoxic activation of a human CD4(+) CTL clone (Een217) specific for an epitope composed of amino acids 410 to 429 of HIV-1 gp120. We report that a natural variant of this epitope induced a functional anergic state rendering the T cells unable to respond to their antigenic ligand and preventing the proliferation and cytotoxic activation normally induced by the original antigenic peptide. Furthermore, the stimulation of Een217 cells with this APL generated altered TCR-proximal signaling events that have been associated with the induction of T-cell anergy in CD4(+) T cells. Importantly, the APL-induced anergic state of the Een217 T cells could be prevented by the addition of interleukin 2, which restored their ability to respond to their nominal antigen. Our data therefore suggest that HIV-1 variants can induce a state of anergy in HIV-specific CD4(+) CTLs. Such a mechanism may allow a viral variant to not only escape the CTL response but also facilitate the persistence of other viral strains that may otherwise be recognized and eliminated by HIV-specific CTLs.

HIV-1 gp120/160 expressing cells upregulate HIV-1 LTR directed gene expression in a cell line transfected with HIV-1 LTR-reporter gene constructs.

The results described in this paper demonstrate that HIV-1 gp120 can upregulate gene expression directed by the HIV-1 LTR. Briefly, exposing responder CD4+CEM-T4 ID5 cells to stimulator CEMgp120/160 expressing cells (stably transfected with HIV-1 LTR-CAT and HIV-1 gp160, respectively) resulted in the increased synthesis of the CAT enzyme. Control non-transfected CEM-T4 cells did not induce the synthesis of CAT. In addition, when the responder cell line, U937-1C5 which also contains stably transfected HIV-1 LTR-CAT plasmid was exposed to irradiated CEM gp120/160 cells, there was no synthesis of the CAT enzyme. Neither recombinant gp120 nor gp160 were able to stimulate the synthesis of CAT in the responder cells. These results indicate that the mechanism by which gp120/160 expressed on transfected cells increase CAT synthesis in responder cells may be dependent on the manner which the protein is presented in association with accessory molecules. Moreover, recombinant soluble CD4 and anti-CD4 monoclonal antibodies inhibited CEM gp120/160 induced expression of HIV-1 LTR-directed expression in CEM-1D5 cells. Based on these results we hypothesize that HIV or its envelope protein, gp120, upon interaction with its receptor, the CD4 molecule on T helper cells, transduces a signal which translates into the upregulation of the gene expression directed by the HIV-1 LTR.

Human immunodeficiency virus type 1 long terminal repeat variants from 42 patients representing all stages of infection display a wide range of sequence polymorphism and transcription activity.

Despite extensive in vitro studies identifying a myriad of cellular transcription factors that bind the human immunodeficiency virus type 1 5' long terminal repeat (LTR), the relative contribution of these factors to human immunodeficiency virus type 1 replication in infected individuals remains obscure. To address this question, we investigated 478 proviral quasispecies derived from uncultured peripheral blood mononuclear cells of 42 patients representing all stages of infection. In addition to highly conserved TATA box, SP-1, and NF-kappaB sites, the Ets core and an adjacent 5'-ACYGCTGA-3' motif were extremely conserved. Importantly, the most frequent naturally occurring length polymorphism (MFNLP) duplicated 5'-ACYGCTGA-3' motifs in LTRs in which this same motif was disrupted or in LTRs in which a single point mutation to the Ets core ablated binding of c-Ets 1 and another factor distinct from both c-Ets 1 and Elf 1. The MFNLP's location was precise (position -121) and surprisingly frequent (38% of patients) and demarcated LTR Nef-coding sequences from LTR noncoding sequences that appear to be evolving independently. Aside from these features, we found no definitive clinical or transcription phenotype common to all MFNLP LTRs. We also found previously described and novel point polymorphisms, including some conferring TAR-dependent and TAR- independent Tat unresponsiveness, and showed that differential binding of nuclear factor(s) to a TCTAA TATA box variant may be the mechanism for the latter.

Human immunodeficiency virus type 1 cellular RNA load and splicing patterns predict disease progression in a longitudinally studied cohort.

We report the results of a longitudinal study of RNA splicing patterns in 31 early-stage human immunodeficiency virus disease patients with an average follow-up time of 3 years. Eighteen patients showed no evidence for disease progression, whereas 13 patients either showed a > or = 50% reduction in baseline CD4 count or developed opportunistic infections. Levels of unspliced, tat, rev, and nef mRNAs in peripheral blood mononuclear cells were measured by a reverse transcriptase-quantitative, competitive PCR assay. Viral RNA was detected in all patients at all time points. All 13 rapid progressors had viral RNA loads that were > or = 1 log unit greater than those of the slow progressors. In addition, seven of the rapid progressors showed a reduction of more than threefold in the ratio of spliced to unspliced RNA over the 3 years of follow-up. Conversely, two slow progressors with intermediate levels of viral RNA showed no splicing shift. These results confirm earlier observations that viral RNA is uniformly expressed in early-stage patients. We further show that cellular RNA viral load is predictive of disease progression. Importantly, the shift from a predominately spliced or regulatory viral mRNA pattern to a predominately unspliced pattern both is associated with disease progression and adds predictive utility to measurement of either RNA class alone.