Developments in Cell-Penetrating Peptides as Antiviral Agents and as Vehicles for Delivery of Peptide Nucleic Acid Targeting Hepadnaviral Replication Pathway.

Alternative therapeutic approaches against chronic hepatitis B virus (HBV) infection need to be urgently developed because current therapies are only virostatic. In this context, cell penetration peptides (CPPs) and their Peptide Nucleic Acids (PNAs) cargoes appear as a promising novel class of biologically active compounds. In this review we summarize different in vitro and in vivo studies, exploring the potential of CPPs as vehicles for intracellular delivery of PNAs targeting hepadnaviral replication. Thus, studies conducted in the duck HBV (DHBV) infection model showed that conjugation of (D-Arg)8 CPP to PNA targeting viral epsilon (ε) were able to efficiently inhibit viral replication in vivo following intravenous administration to ducklings. Unexpectedly, some CPPs, (D-Arg)8 and Decanoyl-(D-Arg)8, alone displayed potent antiviral effect, altering late stages of DHBV and HBV morphogenesis. Such antiviral effects of CPPs may affect the sequence-specificity of CPP-PNA conjugates. By contrast, PNA conjugated to (D-Lys)4 inhibited hepadnaviral replication without compromising sequence specificity. Interestingly, Lactose-modified CPP mediated the delivery of anti-HBV PNA to human hepatoma cells HepaRG, thus improving its antiviral activity. In light of these promising data, we believe that future studies will open new perspectives for translation of CPPs and CPP-PNA based technology to therapy of chronic hepatitis B.

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys)4 (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.

Non-malarial infectious diseases of antenatal care in pregnant women in Franceville, Gabon.

BACKGROUND: In sub-tropical countries, infectious diseases remain one of the main causes of mortality. Because of their lack of active immunity, pregnant women and their unborn children represent the most susceptible people. In Gabon, data on infectious diseases of pregnant women such as syphilis and rubella are either scarce or very old. Few studies have assessed T. gondii infection during pregnancy in the country. Here, we evaluate seroprevalence of HIV, HTVL-1, syphilis and T. gondii and rubella infection during antenatal care among women living in Franceville, Gabon. METHODS: A retrospective study was conducted on data collected from May 2007 to July 2010. After signing an informed written consent form, all pregnant women consulting in two hospitals of Franceville (Gabon) and in offices of maternity and childbirth health centers were included. Demographic and clinical data were collected. Serum samples were collected and analysed using immunological assays relevant for HIV (Genscreen HIV-1 version 2, Bio-Rad®, Marne la Roquette, France).HTLV-1 (Vironostika HTLV-1, Biomérieux®, Marcy l'Etoile, France), T. pallidum (TPHA/VDRL), BIOLABO®SA), rubella virus (Vidas Biomerieux®, Marcy l'Etoile, France) and T. gondii (Vidas Biomerieux®, Marcy l'Etoile, France) diagnoses were performed. Data analysis was done using the Stat view 5.0 software. RESULTS: A total of 973 pregnant women were assessed. The mean age was 25.84 ± 6.9 years, with a minimum age of 14.0 years and a maximum of 45.0 years. Women from 26 to 45 years old and unemployed women were the most prevalent: 41.93% and 77.18%, respectively. The prevalence of studied infectious diseases were 2.50% for syphilis, 2.88% for HTLV-1, 4.00% for HIV with no significant difference between them (p = 0.1). Seropositivity against rubella was higher (87.56%, n = 852) than seropositivity against T. gondii (57.35%, n = 557), (p < 0.0001). Only 5 (0.51%) co-infection cases were found: 2 co-infected with HIVand T. pallidum, 2 co-infected with HIV and HTLV-1, and one co-infected with T. pallidum and HTLV-1. Sixty-two pregnant women were seronegative against toxoplasmosis and rubella (6.37%). CONCLUSION: High levels of seropositivity against T. gondii and the rubella virus were observed. The prevalence of T. pallidum and HTLV-1 were lowest but HIV prevalence in young women was worrying.

Prevalence of malaria, prevention measures, and main clinical features in febrile children admitted to the Franceville Regional Hospital, Gabon.

Recently, major progress has been made in controlling malaria in Africa. However, in Gabon, little information is available on the role of malaria in childhood febrile syndromes, the use and efficacy of preventive measures, and Plasmodium species distribution. Here, we characterized malaria in febrile children in Franceville, Gabon through a cross-sectional study at the pediatric unit of the Franceville Regional Hospital. We registered 940 febrile children. Their general condition was markedly altered in 11.7% of cases (n = 89/760); among them 19 (21.4%) had a severely altered condition. Malaria was the second most frequent etiology (22.0%; n = 162/738), after respiratory tract infections (37.3%; n = 275/738). Children with malaria (63 ± 39 months) were older than children without malaria (40 ± 37 months) (p = 0.0013). Hemoglobin, red blood cell, white blood cell, and platelet values were lower in children with malaria than in those without malaria (p < 0.0001). Anemia was the most common feature of severe malaria (70.6%; n = 12/17), followed by neurological involvement (23.5%; n = 4/17). The prevalence of malaria was significantly higher in children older than 60 months than in younger children (40% vs. 15.5%; p < 0.0001). Plasmodium falciparum accounted for 97.5% of cases (158/162), followed by Plasmodium malariae (2.5%; n = 4/162). Bed net use was high (74.4%; n = 697/936) and contributed to malaria prevention (p = 0.001). Good basic knowledge of malaria also had a preventive effect (p < 0.0001). The prevalence of malaria in children in Franceville did not decrease significantly from 2009 to 2012, remaining at about 20%, highlighting that preventive measures should be reinforced.

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.

Molecular Detection of Fastidious and Common Bacteria as Well as Plasmodium spp. in Febrile and Afebrile Children in Franceville, Gabon.

Malaria was considered as the main cause of fever in Africa. However, with the roll back malaria initiative, the causes of fever in Africa may change. This study aimed to evaluate the prevalence of bacteria and Plasmodium spp. in febrile and afebrile (controls) children from Franceville, Gabon. About 793 blood samples from febrile children and 100 from controls were analyzed using polymerase chain reaction (PCR) coupled with sequencing. Plasmodium spp. was the microorganism most detected in febrile (74.5%, 591/793) and controls (13%, 13/100), P < 0.0001. Its coinfection with bacteria was found only in febrile children (P = 0.0001). Staphylococcus aureus was the most prevalent bacterium in febrile children (2.8%, 22/793) and controls (3%, 3/100). Eight cases of Salmonella spp. (including two Salmonella enterica serovar Paratyphi) and two of Streptococcus pneumoniae were found only among febrile children. Borrelia spp. was found in 2 controls while Rickettsia felis was detected in 10 children (in 8 febriles and 2 afebriles). No DNA of other targeted microorganisms was detected. Plasmodium spp. remains prevalent while Salmonella spp., Staphylococcus aureus, and Streptococcus pneumoniae were common bacteria in Gabon. Two fastidious bacteria, Rickettsia felis and Borrelia spp., were found. Inclusion of controls should improve the understanding of the causes of fever in sub-Saharan Africa.

Clonidine-induced growth hormone and growth-hormone-releasing hormone release is mediated by tachykinin NK2 receptors in sheep.

Clonidine is an α(2)-adrenergic agonist, historically known to treat high blood pressure. Further studies showed that it could be used in the treatment of neuropsychiatric disorders. Afterwards, it has been reported that clonidine stimulated growth hormone (GH) release in many species including man. Using a transnasal surgery technique in awake sheep that allowed accessing hypothalamopituitary portal vessels, our laboratory previously reported that the injection of clonidine in sheep induced a significant, immediate and short-lasting increase in peripheral GH and portal GH-releasing hormone (GHRH) levels. In this study, we show that the clonidine-induced peripheral GH and portal GHRH increase in sheep appears to be mediated by the tachykinin NK2 receptor.

Substance P stimulates Growth Hormone (GH) and GH-Releasing Hormone (GHRH) secretions through tachykinin NK2 receptors in sheep.

Substance P is ubiquitous undecapeptide belonging to the tachykinins family. It has been found in the hypothalamus and is involved in the hypothalamo-hypophysial axis in several mammals, including human. Previous studies have shown that substance P increases GH secretions in rats and human. In this study, we have shown that intravenously infused substance P in sheep caused an increased level of Growth Hormone (GH) and GH-Releasing Hormone (GHRH), and decreased Somatotropin Release Inhibiting Hormone (SRIH) secretions. GH was obtained from peripheral blood. GHRH and SRIH were directly collected from hypophysial portal blood, using a trans-nasal surgery technique in a vigil sheep that allowed accessing to hypothalamo-hypophysial portal vessels. Hormones assays were performed by radioimmunoassay (RIA). Moreover, we showed that substance P-induced GH and GHRH secretion appears to be mediated by NK2 tachykinin receptors, since it is specifically blocked by a non peptidic tachykinin NK2 receptor antagonist (SR48968, Sanofi, Montpellier, France) whereas a non peptidic tachykinin NK1 antagonist (SR140333, Sanofi, Montpellier, France) failed to modify GH and GHRH hormones secretions.

[Vitamin A in Gabonese children not receiving supplements: relation to ocular and nutritional diseases]. / Vitamine A chez les enfants gabonais non supplémentés : relation avec les pathologies oculaires et nutritionnelles.

To define vitamin A status in Gabonese children before the supplementation campaigns by UNICEF and the Gabonese Ministry of Health. We conducted an epidemiological, clinical and laboratory study at two of the main child health centers of Libreville and included 150 children aged from 0 to 156 months. We assessed their nutritional and ophthalmological status, their consumption of food rich in vitamin A and their blood levels of vitamin A (retinol). The latter was below the normal level (0.76 mmol/l) for 70% of the children. We observed the following eye diseases correlated with retinol levels: nyctalopia (16%), conjunctiva xerosis (6.7%), Bitot's spots (1.3%), corneal xerosis (3.3%), leukoma (0.7%) and blindness (1.3%). Moreover, 48.7% of the children had delayed growth, positively correlated with low retinol concentrations (0.643 mmol/l), 10% kwashiorkor and 19.9% marasmus. Among the mothers interviewed, 56% were unemployed and 69.3% knew little about vitamin A-rich food, which indeed is a rare part of the population's diet. This prospective study showed that the vitamin A deficiency is a major public health problem in Libreville. Supplementation campaigns are certainly necessary to fight it, but also and especially improved nutrition, including regular consumption of the vitamin A-rich food available in Gabonese market places.

Is the mannose-6-phosphate/insulin-like growth factor 2 receptor coded by a breast cancer suppressor gene?

The multifunctional growth factor mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2-R) binds proteins sharing M6P signals, including cathepsins and IGF2. It is involved in targeting newly synthesized mannose-6-phosphorylated lysosomal enzymes, activating transforming growth factor beta (TGFbeta), and neutralising the mitogen IGF2 by transporting it to lysosomes. The M6P/IGF2-R was proposed as being coded by a tumor suppressor gene. We measured gene expression at the protein level by quantitative immunohistochemistry, using chicken high affinity IgY antibodies directed against human M6P/IGF2-R. Chicken immunization was performed with human purified M6P/IGF2-R, and IgY antibodies were extracted from egg yolk by polyethylene glycol precipitation method. The biosensor analysis showed that IgY antibodies bind M6P/IGF2-R with high affinity (Kd = 7.5 nM). Quantitative immunohistochemical studies in sections from invasive breast carcinoma and ductal carcinoma in situ (DCIS) indicated various levels (from 5 to 400 units) of the M6P/IGF2-R protein, which did not correlate with tumor size, histological grade, estrogen and progesterone receptors. Moreover, the M6P/IGF2-R level was increased in DCIS relative to adjacent normal tissue (p < 0.005) and then decreased in invasive carcinoma compared with DCIS (p < 0.02). The hypothesis of tumor suppressor gene is not supported by these studies. However, it is not excluded for a small proportion of the tumors. Its assay might help to complement the cathepsin D assay to predict breast cancer prognosis and physiopathology.