Antimicrobial peptide-loaded gold nanoparticle-DNA aptamer conjugates as highly effective antibacterial therapeutics against Vibrio vulnificus.

Vibrio vulnificus causes fatal infections in humans, and antibiotics are commonly used in treatment regimens against V. vulnificus infection. However, the therapeutic effects of antibiotics are limited by multidrug resistance. In this study, we demonstrated that an antimicrobial peptide (AMP), HPA3PHis, loaded onto a gold nanoparticle-DNA aptamer (AuNP-Apt) conjugate (AuNP-Apt-HPA3PHis) is an effective therapeutic tool against V. vulnificus infection in vivo in mice. HPA3PHis induced bacterial cell death through the disruption of membrane integrity of V. vulnificus. The introduction of AuNP-Apt-HPA3PHis into V. vulnificus-infected HeLa cells dramatically reduced intracellular V. vulnificus by 90%, leading to an increase in the viability of the infected cells. Moreover, when V. vulnificus-infected mice were intravenously injected with AuNP-Apt-HPA3PHis, a complete inhibition of V. vulnificus colonization was observed in the mouse organs, leading to a 100% survival rate among the treated mice, whereas all the control mice died within 40 hours of being infected. Therefore, this study demonstrated the potential of an AMP delivered by AuNP-Apt as an effective and rapid treatment option against infection caused by a major pathogen in humans and aquatic animals.

Salud mental de migrantes centroamericanos indocumentados en tránsito por la frontera sur de México / Mental health of undocumented migrants in transit at the southern border of Mexico

Objetivo. Conocer las necesidades percibidas de salud mental de migrantes centroamericanos indocumentados en tránsito por la ciudad de Tapachula, Chiapas. Material y métodos. Estudio cualitativo realizado en Casa de Migrantes de Tapachula, Chiapas. Se realizaron 20 entrevistas semiestructuradas a diez mujeres y diez hombres migrantes. Se exploró el estado de salud mental y las expectativas de atención. Se retomaron nociones teórico-metodológicas de la fenomenología sociológica. Resultados. Los migrantes presentaban signos y síntomas de daños en su salud mental relacionados con experiencias vividas en el lugar de origen y en el tránsito por México. La percepción sobre su salud mental es influida por el modelo biomédico hegemónico. Las expectativas de servicios se relacionaron con la satisfacción de necesidades básicas. Conclusiones. Es necesario fortalecer la respuesta del sistema de atención en salud mental a partir de estrategias de cooperación y emprender acciones que promuevan la superación de una construcción biomédica de salud mental que estigmatiza, medicaliza, segrega y dificulta el acceso a servicios.

Objective. To identify the perception and needs in mental health of Central American migrants in transit through Tapachula, Chiapas. Materials and methods. Qualitative study in a migrant shelter in Tapachula, Chiapas. In 20 semi-structured interviews with migrant men and women, we explored their perceptions on mental health and expectations on care. We used basic notions of phenomenology to guide the analysis. Results. Migrants had several mental health problems related to the conditions at their country of origin and due to their initial transit through Mexico.Their perception on mental health problems was heavily influenced by the biomedical health paradigm. The expectations they had on the provision of services were related to the satisfaction of basic needs. Conclusions. It is necessary to strengthen the governmental response to mental health needs through collaborative strategies. Also, actions are needed to further the understanding of mental health in order to transcend the biomedical notions that stigmatize, segregate and create a barrier to accessing services.

Functional and structural characterization of 2-amino-4-phenylthiazole inhibitors of the HIV-1 nucleocapsid protein with antiviral activity.

The nucleocapsid protein (NC) is a highly conserved protein in diverse HIV-1 subtypes that plays a central role in virus replication, mainly by interacting with conserved nucleic acid sequences. NC is considered a highly profitable drug target to inhibit multiple steps in the HIV-1 life cycle with just one compound, a unique property not shown by any of the other antiretroviral classes. However, most of NC inhibitors developed so far act through an unspecific and potentially toxic mechanism (zinc ejection) and are mainly being investigated as topical microbicides. In an effort to provide specific NC inhibitors that compete for the binding of nucleic acids to NC, here we combined molecular modeling, organic synthesis, biophysical studies, NMR spectroscopy, and antiviral assays to design, synthesize, and characterize an efficient NC inhibitor endowed with antiviral activity in vitro, a desirable property for the development of efficient antiretroviral lead compounds.

Synthesis and antiviral properties of arabino and ribonucleosides of 1,3-dideazaadenine, 4-nitro-1,3-dideazapurine and diketopiperazine.

Different arabinosides and ribosides, viz. Ara-DDA or 9(1-beta-D-arabinofuranosyl) 1,3-dideazaadenine (6), Ara-NDDP or 9(1-beta-D-arabinofuranosyl) 4-nitro-1,3-dideazapurine (7), Ara-DKP or 1(1-beta-D-arabinofuranosyl) diketopiperazine (8), Ribo-DDA or 9(1-beta-D-ribofuranosyl) 1,3-dideazaadenine (9) and Ribo-NDDP or 9(1-beta-D-ribofuranosyl) 4-nitro-1,3-dideazapurine (10) have been synthesized as probable antiviral agents. The arabinosides have been synthesized using the catalyst TDA-1 that causes stereospecific formation of beta-nucleosides while a one-pot synthesis procedure was adopted for the synthesis of the ribonucleosides where beta-anomers were obtained in higher yields. All the five nucleoside analogs have been screened for antiviral property against HIV-1 (IIIB), HSV-1 and 2, parainfluenza-3, reovirus-1 and many others. It was observed that arabinosides had greater inhibitory action than ribosides. The compound 7 or Ara-NDDP has shown maximum inhibition of HIV-1 replication than the rest of the molecules with an IC50 of 79.4 microg/mL.

A telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer.

The catalytic component of human telomerase reverse transcriptase (hTERT) is not expressed in most primary somatic human cells, whereas the majority of cancer cells reactivate telomerase by transcriptional up-regulation of hTERT. Several studies demonstrated that the hTERT promoter can be used to restrict gene expression of E1-deleted replication defective adenoviral vectors to telomerase-positive cancer cells. In this study, a conditionally replicating adenovirus (hTERT-Ad) expressing E1A genes under control of a 255-bp hTERT-promoter was constructed. Additionally, an internal ribosomal entry site-enhanced green fluorescent protein cassette was inserted downstream of the E1B locus to monitor viral replication in vivo. Adenoviral replication of hTERT-Ad and enhancement of enhanced green fluorescent protein expression could be observed in all investigated telomerase-positive tumor cell lines. In contrast, hTERT-Ad infection of telomerase-negative primary human hepatocytes did not result in significant replication. The capability of hTERT-Ad to induce cytopathic effects in tumor cells was comparable with that of adenovirus wild type and significantly higher compared with ONYX-015, regardless of the p53 status of the tumor cells. Single application of low-dose hTERT-Ad to tumor xenografts led to significant inhibition of tumor growth, confirming the potential therapeutic value of conditionally replicative adenoviral vectors. These in vivo experiments also revealed that hTERT-Ad-mediated oncolysis was more efficient than ONYX-015 treatment. These results demonstrate that expression of E1A under transcriptional control of the hTERT promoter is sufficient for effective telomerase-dependent adenovirus replication as a promising perspective for the treatment of the majority of epithelial tumors.

Mood-enhancing antidepressant St. John's wort inhibits the activation of human immunodeficiency virus gene expression by ultraviolet light.

Ultraviolet (UV) radiation is a potent activator of the human immunodeficiency virus (HIV) gene expression in a HeLa cell clone with stably integrated copies of the HIVcat reporter construct. Recently, we have shown that activation of p38 MAP kinase and NF-kappaB is necessary but not sufficient for triggering efficient HIV gene expression in response to UV. Here we demonstrate that St. John's wort is a potent inhibitor of the UV-induced activation of HIV gene expression in HeLa cells. Stably transfected HIVcat/HeLa cells were preincubated with different amounts (25-100 microl) of St. John's wort or gingko biloba extracts for 30 min, then irradiated with UV (30 J/m2). In contrast to ginkgo biloba, St. John's wort inhibited the UV-induced HIV gene expression in a dose-dependent manner. Furthermore, preincubation with St. John's wort (10, 20, and 30 microl) for 30 min before UV (30 J/m2) irradiation, PMA- and UV-induced NF-kappaB activation was completely blocked, whereas ginkgo biloba did not affect the PMA- and UV-induced NF-kappaB activation in HeLa cells. UV activation of p38 MAP kinase was not inhibited by St. John's wort or by ginkgo biloba. However, we found that p38 MAP kinase and JNK1 and -2 were activated by St. John's wort, but p44/42 MAP kinase was not activated by St. John's wort in HeLa cells. Hypericin an active ingredient in St. John's wort also inhibited the UV activation of HIV gene expression in HeLa cells. These results firmly confirm that St. John's wort is a potent inhibitor of the UV-induced activation of HIV gene expression in HeLa cells.

Inhibition of the replication of a hepatitis C virus-like RNA template by interferon and 3'-deoxycytidine.

The development of low molecular weight inhibitors of hepatitis C virus (HCV) replication has been hindered by the lack of a good cell-based system that models the entire HCV replication cycle. To date the only two therapies approved for the treatment of HCV infection are interferon (IFN)-alpha and the nucleoside analogue, ribavirin. We have created a cell-based system that allows for the accurate quantification of the replication of an HCV-like RNA template by proteins that are encoded for by the HCV genome. The system consists of a cell line that constitutively produces luciferase in response to the production of functional HCV replicative proteins. The 293B4alpha cell line has been formatted into a semi-high throughput, cell-based screen for inhibitors of HCV replication. When these cells were treated with either IFN-alpha or -beta, luciferase production decreased in a dose-responsive manner. Counterscreening these molecules in another cell line, 293SVLuc, in which luciferase production in not dependent the presence of functional HCV proteins, showed that the inhibition of luciferase in the 293B4alpha cell line was due to inhibition of the replication of the HCV-like RNA template and not anti-cellular or -luciferase activity. Moreover, when the 293B4alpha cell line was treated with the ribonucleoside analogue, 3'-deoxycytidine, luciferase decreased in a dose-responsive manner. 3'-deoxyguanosine and 3'-deoxyuridine did not inhibit luciferase production and 3'-deoxyadenosine was too cytotoxic to determine if it had any anti-HCV activity.

Antiviral effect of hyperthermic treatment in rhinovirus infection.

Human rhinoviruses (HRV) are recognized as the major etiologic agents for the common cold. Starting from the observation that local hyperthermic treatment is beneficial in patients with natural and experimental common colds, we have studied the effect of brief hyperthermic treatment (HT) on HRV replication in HeLa cells. We report that a 20-min HT at 45 degrees C is effective in suppressing HRV multiplication by more than 90% when applied at specific stages of the virus replication cycle. Synthesis of virus proteins is not affected by HT, indicating that the target for treatment is a posttranslational event. The antiviral effect is a transient cell-mediated event and is associated with the synthesis of the 70-kDa heat shock protein hsp70. Unlike poliovirus, rhinovirus infection does not inhibit the expression of hsp70 induced by heat. The possibility that hsp70 could play a role in the control of rhinovirus replication is suggested by the fact that a different class of HSP inducers, the cyclopentenone prostaglandins PGA1 and delta 12-PGJ2, were also effective in inhibiting HRV replication in HeLa cells. Inhibition of hsp70 expression by actinomycin D prevented the antiviral activity of prostaglandins in HRV-infected cells. These results indicate that the beneficial effect of respiratory hyperthermia may be mediated by the induction of a cytoprotective heat shock response in rhinovirus-infected cells.

In situ localization of PCR-amplified DNA and cDNA.

Combining the high sensitivity of PCR with the cell localizing ability of in situ hybridization allows for the reproducible detection of low copy targets in intact cells. This article describes several key variables that include fixation, protease digestion, the hot start maneuver, stringency, and, for RNA analysis, DNase digestion that are important to successful in situ PCR. Also stressed is the importance of performing and interpreting controls with each experiment. Important controls include omission of key components, use of samples known either to contain or lack the target of interest and, most importantly, the in-built controls invariably present in the heterogeneous component of any given tissue type.

[Antivirus effect of polysaccharides of brewer yeast in vitro].

The antivirus effect of polysaccharides of brewer yeast from yeast mud on 13 kinds of viruses including DNA and RNA virus along with their mechanisms were studied. The result showed that this effect was remarkable on the infections with poliovirus III, adenovirus III, ECHO6 virus, enterovirus 71, vesicular stomatitis virus, herpesvirus I, II, coxsackie A16 virus and coxsackie B3 virus. The polysaccharides of brewer yeast could also inhibit the development of cytopathic effect(CPE) and protect cultural cells from being infected with the above viruses.

[Antiviral activity of hydroalcoholic extract from Haemanthus albiflos on the Moloney murine leukemia virus and the human immunodeficiency virus]. / Activité antivirale d'un extrait hydro-alcoolique d'Haemanthus albiflos sur le virus de la leucémie murine de Moloney et le virus du syndrome de l'immunodéficience acquise.

We have used a biological test on the microplates of cellular cultures in order to investigate the toxicity and the antiviral properties against different viruses: defective Moloney Murine Leukemia virus (MoMLV) derived from the SVX shuttle and expressing resistance to the G418 antimitotic, and Human Immunodeficiency Virus (HIV) of a hydroalcoholic extract from Haemanthus albiflos (Amaryllidacae). The toxicity was assessed through coloric test evaluation of fixed cells stained with crystal violet. In a population of NIH 3T3 cells (Fibroblasts mouse), the toxicity found with 2, 7, 14 and 28 microliters/ml of lyophilisat extract corresponding at: 0.23, 0.81, 1.62 and 3.24 mg of plant dry, was 32, 50, 63 and 70% respectively. With regards to the antiviral properties, the plant extracts showed an inhibition of 88% on the formation of G418 resistant 3T3 clones. The assay on HIV infected lymphotic cells (P4) showed an IC50 of 4 microliters/ml for this extract plant. Therefore, the toxic effect was similar to the antiviral response.

Rhinovirus inhibits antigen-specific T cell proliferation through an intercellular adhesion molecule-1-dependent mechanism.

To determine whether binding of human rhinovirus (HRV) to intracellular adhesion molecule-1 might disrupt airway immune processes, effects of a major HRV group, HRV-16, on T cell proliferation and cytotoxicity were defined. HRV (1-10 TCID50/cell) significantly inhibited T cell proliferation induced by antigen but not proliferation secondary to mitogens, interleukin-2, or an irradiated allogeneic T cell line. Noninfectious (UV-irradiated) HRV had similar effects. Inhibition of T cell proliferation was dependent on HRV binding to intercellular adhesion molecule-1 on monocytes, indicating that the virus interferes with lymphocyte activation indirectly through effects on antigen-presenting cells. In addition, HRV inhibited T cell cytotoxic responses but not NK cell activity. If these effects also occur in vivo, the resulting disturbance in local airway immunity could increase the chances of successful viral replication, and might also be a factor in the pathogenesis of secondary viral or bacterial respiratory tract infections.

Characterization of antiviral activity of a sesquiterpene, triptofordin C-2.

The activities of 13 sesquiterpenes isolated from Tripterygium wilfordii Hook fil. var. regelii Makino were studied against herpes simplex virus type 1 (HSV-1) in vitro. Among these compounds, only triptofordin C-2 showed a selectivity index of more than 10. The compound, which could also inhibit the replication of human cytomegalovirus (HCMV), did not affect either adsorption or penetration of HSV-1 to host cells, but showed moderate virucidal activity against several enveloped viruses including HSV-1, HCMV, measles virus and influenza A virus. Triptofordin C-2 suppressed viral protein synthesis of infected cells when added at early steps of HSV-1 replication and exerted inhibition of translation of the transcripts of the immediate early genes. When acyclovir and triptofordin C-2 were evaluated in combination for antiviral activity against HSV-1 replication, additive antiviral effects were observed for this virus.

Ferrochelatase cDNA delivered by adenoviral vector corrects biochemical defect in protoporphyric cells.

Protoporphyria is generally an autosomal dominant disease characterized genetically by mutations in the ferrochelatase gene. The interaction between the wild-type and mutant ferrochelatase protein is unknown. The aim of this study was to evaluate the ability to correct the enzymatic and biochemical defects in cells from patients with protoporphyria, using a replication-defective human adenovirus for gene transfer. Overexpression of ferrochelatase was accomplished by construction of a vector in which expression of the wild-type ferrochelatase cDNA was driven by the constitutive cytomegalovirus (CMV) promoter, introduction and packaging of the cDNA into human adenovirus dl309, and transduction of normal and protoporphyric fibroblasts. Fibroblasts from controls and patients were infected with the ferrochelatase adenovirus or a control adenovirus and assayed for ferrochelatase activity and the accumulation of protoporphyrin upon challenge with the precursor delta-aminolevulinic acid (ALA). At a multiplicity of infection (moi) of 10, greater than 85% of both the wild-type and protoporphyric fibroblasts were infected. The recombinant adenovirus increased the ferrochelatase protein content and activity in the wild-type and protoporphyric fibroblasts with equal efficiency. Therefore, the presence of the mutant ferrochelatase protein did not inhibit the ferrochelatase activity expressed by the transgene.

[Antiviral activity of tannin from the pericarp of Punica granatum L. against genital Herpes virus in vitro].

Based on cell culture techniques this study has demonstrated that tannin from the pericarp of Punica granatum is an effective component against genital herpes virus (HSV-2). The tannin not only inhibits HSV-2 replication, but also shows stronger effects of killing virus and blocking its absorption to cells.

Suppression of tumorigenesis by transcription units expressing the antisense E6 and E7 messenger RNA (mRNA) for the transforming proteins of the human papilloma virus and the sense mRNA for the retinoblastoma gene in cervical carcinoma cells.

Human cervical carcinoma cell lines that harbor human papilloma virus (HPV) have been reported to express HPV E6 and E7 proteins at least in the beginning stages if not at all stages of the disease. The HPV E6 and E7 proteins bind to and inactivate the products of the p53 and retinoblastoma (Rb) tumor suppressor genes, which thereby allow the cervical carcinoma cells to circumvent the action of these tumor suppressor genes. We observed that the introduction of the antisense HPV 18 E6 and E7 sequences, as well as a sense cDNA for the human wild-type Rb gene into a human cervical carcinoma cell line (HeLa), which is positive for the HPV 18 provirus, decreased the in vitro and in vivo growth rate of the transfected cells if both antisense transcripts for the HPV 18 E6 and E7 and sense transcripts for human Rb were expressed. In addition, overexpression of a complementary DNA (cDNA) for the Rb messenger RNA was sufficient to slow the proliferation of HeLa cells, and the level of Rb cDNA expression was correlated with the degree to which the rate of growth of the tumor was slowed. The results of our experiments show that the presence of HPV E6 and E7 proteins and the resultant inactivation of Rb in cervical carcinoma cells contributes to the neoplastic phenotype even in highly evolved cervical carcinoma cell lines such as HeLa, which have been derived from a cervical carcinoma patient at an advanced stage of the disease process. These data suggest that the HPV proteins play a role not only at the beginning of cervical cancer, but also at advanced stages of this disease. These experiments may lead to genetic approaches to the control of this disease that involve antisense sequences that downregulate the E6 and E7 genes or lead to expression of the Rb gene.