Cranberry dosed extract: an effective therapy for recurrent Escherichia coli cystitis in elderly patients. The GerHogar Cysticlean® study / Extracto dosificado de arándano rojo: una terapia eficaz para la cistitis recurrente por Escherichia coli en pacientes de la tercera edad. El estudio GerHogar Cysticlean®

Abstract Introduction: Cystitis is the most prevalent urinary tract infection (UTI), and antibiotics are its conventional therapy. However, the prevalence rate of antibiotic resistance to uropathogens is significantly increased. Cranberry treatment has been associated with the inhibition of Escherichia coli (Ec) adherence to uroepithelial cells due to the anti-adhesive property related to its proanthocyanidins content, and cysticlean® (CYS) is a cranberry extract which contains 240 mg PACs per capsule. Since elderly people is one of the populations mostly exposed to cystitis and bacteria antibiotic resistance, it was decided to originally study the efficacy and safety of CYS, to treat cystitis instead of antibiotic, in elderly individuals. Material & Methods: Two groups were studied: Group 1 (G1): first cystitis episode was recorded within the last 3 months before the study initiation. Group 2 (G2): frequent cystitis recurrent episodes (1-2/month or more) within the last 3 months before the study initiation. G1 patients were treated with 1 capsule of CYS every 12 h for 1 month, while G2 patients were treated up to 12 months. Comparative evaluation was performed using Student test. Results: 160 elderly ambulatory and nursing home patients suffering from recurrent cystitis were treated with CYS. G1 and G2 had 38 and 122 subjects, respectively. Cranberry-based cystitis treatment was successful in 81.57 % and 81.96 % in G1 and G2 patients, respectively. Conclusion: CYS showed to be an effective alternative therapy to antibiotics to treat cystitis recurrences caused by Ec. Neither side effects nor adverse reactions have been reported.

Resumen Introducción: la cistitis es la infección del tracto urinario más común a nivel mundial y los antibióticos son su terapia convencional; sin embargo, la tasa de prevalencia de la resistencia de los uropatógenos a los antibióticos ha aumentado significativamente en los últimos tiempos. El tratamiento con arándano rojo se ha asociado con la inhibición de la adherencia de la Escherichia coli a las células uroepiteliales debido a la propiedad antiadherente relacionada con su contenido de proantocianidinas. La cysticlean® (CYS) es un extracto de arándano rojo que contiene 240 mg de PAC por cápsula. Objetivo: estudiar la eficacia y seguridad de la CYS en el tratamiento de la cistitis como reemplazo de los antibióticos en personas adultas mayores. Material y métodos: se estudiaron dos grupos, uno (G1) en el que el primer episodio de cistitis se registró dentro de los últimos 3 meses antes del inicio del estudio y otro (G2) en el que se registraron episodios recurrentes de cistitis frecuentes (≥1-2 al mes) en los últimos 3 meses antes del inicio del estudio. Los pacientes del G1 fueron tratados con 1 cápsula de CYS cada 12 horas durante 1 mes, mientras que los del G2 fueron tratados por 12 meses con el mismo esquema. La evaluación comparativa se realizó mediante la prueba de Student. Resultados: en el estudio participaron 160 pacientes ambulatorios de la tercera edad residentes de hogares de ancianos y con diagnóstico de cistitis recurrente. De estos, 38 se incluyeron en G1 y 122, en G2. El tratamiento de la cistitis a base de arándano rojo tuvo éxito en el 81,57 % y el 81,96 % de los pacientes de G1 y G2, respectivamente. Conclusión: la CYS demostró ser una terapia alternativa eficaz a los antibióticos para tratar las recurrencias de cistitis causadas por E. coli al no presentarse efectos secundarios ni reacciones adversas.

ISG15 Is Upregulated in Respiratory Syncytial Virus Infection and Reduces Virus Growth through Protein ISGylation.

UNLABELLED: Human respiratory syncytial virus (RSV), for which neither a vaccine nor an effective therapeutic treatment is currently available, is the leading cause of severe lower respiratory tract infections in children. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is highly increased during viral infections and has been reported to have an antiviral or a proviral activity, depending on the virus. Previous studies from our laboratory demonstrated strong ISG15 upregulation during RSV infection in vitro. In this study, an in-depth analysis of the role of ISG15 in RSV infection is presented. ISG15 overexpression and small interfering RNA (siRNA)-silencing experiments, along with ISG15 knockout (ISG15(-/-)) cells, revealed an anti-RSV effect of the molecule. Conjugation inhibition assays demonstrated that ISG15 exerts its antiviral activity via protein ISGylation. This antiviral activity requires high levels of ISG15 to be present in the cells before RSV infection. Finally, ISG15 is also upregulated in human respiratory pseudostratified epithelia and in nasopharyngeal washes from infants infected with RSV, pointing to a possible antiviral role of the molecule in vivo. These results advance our understanding of the innate immune response elicited by RSV and open new possibilities to control infections by the virus. IMPORTANCE: At present, no vaccine or effective treatment for human respiratory syncytial virus (RSV) is available. This study shows that interferon-stimulated gene 15 (ISG15) lowers RSV growth through protein ISGylation. In addition, ISG15 accumulation highly correlates with the RSV load in nasopharyngeal washes from children, indicating that ISG15 may also have an antiviral role in vivo. These results improve our understanding of the innate immune response to RSV and identify ISG15 as a potential target for virus control.

Negatively charged glyconanoparticles modulate and stabilize the secondary structures of a gp120 V3 loop peptide: toward fully synthetic HIV vaccine candidates.

The third variable region (V3 peptide) of the HIV-1 gp120 is a major immunogenic domain of HIV-1. Controlling the formation of the immunologically active conformation is a crucial step to the rational design of fully synthetic candidate vaccines. Herein, we present the modulation and stabilization of either the α-helix or ß-strand conformation of the V3 peptide by conjugation to negatively charged gold glyconanoparticles (GNPs). The formation of the secondary structure can be triggered by the variation of the buffer concentration and/or pH as indicated by circular dichoism. The peptide on the GNPs shows increased stability toward peptidase degradation as compared to the free peptide. Moreover, only the V3ß-GNPs bind to the anti-V3 human broadly neutralizing mAb 447-52D as demonstrated by surface plasmon resonance (SPR). The strong binding of V3ß-GNPs to the 447-52D mAb was the starting point to address its study as immunogen. V3ß-GNPs elicit antibodies in rabbits that recognize a recombinant gp120 and the serum displayed low but consistent neutralizing activity. These results open up the way for the design of new fully synthetic HIV vaccine candidates.

Safety and immunogenicity of a modified pox vector-based HIV/AIDS vaccine candidate expressing Env, Gag, Pol and Nef proteins of HIV-1 subtype B (MVA-B) in healthy HIV-1-uninfected volunteers: A phase I clinical trial (RISVAC02).

BACKGROUND: To investigate the safety and immunogenicity of a modified vaccinia virus Ankara vector expressing HIV-1 antigens from clade B (MVA-B), a phase-I, doubled-blind placebo-controlled trial was performed. METHODS: 30 HIV-uninfected volunteers at low risk of HIV-1 infection were randomly allocated to receive 3 intramuscular injections (1×10(8)pfu/dose) of MVA-B (n=24) or placebo (n=6) at weeks 0, 4 and 16. All volunteers were followed 48 weeks. Primary end-points were adverse events and immunogenicity. RESULTS: A total of 169 adverse events were reported, 164 of grade 1-2, and 5 of grade 3 (none related to vaccination). Overall 75% of the volunteers showed positive ELISPOT responses at any time point. The magnitude (median) of the total responses induced was 288SFC/10(6)PBMC at week 18. Antibody responses against Env were observed in 95% and 72% of vaccinees at week 18 and 48, respectively. HIV-1 neutralizing antibodies were detected in 33% of volunteers. CONCLUSIONS: MVA-B was safe, well tolerated and elicited strong and durable T-cell and antibody responses in 75% and 95% of volunteers, respectively. These data support further exploration of MVA-B as an HIV-1 vaccine candidate. Clinical Trials.gov identifier: NCT00679497.

Broadly neutralizing antibodies and their significance for HIV-1 vaccines.

Despite extensive efforts, a preventive HIV vaccine has not yet been obtained and remains the main challenge in the field of AIDS research. Empirical approaches which have proved successful for many vaccines are not sufficient to tackle HIV-1 and new strategies to design effective preventive AIDS vaccines are critical. To this aim, further understanding of the mechanisms of action of neutralizing antibodies is required. In this review we summarize our current knowledge on the structure of the gp160 viral envelope and the dynamics of viral entry, the evolution of humoral response in HIV-infected patients and the mechanisms of viral escape. Finally, we describe the few neutralizing antibodies with activity against a broad spectrum of circulating HIV strains and their relevance in the design of new candidates to HIV-1 vaccines.

A sensitive phenotypic assay for the determination of human immunodeficiency virus type 1 tropism.

OBJECTIVES: To develop a sensitive phenotypic assay based on recombinant viruses (RVs) for characterizing HIV-1 tropism. METHODS: Viral tropism was assessed in 159 plasma samples. The env gene was amplified and ligated into pNL-lacZ/env-Ren, which carries a luciferase reporter gene. Resulting constructs were transfected into HEK293T cells to generate RVs. To assess co-receptor tropism, U87.CD4.CXCR4/CCR5 cells were infected and luciferase activity was measured. RESULTS: RVs containing env from different HIV-1 subtypes were replication competent. Minor variants were detectable in 1% of the viral population. Tropism was determined in 65% of samples with a viral load of <1000 copies/mL. The phenotypic assay described here was validated with the Trofile™ and Trofile™ES assays. Considering the Trofile™ assay as a reference, the sensitivity for R5 and R5X4/X4 detection was 90% and 77%, and the specificity was 77% and 90%, respectively. Our assay was 86% concordant with Trofile™ (90% for R5 and 77% for R5X4/X4). When our system was compared with Trofile™ES, the concordance was 89% (86% for R5 and 92% for R5X4/X4), the sensitivity for R5 was 86% and for R5X4/X4 was 92%, and the specificity for R5 was 92% and for R5X4/X4 was 86%. The phenotypic results were compared with those obtained using the following V3 genotypic prediction tools: position-specific scoring matrix; geno2pheno[coreceptor]; C4.5; C4.5 using positions 8 and 12; PART; support vector machines; and the charge rule. CONCLUSIONS: We describe a system to assess co-receptor tropism based on the generation of chimeric replication-competent viruses with high sensitivity in the detection of minor populations. A good correlation of our results with Trofile™ assays was found.

SJ23B, a jatrophane diterpene activates classical PKCs and displays strong activity against HIV in vitro.

Existence of virus reservoirs makes the eradication of HIV infection extremely difficult. Current drug therapies neither eliminate these viral reservoirs nor prevent their formation. Consequently, new strategies are needed to target these reservoirs with the aim of decreasing their size. We analysed a series of jatrophane diterpenes isolated from Euphorbia hyberna and we found that one of them, SJ23B, induces the internalization of the HIV-1 receptors CD4, CXCR4 and CCR5 and prevents R5 and X4 viral infection in human primary T cells at the nanomolar range. Moreover, SJ23B is a potent antagonist of HIV-1 latency. Using Jurkat-LAT-GFP cells, a model for HIV-1 latency, we found that prostratin and SJ23B activate HIV-1 gene expression, with SJ23B being at least 10-fold more potent than prostratin. SJ23B did not elicit transforming foci activity in NIH 3T3 cells but is a potent activator of PKCalpha and delta as measured by in vitro kinase assays and by cellular translocation experiments. By using isoform-specific PKC inhibitors we found that cPKCs are critical for SJ23B-induced HIV-1 reactivation. We also showed that both SJ23B-induced IkappaBalpha degradation and NF-kappaB activation were inhibited by the classical PKC inhibitor, Gö6976. Accordingly, SJ23B synergizes with ionomycin to translocate PKCalpha to the plasma membrane and to activate the NF-kappaB pathway. Moreover, SJ23B activates both NF-kappaB and Sp1-dependent transcriptional activities in primary T cells. We have shown that diterpene jatrophanes represent a new member of anti-AIDS agents that could be developed for mitigating HIV reactivation.

Guatemalan plants extracts as virucides against HIV-1 infection.

Prevention methods to avoid transmission of pathogens, including HIV, are crucial in the control of infectious diseases, not only to block epidemic spread but to avoid long-term treatments leading to emergence of resistances and drug associated side effects. Together with vaccine development, the discovery of new virucidal agents represents a research priority in this setting. In the screening of new compounds with antiviral activity, three Guatemalan plant extracts from Justicia reptans, Neurolaena lobata and Pouteria viridis were evaluated with a classic antiviral assay and were found to inhibit HIV replication. This activity was corroborated by an original recombinant virus assay, leading us to perform a deeper study of the virucidal activity. Active fractions were non-toxic in vitro and also inhibited other enveloped viruses. Moreover, these fractions were able to inhibit the transfer of HIV from dendritic cells (DCs) to lymphocytes, that represents the main way of HIV spread in vivo.

Contracaecum sp. infection in Hoplias malabaricus (moncholo) from rivers and marshes of Colombia.

Nematode infection indices were recorded in Hoplias malabaricus captured in six different rivers and a marsh belonging to the North Coast Basin of Colombia, and from the Amazon River, during February 2003-December 2004. Preliminary morphological analysis of nematodes indicated the presence of Contracaecum sp. Parasites were mostly found in the intestinal mesenteries and a very low percentage in muscle. Parasite prevalence in all sampling locations at the north of Colombia was 100%, whereas in the Amazon River it was 6.12%. The mean intensity in the different stations were as follows: Magdalena River at the City of Magangué (58.92+/-7.59), Magdalena river at the city of Zambrano (128.9+/-7.08), Sinú River (53.88+/-4.92), Dique Channel (207.3+/-59.52), Cauca River (77.26+/-9.35), Atrato River (21.11+/-2.6), San Jorge River (39.5+/-7.13), and Totumo Marsh (62.5+/-6.38). In average, all specimens of Hoplias malabaricus from the north coast basin of Colombia were infected with a mean intensity of 77.82+/-4.81 (1-466 parasites per host) whereas in fish from the Amazon River this value was significantly lower (intensity 1.0+/-0.0). Size and weight correlated significantly with parasite intensity in fish collected from sampling locations at the north of Colombia (R=0.240, P<0.001 and R=0.199, P=0.008, respectively). Moreover, a significant, but low and negative correlation was found between condition factor and parasite intensity (R=-0.159, P=0.034), suggesting a possible impact of parasites on fish health. These results suggest, for the first time, that the parasitism in Moncholo is a widespread phenomenon in Colombian rivers and could represent a risk factor for human consumers.

Variability in the P6gag domains of HIV-1 involved in viral budding.

The genetic variability within PT/SAP, LYP and LXXLF HIV-1 P6gag motifs, required for the binding to Tsg101 and AIP1 cellular host proteins during viral budding, was examined in 122 HIV-infected subjects. PT/SAP duplications were statistically more frequent in B versus non-B subtypes. Substitutions at LYP were fourfold less frequent in antiretroviral-experienced only in clade B. P6gag variability across HIV-1 subtypes and after antiretroviral exposure may influence interactions with host cells involved in viral budding.

Differences in the length of gag proteins among different HIV type 1 subtypes.

The effect of HIV-1 subtype on Gag protein length was examined in 122 individuals infected with different HIV-1 clades. Except for the P1 protein, a wide variation in the Gag proteins length was noticed. P2 was significantly shorter in 68 non-B with respect to 54 subtype B viruses. Nearly 85% of subtype B gag sequences harbored P2 with 14 or more amino acid (aa) residues, while 75% of non-B subtypes had P2 with 13 or less aa (p < 0.0001). The P7 protein was one residue shorter in 64.2% of non-B specimens but only in 9.3% of subtype B isolates (p = 0.0001). Overall, the P6gag protein length was modified by the presence of insertions, deletions, and stop codons in 89 (73%) of the tested population, but was mainly dependent of changes in non- B compared to B viruses (97% vs. 42.6%, p < 0.0001). However, insertions at P6(gag) (from 1 to 9 aa) were significantly more frequent in B than in non-B viruses (33.3% vs. 4.4%; p = 0.00002). Overall, conserved Gag residues and aa motifs, regardless of the genetic subtype, were 68.7% in P1, 54% in P7, 33.3% in P2, and 25% in P6(gag) proteins. In summary, length variation in Gag proteins is extensive across different HIV-1 subtypes, and could influence protein structure and function. The effect of Gag variation on the viral cycle among different HIV-1 clades needs to be further investigated.

Heterogeneous nature of HIV-1 recombinants spreading in Spain.

HIV-1 infections due to non-B subtypes are increasing rapidly in number and spreading across Europe. The genetic nature of HIV-1 non-B variants containing subtype G sequences at the protease (PR)-coding region are described from 48 unrelated subjects living in Spain. Phylogenetic analyses of the HIV-1 reverse transcriptase (RT) and envelope (env) genes (including the V3 loop) were performed. Up to 32 (66.6%) of samples carried inter-subtype recombinant viruses. Although double recombinants were found most frequently (G/A in 20; G/B in 8; G/K in 2), two individuals harbored triple recombinant viruses (GPR/BRT/Aenv and GPR/KRT/Aenv, respectively). Only 33 (68.7%) and 9 (18.7%) sequences clustered with clade G when examining the RT and env genes, respectively. Nearly 70% of samples with pol sequences (PR/RT) belonging to subtype G harbored env sequences ascribed to other clades: A (55.6%), B (11.1%), or K (3.7%). Of note, most recombinant viruses clustered with CRF02_AG, although CRF14_BG recombinants were also found. This study demonstrates that most viruses circulating in Spain with clade G sequences at the pol-coding region are in fact inter-subtype recombinants, with CRF02_AG being the most prevalent virus.

Prevalence of HIV-1 non-B subtypes, syphilis, HTLV, and hepatitis B and C viruses among immigrant sex workers in Madrid, Spain.

Sexually transmitted disease (STD) remains a major public health challenge in developed countries, exacerbated by the advent of the HIV epidemic. The objectives of this study were to assess the prevalence of serological markers of syphilis, HIV-1/2, HTLV-I/II, HBV, and HCV infections among immigrant sex workers in Madrid, Spain and to characterize the HIV-1 variants in seropositive individuals. Sera from 762 immigrant commercial sex workers (75.3% from sub-Saharan Africa, 18.2% from South America, and 6.4% from Eastern Europe) were collected between 1998 and 2003 in Madrid and examined. Antibody detection was performed by screening assays (RPR, ELISAs) and confirmed by FTA-Abs, LIAs and Western-blot tests. HIV-1 subtyping was carried out by phylogenetic analyses of the protease and envelope genes. Antibodies to HIV-1 were found in 5.2%, while 3.5% tested positive for HBsAg, 3% for syphilis antibodies, 0.8% for HCV antibodies, and 0.2% for HTLV-I antibodies. None were reactive for HIV-2 or HTLV-II antibodies. HIV-1 seroprevalence among Africans and Ecuadorians was 4.5 and 10.9%, respectively. All HIV-1 seropositive Ecuadorians were transsexual men, and 28.6% had active syphilis infection. Up to 80% of HIV-1 positive specimens were characterized as non-B subtypes, with subtypes G, A, and G/A recombinants being the most frequent among African individuals. In contrast, South Americans with HIV-1 infection carried exclusively subtype B variants. A relatively high proportion of immigrant sex workers in Madrid were infected with HIV-1 and syphilis, whereas infections with hepatitis viruses or HTLV were uncommon.

HIV-positive immigrants in the Canary Islands, Spain: implications for public health in Europe.

BACKGROUND: The Canary Islands face the northwest coast of Africa and belong administratively to Spain. They represent a frequent step for the entrance of Africans into Spain and, from there, to all the European Union. The presence of HIV-1 non-B variants has already been reported in Spain and other European countries, mostly among African immigrants. PURPOSE: The aim of this study was to exam the genetic diversity of HIV-1 among immigrants attending a reference hospital in the Canary Islands during 2000. METHOD: Phylogenetic analyses of the reverse transcriptase (RT), protease, and env genes were carried out in 33 immigrants found to be HIV-1 positive. RESULTS: HIV-1 non-B subtypes were recognized in 21 (63.6%) of the 33 infected participants. Phylogenetic analyses showed non-B sequences in 60%, 60.6%, and 48.3% of specimens, depending on the genomic region examined (RT, protease, and env, respectively). Overall, 15 viruses (45%) were found to be inter-subtype recombinants: AG in 8 (53%), GB in 4 (27%), and AB in 3 (20%). CONCLUSION: Nearly two thirds of HIV-infected immigrants arriving to the Canary Islands carry non-B subtypes. Thus, the Canary Islands may be a frequent entry point for new HIV-1 variants into the European Union.

High prevalence of HIV-1 subtype G and natural polymorphisms at the protease gene among HIV-infected immigrants in Madrid.

OBJECTIVES: Genetic characterization of HIV-1 subtypes among immigrants and natives infected overseas. METHODS: Phylogenetic analysis of HIV-1 protease sequences obtained from 109 foreigners (mainly Africans) and 32 native individuals infected overseas attending a reference HIV/AIDS centre located in Madrid, Spain. RESULTS: The overall rate of infection with HIV-1 non-B subtypes was 50.3% (71/141). Whereas 94.3% (67/71) belonged to immigrants (mostly Africans, 60/67), only 5.6% (4/71) were from native individuals (P < 0.05). The distribution of non-B subtypes was: 49 G, eight C, six A, four D, two F and two H. The high prevalence of subtype G was mainly related to individuals from west-central Africa. Interestingly, substitutions at three or more positions associated with protease inhibitor (PI) resistance were recognized in 52.6% of naive subjects carrying non-B subtypes, but only in 8% of those infected with B viruses (P < 0.05). The genotypes most frequently recognized among non-B and B subtypes occurred, respectively, at positions 36 (100 versus 12%), 20 (77.2 versus 0%), 63 (40.3 versus 64%), 82 (17.5 versus 0%), 10 (14 versus 12%), 77 (3.5 versus 34%), and 71 (0 versus 2%). Accordingly, changes I-36 and I-20 may be considered specific genetic markers for non-B, group M variants and subtype G infections, respectively. CONCLUSION: Nearly two-thirds of foreigners with HIV-1 infection in Madrid carry non-B subtypes, subtype G (protease) being the most common among west-central African immigrants. The high rate of natural polymorphisms at the protease gene in non-B viruses may compromise the response to PI. Therefore, HIV subtyping should be considered in treatment guidelines.

HIV-1 subtype J recombinant viruses in Spain.

Genetic characterization of HIV-1 was carried out in two women who originated in Cameroon and Equatorial Guinea, respectively, and who were diagnosed more recently as HIV-1 seropositive in Madrid, Spain. Phylogenetic studies showed that the protease-encoding gene from both individuals clustered with subtype J sequences with a high bootstrap. However, env sequences clustered with subtypes A and C, respectively. This work represents the first characterization of HIV-1 containing subtype J-like genomic regions in Spain.