Pyrazoline derivatives as promising novel antischistosomal agents.

Praziquantel is the only available drug to treat schistosomiasis, a parasitic disease that currently infects more than 240 million people globally. Due to increasing concerns about resistance and inadequate efficacy there is a need for new therapeutics. In this study, a series of 17 pyrazolines (15-31) and three pyrazoles (32-34) were synthesized and evaluated for their antiparasitic properties against ex vivo adult Schistosoma mansoni worms. Of the 20 compounds tested, six had a 50% effective concentration (EC50) below 30 µM. Our best hit, pyrazoline 22, showed promising activity against adult schistosomes, with an EC50 < 10 µM. Additionally, compound 22 had low cytotoxicity, with selectivity index of 21.6 and 32.2 for monkey and human cell lines, respectively. All active pyrazolines demonstrated a negative effect on schistosome fecundity, with a marked reduction in the number of eggs. Structure-activity relationship analysis showed that the presence of the non-aromatic heterocycle and N-substitution are fundamental to the antischistosomal properties. Pharmacokinetics, drug-likeness and medicinal chemistry friendliness studies were performed, and predicted values demonstrated an excellent drug-likeness profile for pyrazolines as well as an adherence to major pharmaceutical companies' filters. Collectively, this study demonstrates that pyrazoline derivatives are promising scaffolds in the discovery of novel antischistosomal agents.

Nebulization of Vancomycin Provides Higher Lung Tissue Concentrations than Intravenous Administration in Ventilated Female Piglets with Healthy Lungs.

BACKGROUND: Intravenous vancomycin is used to treat ventilator-associated pneumonia caused by methicillin-resistant Staphylococcus aureus, but achieves high rates of failure. Vancomycin nebulization may be efficient to provide high vancomycin lung tissue concentrations. The aim of this study was to compare lung tissue and serum concentrations of vancomycin administered intravenously and by aerosol in mechanically ventilated and anesthetized healthy piglets. METHODS: Twelve female piglets received a single intravenous dose of vancomycin (15 mg/kg) and were killed 1 (n = 6) or 12 h (n = 6) after the end of administration. Twelve piglets received a single nebulized dose of vancomycin (37.5 mg/kg) and were killed 1 (n = 6) or 12 h (n = 6) after the end of the aerosol administration. In each group, vancomycin lung tissue concentrations were assessed on postmortem lung specimens using high-performance liquid chromatography. Blood samples were collected for serum vancomycin concentration measurement 30 min and 1, 2, 4, 6, 8, and 12 h after the end of vancomycin administration. Pharmacokinetics was analyzed by nonlinear mixed effect modeling. RESULTS: One hour after vancomycin administration, lung tissue concentrations in the aerosol group were 13 times the concentrations in the intravenous group (median and interquartile range: 161 [71, 301] µg/g versus 12 [4, 42] µg/g; P < 0.0001). Twelve hours after vancomycin administration, lung tissue concentrations in the aerosol group were 63 (23, 119) µg/g and 0 (0, 19) µg/g in the intravenous group (P < 0.0001). A two-compartment weight-scaled allometric model with first-order absorption and elimination best fit serum pharmacokinetics after both routes of administration. Area under the time-concentration curve from 0 to 12 h was lower in the aerosol group in comparison to the intravenous group (56 [8, 70] mg · h · l vs. 121 [103, 149] mg · h · l, P = 0.002). Using a population model, vancomycin bioavailability was 13% (95% CI, 6 to 69; coefficient of variation = 85%) and absorption rate was slow (absorption half life = 0.3 h). CONCLUSIONS: Administration of vancomycin by nebulization resulted in higher lung tissue concentrations than the intravenous route.

Antiparasitic activity of piplartine (piperlongumine) in a mouse model of schistosomiasis.

Schistosomiasis is one of the most important parasitic infections in terms of its negative effects on public health and economics. Since praziquantel is currently the only drug available to treat schistosomiasis, there is an urgent need to identify new anthelmintic agents. Piplartine, also known as piperlongumine, is a biologically active alkaloid/amide from peppers that can be detected in high amounts in the roots of Piper tuberculatum. Previously, it has been shown to have in vitro schistosomicidal effects. However, its anthelmintic activity in an animal host has not been reported. In the present work, in vivo antischistosomal properties of isolated piplartine were evaluated in a mouse model of schistosomiasis infected with either adult (patent infection) or juvenile (pre-patent infection) stages of Schistosoma mansoni. A single dose of piplartine (100, 200 or 400 mg/kg) or daily doses for five consecutive days (100 mg/kg/day) administered orally to mice infected with schistosomes resulted in a reduction in worm burden and egg production. Treatment with the highest piplartine dose (400 mg/kg) caused a significant reduction in a total worm burden of 60.4% (P < 0.001) in mice harbouring adult parasites. S. mansoni egg production, a process responsible for pathology in schistosomiasis, was also significantly inhibited by piplartine. Studies using scanning electron microscopy revealed substantial tegumental alterations in parasites recovered from mice. Since piplartine has well-characterized mechanisms of toxicity, is easily available, and is cost-effective, our results indicate that this bioactive molecule derived from medicinal plants could be a potential lead compound for novel antischistosomal agents.

Promethazine exhibits antiparasitic properties in vitro and reduces worm burden, egg production, hepato-, and splenomegaly in a schistosomiasis animal model.

The treatment and control of schistosomiasis, a neglected disease that affects more than 200 million people worldwide, rely on the use of a single drug, praziquantel. A vaccine has yet to be developed and since new drug design and development is a lengthy and costly process, drug repurposing is a promising strategy. In this study, the efficacy of promethazine, a first-generation antihistamine, was evaluated against Schistosoma mansoni ex vivo and in a murine model of schistosomiasis. In vitro assays demonstrated that promethazine affected parasite motility, viability, and it induced severe tegumental damage in schistosomes. The LC50 of the drug was 5.84 µM. Similar to promethazine, schistosomes incubated with atropine, a classical anticholinergic drug, displayed reduced motor activity. In an animal model, promethazine treatment was introduced at an oral dose of 100 mg/kg for five successive days at different intervals from the time of infection, for the evaluation of the stage-specific susceptibility (pre-patent and patent infections). Various parasitological criteria indicated the in vivo antischistosomal effects of promethazine: there were significant reductions in worm burden, egg production, and hepato- and splenomegaly. The highest worm burden reduction was achieved with promethazine in patent infections (> 90%). Taken together, considering the importance of the repositioning of drugs in infectious diseases, especially those related to poverty, our data revealed the possibility of promethazine repositioning as an antischistosomal agent.

Allele-specific antibodies to Plasmodium vivax merozoite surface protein-1: prevalence and inverse relationship to haemoglobin levels during infection.

BACKGROUND: Antigenic polymorphisms are considered as one of the main strategies employed by malaria parasites to escape from the host immune responses after infections. Merozoite surface protein-1 (MSP-1) of Plasmodium vivax, a promising vaccine candidate, is a highly polymorphic protein whose immune recognition is not well understood. METHODS AND RESULTS: The IgG responses to conserved (MSP-119) and polymorphic (block 2 and block 10) epitopes of PvMSP-1 were evaluated in 141 P. vivax infected patients. Ten recombinant proteins corresponding to block 2 (variants BR07, BP29, BP39, BP30, BEL) and block 10 (BR07, BP29, BP39, BP01, BP13) often observed in Brazilian P. vivax isolates were assessed by ELISA in order to determine levels of specific antibodies and their respective seroprevalence. The magnitude and the frequency of variant-specific responses were very low, except for BR07 variant (>40%), which was the predominant haplotype as revealed by block 10 PvMSP-1 gene sequencing. By contrast, 89% of patients had IgG against the C-terminal conserved domain (PvMSP-119), confirming the high antigenicity of this protein. Using multiple linear and logistic regression models, there was evidence for a negative association between levels of haemoglobin and several IgG antibodies against block 2 variant antigens, with the strongest association being observed for BP39 allelic version. This variant was also found to increase the odds of anaemia in these patients. CONCLUSIONS: These findings may have implications for vaccine development and represent an important step towards a better understanding of the polymorphic PvMSP-1 domain as potential targets of vaccine development. These data highlight the importance of extending the study of these polymorphic epitopes of PvMSP-1 to different epidemiological settings.

Interleukin-17 producing T helper cells are increased during natural Plasmodium vivax infection.

Recent evidences have demonstrated the importance of Th17 cells in host defense against infectious diseases. However, little is known about their role in parasitic infections. Here, we showed that uncomplicated acute vivax malaria induce a significant expansion of IL-17-producing CD4(+) T cells associated to a pro-inflammatory cytokine profile. Furthermore, we demonstrated a correlation between numbers of IL-17(+)CD4(+) T cells and circulating CD4(+) T-cells producing IFN-γ, IL-10 and TGF-ß. Finally, correlations between number of these cells and morbidity or parasitemia were not detected. Further studies are underway to investigate whether IL-17-producing CD4(+) T cells are critically involved in the immunity against Plasmodium vivax infection.

Naturally acquired antibodies to Plasmodium vivax blood-stage vaccine candidates (PvMSP-119 and PvMSP-3α359₋798 and their relationship with hematological features in malaria patients from the Brazilian Amazon.

An important step when designing a vaccine is identifying the antigens that function as targets of naturally acquired antibodies. We investigated specific antibody responses against two Plasmodium vivax vaccine candidates, PvMSP-119 and PvMSP-3α359₋798. Moreover, we assessed the relationship between these antibodies and morbidity parameters. PvMSP-119 was the most immunogenic antigen and the frequency of responders to this protein tended to increase in P. vivax patients with higher parasitemia. For both antigens, IgG antibody responses tended to be lower in patients who had experienced their first bout of malaria. Furthermore, anemic patients presented higher IgG antibody responses to PvMSP-3α359₋798. Since the humoral response involves a number of antibodies acting simultaneously on different targets, we performed a Principal Component Analysis (PCA). Anemic patients had, on average, higher first principal component scores (IgG1/IgG2/IgG3/IgG4 anti-MSP3α), which were negatively correlated with hemoglobin levels. Since antibodies against PfMSP-3 have been strongly associated with clinical protection, we cannot exclude the possibility of a dual role of PvMSP-3 specific antibodies in both immunity and pathogenesis of vivax malaria. Our results confirm the high immunogenicity of the conserved C terminus of PvMSP-1 and points to the considerable immunogenicity of polymorphic PvMSP-3α359₋798 during natural infection.

Identification of a highly antigenic linear B cell epitope within Plasmodium vivax apical membrane antigen 1 (AMA-1).

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

Immunoglobulin GM 3 23 5,13,14 phenotype is strongly associated with IgG1 antibody responses to Plasmodium vivax vaccine candidate antigens PvMSP1-19 and PvAMA-1.

BACKGROUND: Humoral immune responses play a key role in the development of immunity to malaria, but the host genetic factors that contribute to the naturally occurring immune responses to malarial antigens are not completely understood. The aim of the present investigation was to determine whether, in subjects exposed to malaria, GM and KM allotypes--genetic markers of immunoglobulin gamma and kappa-type light chains, respectively--contribute to the magnitude of natural antibody responses to target antigens that are leading vaccine candidates for protection against Plasmodium vivax. METHODS: Sera from 210 adults, who had been exposed to malaria transmission in the Brazilian Amazon endemic area, were allotyped for several GM and KM determinants by a standard hemagglutination-inhibition method. IgG subclass antibodies to P. vivax apical membrane antigen 1 (PvAMA-1) and merozoite surface protein 1 (PvMSP1-19) were determined by an enzyme-linked immunosorbent assay. Multiple linear regression models and the non-parametric Mann-Whitney test were used for data analyses. RESULTS: IgG1 antibody levels to both PvMSP1-19 and PvAMA-1 antigens were significantly higher (P = 0.004, P = 0.002, respectively) in subjects with the GM 3 23 5,13,14 phenotype than in those who lacked this phenotype. CONCLUSIONS: Results presented here show that immunoglobulin GM allotypes contribute to the natural antibody responses to P. vivax malaria antigens. These findings have important implications for the effectiveness of vaccines containing PvAMA-1 or PvMSP1-19 antigens. They also shed light on the possible role of malaria as one of the evolutionary selective forces that may have contributed to the maintenance of the extensive polymorphism at the GM loci.

Plasmodium vivax: induction of CD4+CD25+FoxP3+ regulatory T cells during infection are directly associated with level of circulating parasites.

Circulation CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-beta) as well as pro-inflammatory (IFN-gamma, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4(+)CD25(+)FoxP3(+)GITR(+) or CD4(+)CD25(+)FoxP3(+)CTLA-4(+) and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.

Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells.

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naïve subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and FcgammaRI (CD64) receptor (P < or = 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells (P=0.0356 and P=0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-alpha, IL-12p40 and IFN-gamma (P < or = 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection.

Antibodies to Plasmodium vivax apical membrane antigen 1: persistence and correlation with malaria transmission intensity.

The antibody responses to the apical membrane antigen 1 of the Plasmodium vivax (PvAMA-1) were investigated in subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the levels of IgG to PvAMA-1 increased with the time of exposure. The frequency of a positive response and the mean IgG level were higher in areas where malaria prevalence was more intense, especially among non-infected subjects exposed to moderate transmission over a period of 20 years. The proportions and levels of IgG1and IgG3 isotypes were significantly higher among those subjects with long-term exposure. Antibodies, mainly IgG1, to PvAMA-1 persisted for seven years among subjects briefly exposed to malaria in an outbreak outside the Brazilian malaria-endemic area. These data show the highly immunogenic properties of PvAMA-1 and emphasize its possible use as a malaria vaccine candidate.

Profilaxia para úlcera de estresse nas unidades de terapia intensiva: estudo observacional multicêntrico / Stress ulcer prophylaxis in intensive care units: an observational multicenter study

JUSTIFICATIVA E OBJETIVOS: O sangramento digestivo por úlcera de estresse (SDUE) é uma complicação grave dos pacientes criticamente doentes e com necessidade de profilaxia baseada em critérios literários definidos. O objetivo deste estudo foi revisar o uso de profilaxia para SDUE em UTI do Estado do Rio Grande do Sul, comparando os resultados com as evidências atuais da literatura. MÉTODO: Estudo transversal realizado em um único dia, com coleta de dados de todos os pacientes internados em 21 unidades de terapia intensiva (UTI). Para análise dos dados, os pacientes foram distribuídos em três subgrupos (alto, médio e baixo risco de SDUE). RESULTADOS: Foram analisados 235 pacientes internados, com média de idade de 57,7 ± 19,5 anos e tempo médio de internação em UTI de 13 ± 19,7 dias. Os motivos de internação mais freqüentes foram sepse (26 por cento) e pós-operatório de grandes cirurgias (16,2 por cento). Da totalidade, 73 por cento eram de alto risco para SDUE, 21,5 por cento de risco intermediário e 5,5 por cento de baixo risco. Dos 187 pacientes de alto risco, 139 estavam usando bloqueadores para SDUE (60 por cento com bloqueadores histaminérgicos (BH2) e 39 por cento com inibidor de bomba de prótons (IBP) para profilaxia (60 por cento). Não recebiam profilaxia, apesar de indicada, 25,7 por cento destes pacientes de alto risco. Dos 55 pacientes de risco intermediário para SDUE, 70,9 por cento recebiam profilaxia (22 com BH2 e 17 com IBP) e dos 14 pacientes de baixo risco, 71 por cento recebiam profilaxia (6 com BH2 e 4 com IBP). CONCLUSÕES: Este artigo traduziu a ausência de estratificação de risco para SDUE nas UTI do Estado, além da indicação de fármacos gastro-protetores sem critérios precisos para o seu emprego.

BACKGROUND AND OBJECTIVES: Gastrointestinal bleeding due to stress ulcer (GB) complicates critical disease, and must be received prophylaxis based on defined criteria. To evaluate the GB prophylaxis in Intensive Care Units (ICU), and to compare with the guidelines. METHODS: We carried out a cross-sectional multicenter study in 21 medical-surgical ICU in Brazil to investigate this issue. For data analysis, these were distributed in 3 sub-groups (high, moderate and low risk for GB). RESULTS: 235 patients were evaluated, with mean age of 57.7 ± 19.5 years and days on ICU 13 ± 19.7. The more common admission ICU diagnoses were sepsis (26 percent) and postoperative (16.2 percent) patients. Seventy-three (73 percent) of the patients were GB high risk, 21.5 percent moderate and 5.5 percent low risk. Of the 187 high risk patients, 139 were receiving GB prophylaxis (60 percent with histamine blockers (HB2) and 39 percent with proton pump inhibitors (PPI). Of these patients, 25.7 percent did not receive GU prophylaxis, although indicated it. Of the 55 moderate risk patients, 70.9 percent wer e receiving GU prophylaxis (22 with HB2 and 17 with PPI). Of the 14 low risk patients, 71 percent were using GU prophylaxis (6 with HB2 and 4 with PPI). CONCLUSIONS: Almost 80 percent of the patients made use of GB prophylactic drugs, with no agreement GU risk stratification. This study demonstrated the no adequate GU prophylaxis in the Brazilian ICU.

IgG isotype to C-terminal 19 kDa of Plasmodium vivax merozoite surface protein 1 among subjects with different levels of exposure to malaria in Brazil.

Subclasses of antibodies to the C-terminal 19 kDa fragment of the Plasmodium vivax merozoite surface protein 1 (PvMSP-1(19)) were assessed among subjects with distinct degrees of malaria exposure in the Brazilian endemic area. The PvMSP-1(19) specific IgG1and IgG3 levels were low among subjects with long-term exposure (approximately 19 years) when compared to subjects less and sporadically exposed (<1 year). No statistically difference was observed in IgG subclass distribution of antibodies from symptomatic Plasmodium-infected patients, asymptomatic parasite carriers and non-infected subjects living in a same mesoendemic area. Subjects briefly exposed to a P. vivax outbreak living in a rural community outside the endemic area were also evaluated to measure the persistence of specific antibodies. IgG anti-PvMSP-1(19) antibodies persisted in 40% of the subjects who had had malarial symptoms 8 months before and decreased after 7 years (28%). Specific IgG1 were the predominant isotype. Our study emphasizes the highly immunogenicity of the PvMSP-1(19) and points toward its possible use as a potential malaria vaccine.

Association of the IgG response to Plasmodium falciparum merozoite protein (C-terminal 19 kD) with clinical immunity to malaria in the Brazilian Amazon region.

The antibody response to the C-terminal 19-kD fragment of Plasmodium falciparum merozoite surface protein-1 (PfMSP1-19) was investigated in groups of subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the levels of IgG to PfMSP1-19 increased with the time of exposure and were positively correlated with the absence of clinical symptoms in parasitemic patients. The frequency of positive response and the mean level of IgG were higher in areas where malaria prevalence was more intense, especially among asymptomatic patients. The serum absorbance values of the IgG1 isotype were significantly higher among subjects with long-term exposure and in asymptomatic infections. These data suggest a protective role of IgG1 in naturally acquired immunity in spite of the unstable transmission levels in the Brazilian Amazon.