Plasmodium exposure alters midgut epithelial cell dynamics during the immune memory in Anopheles albimanus.

Immunological priming in insects is defined as a previous contact with non-virulent pathogens, which induces protection after a second virulent infection. The mechanism of this process is not well understood. We have observed midgut DNA synthesis (endoreplication) in Plasmodium berghei exposure mosquitoes (primed) and after the immune challenge, which could be an essential component of the priming response in the mosquito. Endoreplication requires cell cycle components re-direction to make multiple DNA copies. Therefore, it is fundamental to understand the role of cell cycle components in priming. Here, we analyzed the expression of the cyclins A, B, E, and AurkA, and the endoreplication components NOTCH and HNT in the mosquito Anopheles albimanus; after priming with non-infective Plasmodium berghei and challenged with an infective P. berghei. The overexpression of cell cycle elements occurred seven days after priming with a quick reduction 24 h after the challenge. Hnt and NOTCH overexpression occurred 24 h after priming. Antimicrobial peptide cecropin is quickly overexpressed after 24 h in primed mosquitoes, then is downregulated at day seven and overexpressed again after parasite challenge. We also found that DNA synthesis occurs in cells with different nuclear sizes, suggesting a change in midgut epithelial dynamics after Plasmodium exposure. Inhibition of DNA synthesis via cisplatin revealed that DNA synthesis is required for priming to limit Plasmodium infection. Our results indicate the importance of cell cycle components on DNA synthesis and Notch pathway during priming response in An. albimanus mosquitoes.

Mosquito metallomics reveal copper and iron as critical factors for Plasmodium infection.

Iron and copper chelation restricts Plasmodium growth in vitro and in mammalian hosts. The parasite alters metal homeostasis in red blood cells to its favor, for example metabolizing hemoglobin to hemozoin. Metal interactions with the mosquito have not, however, been studied. Here, we describe the metallomes of Anopheles albimanus and Aedes aegypti throughout their life cycle and following a blood meal. Consistent with previous reports, we found evidence of maternal iron deposition in embryos of Ae. aegypti, but less so in An. albimanus. Sodium, potassium, iron, and copper are present at higher concentrations during larval developmental stages. Two An. albimanus phenotypes that differ in their susceptibility to Plasmodium berghei infection were studied. The susceptible white stripe (ws) phenotype was named after a dorsal white stripe apparent during larval stages 3, 4, and pupae. During larval stage 3, ws larvae accumulate more iron and copper than the resistant brown stripe (bs) phenotype counterparts. A similar increase in copper and iron accumulation was also observed in the susceptible ws, but not in the resistant bs phenotype following P. berghei infection. Feeding ws mosquitoes with extracellular iron and copper chelators before and after receiving Plasmodium-infected blood protected from infection and simultaneously affected follicular development in the case of iron chelation. Unexpectedly, the application of the iron chelator to the bs strain reverted resistance to infection. Besides a drop in iron, iron-chelated bs mosquitoes experienced a concomitant loss of copper. Thus, the effect of metal chelation on P. berghei infectivity was strain-specific.

DNA synthesis increases during the first hours post-emergence in Anopheles albimanus mosquito midgut.

In hematophagous insects, the midgut is a fundamental barrier against infections and limits the development and transmission of pathogens. However, in mosquitoes, cell differentiation, proliferation, and cell cycle process in the midgut have not been characterized. Here we provide evidence of how cell cycle progression occurs in the newly emerged Anopheles albimanus mosquito midgut and describing cyclins expression as mediators of the cell cycle. The cell cycle at different post-emergence times was evaluated in disaggregated cells from midgut tissue using flow cytometry. Also, cyclins A, B, and E were identified by bioinformatics tools. These cyclins were used to analyze cell cycle progression. Flow cytometry data and the expression-pattern of the cyclins by qRT-PCR supported a polyploidy process, besides mitosis marker was marginally detected and only in newly emerged mosquitoes. Our results suggest that DNA increment in midguts occurs by polyploidy during the first hours post-emergence.

Ecdysis-related pleiotropic neuropeptides expression during Anopheles albimanus development / Expresión de neuropéptidos pleiotrópicos asociados con ecdisis durante el desarrollo del mosquito Anopheles albimanus

Abstract: Objective: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. Materials and methods: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. Results: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. Conclusion: The CCAP, corazonin and ETH neuropeptides are potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

Resumen: Objetivo: Describir la expresión de neuropéptidos durante la ontogenia del mosquito vector de la malaria Anopheles albimanus. Material y métodos: Se midió la expresión de CCAP, corazonina, ETH, allatostatina, orcokinina, ILP2, ILP5 y bursicon en larvas de primer (2mm), segundo (4mm), tercer (5mm) y cuarto (6mm) estadio, pupas y mosquitos adultos, mediante qPCR. Resultados. A diferencia de otros insectos en donde, CCAP, corazonina y ETH se expresan principalmente en estadios pupales, en An. albimanus se expresaron mayoritariamente en larvas de cuarto estadio, CCAP tuvo 70.8% de expresión relativa, corazonina 76.5% y ETH 60.2%. ILP2 fue el neuropéptido que más se expresó en el primer, segundo y tercer estadio y orcokinina disminuyó durante el desarrollo del mosquito. Conclusión. Los péptidos estudiados se expresaron en todos los estadios de desarrollo del mosquito. Sin embargo, su expresión varió en cada uno de ellos. Los neuropéptidos CCAP, corazonina y ETH, que son esenciales para la transformación de lavas a pupas, pueden ser blancos potenciales para el diseño de estrategias de control dirigidas a interrumpir el desarrollo larvario de An. albimanus.

Análisis de la evidencia sobre eficacia y seguridad de la vacuna de dengue CYD-TDV y su potencial registro e implementación en el Programa de Vacunación Universal de México / Analysis of the evidence on the efficacy and safety of CYD-TDV dengue vaccine and its potential licensing and implementation through Mexico's Universal Vaccination Program

El dengue es un importante problema de salud pública global, que afecta a América Latina y México. Las medidas de prevención y control centradas en vigilancia epidemiológica y control de vectores han resultado parcialmente efectivas y costosas, por lo que el desarrollo de una vacuna contra el dengue ha creado grandes expectativas entre las autoridades sanitarias y las comunidades científicas en el mundo. Sólo la vacuna CYD-TDV, producida por Sanofi-Pasteur, ha sido evaluada en ensayos clínicos controlados fase 3. No obstante a pesar de la importante contribución que esto significa para el desarrollo de una vacuna contra el dengue, los tres estudios clínicos fase 3 de CYD-TDV y el metaanálisis de seguimiento a largo plazo derivado de los mismos proporcionan evidencia de que esta vacuna tiene una eficacia parcial para proteger contra dengue virológicamente confirmado. Al respecto, surgen cuatro consideraciones: a) eficacia adecuada contra infecciones por virus de dengue (DENV) 3 y 4, menor eficacia contra infecciones por DENV 1 y prácticamente nula protección contra infecciones por DENV 2; b) disminución de la eficacia en individuos seronegativos a dengue al inicio de la vacunación; c) 83 y 90% de protección contra hospitalizaciones y formas de dengue grave, respectivamente, a 25 meses de seguimiento, y d) incremento de hospitalización por dengue, en el grupo de vacunados, en niños menores de nueve años de edad al momento de la vacunación, detectado a partir del tercer año de seguimiento. El beneficio de la vacuna CYD-TDV se puede resumir en la protección contra infecciones por DENV 3 y 4, así como en la protección de hospitalizaciones y casos graves en individuos mayores de nueve años y en quienes han tenido infección previa por dengue, pues funciona principalmente como una vacuna de refuerzo. En esta revisión se identificaron elementos sobre eficacia y seguridad de esta vacuna que deben ser tomados en cuenta ante el potencial registro e inclusión en el programa de vacunación en la población mexicana. La evidencia científica disponible sobre la vacuna CYD-TDV demuestra méritos, pero también da lugar a preguntas relevantes que deberían ser contestadas para evaluar apropiadamente el perfil de seguridad del producto, así como las poblaciones blanco de potencial beneficio. Al respecto, consideramos que sería informativo completar el seguimiento indicado de seis años después de iniciar la vacunación, de acuerdo con el protocolo propuesto en los propios estudios del fabricante como una recomendación de la Organización Mundial de la Salud. Al igual que con cualquier nueva vacuna, el potencial registro e implementación de uso de CYD-TDV en el programa nacional de vacunación de México requiere una definición clara de cuál es el balance entre los beneficios y riesgos esperados. En particular, ante una vacuna con eficacia variable y algunas señales de riesgo, en caso de aprobar el registro, se deben desarrollar protocolos de manejo de riesgos detallados que permitan identificar de manera oportuna cualquier evento de salud asociado con la vacunación.

Dengue is a major global public health problem affecting Latin America and Mexico Prevention and control measures, focusing on epidemiological surveillance and vector control, have been partially effective and costly, thus, the development of a vaccine against dengue has created great expectations among health authorities and scientific communities worldwide. The CYD-TDV dengue vaccine produced by Sanofi-Pasteur is the only dengue vaccine evaluated in phase 3 controlled clinical trials. Notwithstanding the significant contribution to the development of a vaccine against dengue, the three phase 3 clinical studies of CYD-TDV and the meta-analysis of the long-term follow up of those studies, have provided evidence that this vaccine exhibited partial vaccine efficacy to protect against virologically confirmed dengue and lead to four considerations: a) adequate vaccine efficacy against dengue virus (DENV) infections 3 and 4, less vaccine efficacy against DENV 1 and no protection against infection by DENV 2; b) decreased vaccine efficacy in dengue seronegative individuals at the beginning of the vaccination; c) 83% and 90% protection against hospitalizations and severe forms of dengue, respectively, at 25 months follow-up; and d) increased hospitalization for dengue in the vaccinated group, in children under nine years of age at the time of vaccination, detected since the third year of follow-up. The benefit of the CYD-TDV vaccine can be summarized in the protection against infection by DENV 3 and 4, as well as protection for hospitalizations and severe cases in people over nine years, who have had previous dengue infection, working mainly as a booster. In this review we identified elements on efficacy and safety of this vaccine that must be taken into account in the licensing process and potential inclusion in the national vaccination program of Mexico. The available scientific evidence on the CYD-TDV vaccine shows merits, but also leads to relevant questions that should be answered to properly assess the safety profile of the product and the target populations of potential benefit. In this regard we consider it would be informative to complete the 6-year follow-up after starting vaccination, according to the company's own study protocol recommended by the World Health Organization. As with any new vaccine, the potential licensing and implementation of the CYD-TDV as part of Mexico's vaccination program, requires a clear definition of the balance between the expected benefits and risks. Particularly with a vaccine with variable efficacy and some signs of risk, in the probable case of licensing, the post-licensed period must involve the development of detailed protocols to immediately identify risks or any health event associated with vaccination.

Nitric oxide participates in the toxicity of Bacillus thuringiensis Cry1Ab toxin to kill Manduca sexta larvae.

Nitric oxide (NO) produced by the nitric oxide synthase (NOS) enzyme is a reactive oxygen molecule widely considered as important participant in the immune system of different organisms to confront microbial infections. In insects the NO molecule has also been implicated in immune response against microbial pathogens. Bacillus thuringiensis (Bt) is an insect-pathogenic bacterium that produces insecticidal proteins such as Cry toxins. These proteins kill insects because they form pores in the larval-midgut cells. Here we show that intoxication of Manduca sexta larvae with Cry1Ab activates expression of NOS with a corresponding increase in NO. This effect is not observed with a non-toxic mutant toxin Cry1Ab-E129K that is affected in pore formation. The increased production of NO triggered by intoxication with LC50 dose of Cry1Ab toxin is not associated with higher expression of antimicrobial peptides. NO participates in Cry1Ab toxicity since inhibition of NOS by selective l-NAME inhibitor prevented NO production and resulted in reduced mortality of the larvae. The fact that mortality was not completely abolished by L-NAME indicates that other processes participate in toxin action and induction of NO production upon Cry1Ab toxin administration accounts only for a part of the toxicity of this protein to M. sexta larvae.

The role of cell proteins in dengue virus infection.

Despite 70 years of study, dengue disease continues to be a global health burden. Treatment is only supportive based on presenting symptoms. To date, there is no licensed prophylactic vaccine and no specific antiviral drugs available. The pathogenesis mechanisms during dengue virus infections remain poorly understood, and the complete picture on risk factors for developing severe clinical illness is still unknown. Viruses as obligate intracellular parasites depend on the host cell machinery for replication. As a result of a co-evolution process for million years, viruses have developed sophisticated strategies to hijack and use cellular factors for entry, replication and propagation, alternate host transmission and to combat host cell defenses. This review focuses on recent reports about cellular proteins involved along the dengue virus replication cycle, in prime cellular targets during the infection of both humans and mosquito hosts and also on the proteomics and other approaches that are being used to reveal the entire orchestration and most significant processes altered during infection. Identification of the key host cell factors involve in these processes will provide a better understanding of how viruses replicate and cause disease, and how to develop more effective therapeutic interventions. BIOLOGICAL SIGNIFICANCE: Dengue disease is as a global health problem. The treatment is only supportive based on presenting symptoms. To date, there is no licensed prophylactic vaccine and no specific antiviral drugs available. The study of the interactions between virus and host cell proteins will provide a better understanding of how viruses replicate and cause disease. Here, we focus on the current knowledge about the cellular proteins involved during DENV infection in different target cells in the two hosts, mosquito and human.

AdDLP, a bacterial defensin-like peptide, exhibits anti-Plasmodium activity.

Antimicrobial defensins with the cysteine-stabilized alpha-helical and beta-sheet (CSalphabeta) motif are widely distributed in three eukaryotic kingdoms. However, recent work suggests that bacteria could possess defensin-like peptides (DLPs). Here, we report recombinant expression, in vitro folding, structural and functional characterization of a DLP from the myxobacterium Anaeromyxobacter dehalogenans (AdDLP). Circular dichroism analysis indicates that recombinant AdDLP adopts a typical structural feature of eukaryotic defensins, which is also consistent with an ab initio structure model predicted using I-TASSER algorithm. We found that AdDLP is an antimalarial peptide that led to more than 50% growth inhibition on sexual stages of Plasmodium berghei at micromolar concentrations and killed 100% intraerythrocytic Plasmodium falciparum at 10 microM in a time-dependent manner. These results provide functional evidence for myxobacterial origin of eukaryotic defensins. High-level production of the pure anti-Plasmodium peptide without harming mammalian red blood cells in Escherichia coli makes AdDLP an interesting candidate for antimalarial drug design.

La importancia de la proteómica en la salud pública: The significance of proteomics in public health

La salud pública en el nuevo milenio tiene como reto integrar los avances de la genómica al derecho fundamental de la salud de todos los seres humanos. La proteómica, entendida como la disciplina científica que estudia los proteomas, es de vital importancia en la investigación en salud, ya que el conocimiento de las proteínas y moléculas efectoras de la función celular permitirá un mejor entendimiento de la fisiología humana. En este trabajo se describen los antecedentes y los conocimientos básicos del análisis proteómico basado en la espectrometría de masas y se comentan los usos de la proteómica en la búsqueda de biomarcadores para el diagnóstico y pronóstico de diferentes enfermedades, los avances en la comprensión de los trastornos crónicos y algunas enfermedades infecciosas. De manera adicional, se delinean las ventajas de la espectrometría de masas en la genotipificación de patógenos y el estudio de los polimorfismos de una sola base (SNP, por sus siglas en inglés).

The proteome is defined as the entirety of proteins expressed by a genome in a given time under specific physiological conditions. In an organism, the cells contain the same genome; however, they express different proteins in response to a specific micro-environment. Proteomics is responsible for the study of proteomes, using a wide range of methodological techniques. Actually, proteomics is a key tool in health research because it has made possible systematic analysis of hundreds of proteins in clinical samples with the promise of discovering new protein biomarkers for different disease conditions. Finally, proteomic strategy is a technology well-suited to provide a better understanding of systems biology and human health.

Cloning and functional characterization of the Anopheles albimanus DMT1/NRAMP homolog: implications in iron metabolism in mosquitoes.

In addition to its wide role in metabolism, iron in insects has been implicated in vitellogenesis and the immune response. The NRAMP family comprises a well-conserved family of divalent cation transporters in metazoans. To gain insight on the role of NRAMP in Anopheles albimanus, we cloned a cDNA encoding a 571-residue protein (AnaNRAMP) with the structural features defining the NRAMP family. AnaNRAMP mRNA induced (59)Fe(2+) incorporation when injected into Xenopus oocytes. Western blot analysis revealed that AnaNRAMP is expressed in the head, midgut and at high levels in Malpighian tubules of unfed female mosquito. Upon blood feeding, AnaNRAMP levels were reduced in the midgut whereas they increased in the Malpighian tubules. Using immuno-localization by transmission electron microscopy, AnaNRAMP was localized in the membrane of the intra-cellular concretions or spherites of the Malpighian tubule principal cells. Taken together, our results suggest an important role of AnaNRAMP in iron transport and indicate a role of the mosquito Malpighian tubule as an important organ for iron homeostasis.

Carminic acid dye from the homopteran Dactylopius coccus hemolymph is consumed during treatment with different microbial elicitors.

The activation of Dactylopius coccus (Costa) hemolymph with microbial polysaccharide molecules was studied. Hemolymph incubated in the presence of laminarin, zymosan, and N-acetyl glucosamine produced a dark fibrillar precipitated, and the red pigment (carminic acid) was consumed (measured spectrophotometrically at 495 nm). Lipopolysaccharide (LPS) did not induce any response. The reaction was inhibited with millimolar concentrations of serine and cysteine protease inhibitors, EGTA and phenyl thiourea. It was also diminished by prostaglandin synthesis inhibitors: dexamethasone, acetylsalicylic acid, and indomethacin. However, Mg2+ chelator EDTA did not inhibit hemolymph activation. Hemolymph proteins were depleted from soluble phase during treatment with laminarin, but a group of around 34 kDa remained unmodified. These results showed that D. coccus hemolymph is activated by microbial elicitors, its activation depends on eicosanoids, and suggest participation of a prophenoloxidase (PPO)-like activation system that could consume carminic acid. We are currently dissecting the molecular factors involved in D. coccus hemolymph activation to determine homologies and differences with other arthropods immune response pathways.