Jesús Martínez-Barnetche

A research agenda for malaria eradication: basic science and enabling technologies

The Malaria Eradication Research Agenda (malERA) consultative group on Basic Science and Enabling Technologies present a research and development agenda for basic research required for malaria eradication.

Cervical Microbiome and Cytokine Profile at Various Stages of Cervical Cancer: A Pilot Study

Cervical cancer (CC) is caused by high-risk human papillomavirus persistence due to the immunosuppressive tumor microenvironment mediated by cytokines. Vaginal microbiota determines the presence of certain cytokines locally. We assessed the association between cervical microbiota diversity and the histopathological diagnosis of each stage of CC, and we evaluated mRNA cervical expression levels of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ across the histopathological diagnosis and specific bacterial clusters. We determined the cervical microbiota by high throughput sequencing of 16S rDNA amplicons and classified it in community state types (CST). Mean difference analyses between alpha-diversity and histopathological diagnosis were carried out, as well as a β-diversity analysis within the histological diagnosis. Cervical cytokine mRNA expression was analyzed across the CSTs and the histopathological diagnoses. We found a significant difference in microbiotas diversity in NCL-HPV negative women vs those with squamous intraepithelial lesions (SIL) and CC(p = 0.006, p = 0.036).When β-diversity was evaluated, the CC samples showed the highest variation within groups (p<0.0006) and the largest distance compared to NCL-HPV negative ones (p<0.00001). The predominant bacteria in women with normal cytology were L. crispatus and L. iners, whereas for SIL, it was Sneathia spp. and for CC, Fusobacterium spp. We found higher median cervical levels of IL-4 and TGF-β1 mRNA in the CST dominated by Fusobacterium spp. These results suggest that the cervical microbiota may be implicated in cervical cancer pathology. Further cohort studies are needed to validate these findings.

Transcriptome of the adult female malaria mosquito vector Anopheles albimanus

BackgroundHuman Malaria is transmitted by mosquitoes of the genus Anopheles. Transmission is a complex phenomenon involving biological and environmental factors of humans, parasites and mosquitoes. Among more than 500 anopheline species, only a few species from different branches of the mosquito evolutionary tree transmit malaria, suggesting that their vectorial capacity has evolved independently. Anopheles albimanus (subgenus Nyssorhynchus) is an important malaria vector in the Americas. The divergence time between Anopheles gambiae, the main malaria vector in Africa, and the Neotropical vectors has been estimated to be 100 My. To better understand the biological basis of malaria transmission and to develop novel and effective means of vector control, there is a need to explore the mosquito biology beyond the An. gambiae complex.ResultsWe sequenced the transcriptome of the An. albimanus adult female. By combining Sanger, 454 and Illumina sequences from cDNA libraries derived from the midgut, cuticular fat body, dorsal vessel, salivary gland and whole body, we generated a single, high-quality assembly containing 16,669 transcripts, 92% of which mapped to the An. darlingi genome and covered 90% of the core eukaryotic genome. Bidirectional comparisons between the An. gambiae, An. darlingi and An. albimanus predicted proteomes allowed the identification of 3,772 putative orthologs. More than half of the transcripts had a match to proteins in other insect vectors and had an InterPro annotation. We identified several protein families that may be relevant to the study of Plasmodium-mosquito interaction. An open source transcript annotation browser called GDAV (Genome-Delinked Annotation Viewer) was developed to facilitate public access to the data generated by this and future transcriptome projects.ConclusionsWe have explored the adult female transcriptome of one important New World malaria vector, An. albimanus. We identified protein-coding transcripts involved in biological processes that may be relevant to the Plasmodium lifecycle and can serve as the starting point for searching targets for novel control strategies. Our data increase the available genomic information regarding An. albimanus several hundred-fold, and will facilitate molecular research in medical entomology, evolutionary biology, genomics and proteomics of anopheline mosquito vectors. The data reported in this manuscript is accessible to the community via the VectorBase website (http://www.vectorbase.org/Other/AdditionalOrganisms/).

The effect of nitric oxide and hydrogen peroxide in the activation of the systemic immune response of Anopheles albimanus infected with Plasmodium berghei

The expression of genes encoding the antimicrobial peptides (AMPs) attacin, cecropin and gambicin, as well as the effects of NO and H(2)O(2) on their expression was investigated in midguts and fat bodies of Anopheles albimanus during the midgut infection with Plasmodium berghei. Midgut infection induced an increase in the expression of the three AMPs in both tissues; while NO and H(2)O(2) were present in haemolymph. Treatment with L-NAME and vitamin C reduced the effect of P. berghei infection on the AMPs expression, and exogenous NO and H(2)O(2) induced their expression in the mosquito fat body. The induction of AMPs in abdominal tissues, while the malaria parasites are in the mosquito midgut, suggests communication between the midgut epithelial cells and the abdominal tissue which has not yet had direct contact with the parasites. Free radical production in mosquito midgut and haemolymph during Plasmodium infection and their inductive effect on AMPs in abdominal tissues indicates the possible participation of these radicals in mediating a systemic immune response in this mosquito.

Development of a Multiplex-PCR probe system for the proper identification of Klebsiella variicola

BackgroundKlebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.ResultThis work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.ConclusionsThis multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.

A bioinformatics approach for integrated transcriptomic and proteomic comparative analyses of model and non-sequenced anopheline vectors of human malaria parasites

Malaria morbidity and mortality caused by both Plasmodium falciparum and Plasmodium vivax extend well beyond the African continent, and although P. vivax causes between 80 and 300 million severe cases each year, vivax transmission remains poorly understood. Plasmodium parasites are transmitted by Anopheles mosquitoes, and the critical site of interaction between parasite and host is at the mosquitos luminal midgut brush border. Although the genome of the “model” African P. falciparum vector, Anopheles gambiae, has been sequenced, evolutionary divergence limits its utility as a reference across anophelines, especially non-sequenced P. vivax vectors such as Anopheles albimanus. Clearly, technologies and platforms that bridge this substantial scientific gap are required in order to provide public health scientists with key transcriptomic and proteomic information that could spur the development of novel interventions to combat this disease. To our knowledge, no approaches have been published that address this issue. To bolster our understanding of P. vivax–An. albimanus midgut interactions, we developed an integrated bioinformatic-hybrid RNA-Seq-LC-MS/MS approach involving An. albimanus transcriptome (15,764 contigs) and luminal midgut subproteome (9,445 proteins) assembly, which, when used with our custom Diptera protein database (685,078 sequences), facilitated a comparative proteomic analysis of the midgut brush borders of two important malaria vectors, An. gambiae and An. albimanus.

CD3ζ Expression and T Cell Proliferation are Inhibited by TGF-β1 and IL-10 in Cervical Cancer Patients

IntroductionCervical cancer development from a squamous intraepithelial lesion is thought to be favored by an impaired T cell immunity. We evaluated parameters of T cell alterations such as proliferation, cytokine, and CD3ζ expression in peripheral blood and tumor-infiltrating T lymphocytes from women with squamous intraepithelial lesions (SIL) or cervical cancer (CC).Results and DiscussionT cell proliferation and cytokine messenger RNA (mRNA) expression were similar in women with SIL and healthy donors, whereas low T cell proliferation and lower mRNA expression of IL-2, IL-10 and IFN-γ were observed in women with CC. Moreover, infiltrating cells showed marginal responses. We also found that CD3ζ mRNA expression, whose protein is required for T cell activation, correlated with a decreased proliferation in advanced stages of the disease.ConclusionExperiments with T cells from healthy donors in the presence TGF-β1 or IL-10 suggest that these cytokines have a relevant role in T cell responses during CC progression.

Reconstructing and mining the B cell repertoire with ImmunediveRsity.

The B cell antigen receptor repertoire is highly diverse and constantly modified by clonal selection. High-throughput DNA sequencing (HTS) of the lymphocyte repertoire (Rep-Seq) represents a promising technology to explore such diversity ex-vivo and assist in the identification of antigen-specific antibodies based on molecular signatures of clonal selection. Therefore, integrative tools for repertoire reconstruction and analysis from antibody sequences are needed. We developed ImmunediveRity, a stand-alone pipeline primarily based in R programming for the integral analysis of B cell repertoire data generated by HTS. The pipeline integrates GNU software and in house scripts to perform quality filtering, sequencing noise correction and repertoire reconstruction based on V, D and J segment assignment, clonal origin and unique heavy chain identification. Post-analysis scripts generate a wealth of repertoire metrics that in conjunction with a rich graphical output facilitates sample comparison and repertoire mining. Its performance was tested with raw and curated human and mouse 454-Roche sequencing benchmarks providing good approximations of repertoire structure. Furthermore, ImmunediveRsity was used to mine the B cell repertoire of immunized mice with a model antigen, allowing the identification of previously validated antigen-specific antibodies, and revealing different and unexpected clonal diversity patterns in the post-immunization IgM and IgG compartments. Although ImmunediveRsity is similar to other recently developed tools, it offers significant advantages that facilitate repertoire analysis and repertoire mining. ImmunediveRsity is open source and free for academic purposes and it runs on 64 bit GNU/Linux and MacOS. Available at: https://bitbucket.org/ImmunediveRsity/immunediversity/

Molecular epidemiology of Plasmodium vivax in Latin America: polymorphism and evolutionary relationships of the circumsporozoite gene.

BackgroundThe origins and dispersal of Plasmodium vivax to its current worldwide distribution remains controversial. Although progress on P. vivax genetics and genomics has been achieved worldwide, information concerning New World parasites remains fragmented and largely incomplete. More information on the genetic diversity in Latin America (LA) is needed to better explain current patterns of parasite dispersion and evolution.MethodsPlasmodium vivax circumsporozoite protein gene polymorphism was investigated using polymerase chain reaction amplification and restriction fragment length polymorphism (PCR-RFLP), and Sanger sequencing in isolates from the Pacific Ocean coast of Mexico, Nicaragua, and Peru. In conjunction with worldwide sequences retrieved from the Genbank, mismatch distribution analysis of central repeat region (CRR), frequency estimation of unique repeat types and phylogenetic analysis of the 3′ terminal region, were performed to obtain an integrative view of the genetic relationships between regional and worldwide isolates.ResultsFour RFLP subtypes, vk210a, b, c and d were identified in Southern Mexico and three subtypes vk210a, e and f in Nicaragua. The nucleotide sequences showed that Mexican vk210a and all Nicaraguan isolates were similar to other American parasites. In contrast, vk210b, c and d were less frequent, had a domain ANKKAEDA in their carboxyl end and clustered with Asian isolates. All vk247 isolates from Mexico and Peru had identical RFLP pattern. Their nucleotide sequences showed two copies of GGQAA GGNAANKKAGDAGA at the carboxyl end. Differences in mismatch distribution parameters of the CRR separate vk247 from most vk210 isolates. While vk247 isolates display a homogeneous pattern with no geographical clustering, vk210 isolates display a heterogeneous geographically clustered pattern which clearly separates LA from non-American isolates, except vk210b, c and d from Southern Mexico.ConclusionsThe presence of vk210a in Mexico and vk210e, f and g in Nicaragua are consistent with other previously reported LA isolates and reflect their circulation throughout the continent. The vk210b, c and d are novel genotypes in LA. Their genetic relationships and low variability within these vk210 and/or within the vk247 parasites in Southern Mexico suggest its recent introduction and/or recent expansion to this region. The global analysis of P. vivax csp suggests this parasite introduction to the region and likely LA by different independent events.

Sequence variation of ookinete surface proteins Pvs25 and Pvs28 of Plasmodium vivax isolates from Southern Mexico and their association to local anophelines infectivity

The polymorphism of Pvs25 and Pvs28 ookinete surface proteins, their association to circumsporozoite protein repeat (CSPr) genotypes (Vk210 and Vk247) and their infectivity to local Anopheles albimanus and Anopheles pseudopunctipennis were investigated in Plasmodium vivax-infected blood samples obtained from patients in Southern Mexico. The pvs25 and pvs28 complete genes were amplified, cloned and sequenced; and the CSPr genotype was determined by PCR amplification and hybridization. The amino acid Pvs25 and Pvs28 polymorphisms were mapped to their corresponding protein structure. Infected blood samples were simultaneously provided through artificial feeders to both mosquito species; the ratio of infected mosquitoes and oocyst numbers were recorded. The polymorphism of pvs25 and pvs28 was limited to few nucleotide positions, and produced three haplotypes: type A/A parasites presented Pvs25 and Pvs28 amino acid sequences identical to that of Sal I reference strain; parasites type B1 presented a mutation 130 Ile-->Thr in Pvs25, while type B2 presented 87 Gln-->Lys/130 Ile-->Thr in the same molecule. Both types B1 and B2 parasites presented changes in Pvs28 at 87 Asn-->Asp, 110 Tyr-->Asn and five GSGGE/D repeat sequences between the fourth EGF-like domain and the GPI. Most P. vivaxparasites from the coastal plains and the overlapping region were Pvs25/28 A/A, CSPrVk210 and were infective only to An. albimanus (p< or =0.0001). Parasites originating in foothills were Pvs25/28 type B1/B or B2/B and CSPrVk210 or Vk247, and were more infective to An. pseudopunctipennis than to An. albimanus (p< or =0.001). These results and the analysis of Pvs25/28 from other parts of the world indicated that non-synonymous variations in these proteins occur in amino acid residues exposed on the surface of the proteins, and are likely to interact with midgut mosquito ligands. We hypothesize that these molecules have been shaped by co-evolutionary adaptations of parasites to their susceptible vectors.