Integrative literature review of the reported uses of serological tests in leprosy management

Abstract: An integrative literature review was conducted to synthesize available publications regarding the potential use of serological tests in leprosy programs. We searched the databases Literatura Latino-Americana e do Caribe em Ciências da Saúde, Índice Bibliográfico Espanhol em Ciências da Saúde, Acervo da Biblioteca da Organização Pan-Americana da Saúde, Medical Literature Analysis and Retrieval System Online, Hanseníase, National Library of Medicine, Scopus, Ovid, Cinahl, and Web of Science for articles investigating the use of serological tests for antibodies against phenolic glycolipid-I (PGL-I), ML0405, ML2331, leprosy IDRI diagnostic-1 (LID-1), and natural disaccharide octyl-leprosy IDRI diagnostic-1 (NDO-LID). From an initial pool of 3.514 articles, 40 full-length articles fulfilled our inclusion criteria. Based on these papers, we concluded that these antibodies can be used to assist in diagnosing leprosy, detecting neuritis, monitoring therapeutic efficacy, and monitoring household contacts or at-risk populations in leprosy-endemic areas. Thus, available data suggest that serological tests could contribute substantially to leprosy management.

Phage display as a tool for generation of bio-therapeutic agents for breast cancer

Notch is a conserved signalling pathway, which plays a crucial role in a multiple cellular processes such as stem cell self-renewal, cell division, proliferation and apoptosis. In mammalian, four Notch receptors and five ligands are described, where interaction is achieved through their extracellular domains, leading to a transcription activation of different target genes. Increased expression of Notch ligands has been detected in several types of cancer, including breast cancer suggesting that these proteins represent possible therapeutic targets. The goal of this work was to generate quality protein targets and, by phage display technology, select function-blocking antibodies specific for Notch ligands. Phage display is a powerful technique that allows the generation of highly specific antibodies to be used for therapeutics, and it has also proved to be a reliable approach in identifying and validating new cancer-related targets. Also, we aimed at solving the tri-dimensional structure of the Notch ligands alone and in complex with selected antibodies. In this work, the initial phase focused on the optimization of the expression and purification of a human Delta-like 1 ligand mutant construct (hDLL1-DE3), by refolding from E. coli inclusion bodies. To confirm the biological activity of the produced recombinant protein cellular functional studies were performed, revealing that treatment with hDLL1-DE3 protein led to a modulation of Notch target genes. In a second stage of this study, Antibody fragments (Fabs) specific for hDLL1-DE3 were generated by phage display, using the produced protein as target, in which one good Fab candidate was selected to determine the best expression conditions. In parallel, multiple crystallization conditions were tested with hDLL1-DE3, but so far none led to positive results.

Epidermis recreation in spongy-like hydrogels: New opportunities to explore epidermis-like analogues

[Excerpt] On the road to successfully achieving skin regeneration, 3D matrices/scaffolds that provide the adequate physico-chemical and biological cues to recreate the ideal healing environment are believed to be a key element [1], [2] and [3]. Numerous polymeric matrices derived from both natural [4] and [5] and synthetic [6], [7] and [8] sources have been used as cellular supports; nowadays, fewer matrices are simple carriers, and more and more are ECM analogues that can actively participate in the healing process. Therefore, the attractive characteristics of hydrogels, such as high water content, tunable elasticity and facilitated mass transportation, have made them excellent materials to mimic cells’ native environment [9]. Moreover, their hygroscopic nature [10] and possibility of attaining soft tissues-like mechanical properties mean they have potential for exploitation as wound healing promoters [11], [12], [13] and [14]. Nonetheless, hydrogels lack natural cell adhesion sites [15], which limits the maximization of their potential in the recreation of the cell niche. This issue has been tackled through the use of a range of sophisticated approaches to decorate the hydrogels with adhesion sequences such as arginine-glycine-aspartic acid (RGD) derived from fibronectin [16], [17] and [18], and tyrosine-isoleucine-glycine-serine-arginine (YIGSR) derived from laminin [18] and [19], which not only aim to modulate cell adhesion, but also influencing cell fate and survival [18]. Nonetheless, its widespread use is still limited by significant costs associated with the use of recombinant bioactive molecules.

Ashbya gossypii beyond industrial riboflavin production: a historical perspective and emerging biotechnological applications

The filamentous fungus Ashbya gossypii has been safely and successfully used for more than two decades in the commercial production of riboflavin (vitamin B2). Its industrial relevance combined with its high genetic similarity with Saccharomyces cerevisiae together promoted the accumulation of fundamental knowledge that has been efficiently converted into a significant molecular and in silico toolbox for its genetic engineering. This synergy has enabled a directed and sustained exploitation of A. gossypii as an industrial riboflavin producer. Although there is still room for optimizing riboflavin production, the recent years have seen an abundant advance in the exploration of A. gossypii for other biotechnological applications, such as the production of recombinant proteins, single cell oil and flavour compounds. Here, we will address the biotechnological potential of A. gossypii beyond riboflavin production by presenting (a) a physiological and metabolic perspective over this fungus; (b) the molecular toolbox available for its manipulation; and (c) commercial and emerging biotechnological applications for this industrially important fungus, together with the approaches adopted for its engineering.

Analysis of a Local HIV-1 Epidemic in Portugal Highlights Established Transmission of non-B and -G Subtypes

Existing data supports Portugal as the Western Europe country with highest HIV-1 subtype diversity. However, detailed phylogenetic studies of Portuguese HIV-1 epidemics are still scarce. Thus, our main goal was to analyze the phylodynamics of a local HIV-1 infection in the Portuguese region of Minho. Molecular epidemiological analysis was applied to data from 289 HIV-1 infected individuals followed in the reference Hospital of the province of Minho, Portugal, in which isolated viruses had been sequenced between 2000 and 2012. Viruses of the G (29.1%) and B (27.0%) subtypes were the most frequent, followed by recombinant forms (17.6%), C (14.5%), F1 (7.3%) and A1 (4.2%) subtypes. Multinomial logistic regression revealed that the odds of being infected with A1 and F1 subtype increased over the years when compared with B, G, C or recombinant viruses. As expected, polyphyletic patterns suggesting multiple and old introductions of subtypes B and G were found. However, transmission clusters of non-B and -G viruses among native individuals were also found with the dates of the most recent common ancestor estimated to the early 2000s. Our study supports that the HIV-1 subtype diversity in the Portuguese region of Minho is high and has been increasing in a manner that is apparently driven by factors other than immigration and international travel. Infections with A1 and F1 viruses in the region of Minho are becoming established and were mainly found in sexually transmitted clusters, reinforcing the need for more efficacious control measures targeting this infection route.

Clivagem e caracterização de frutalina recombinante produzida em Escherichia coli

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Expressão e caracterização de uma lectina recombinante de interesse biomédico

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Development of elastin-like recombinamer films with antimicrobial activity

In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.

Exploring silk based biomaterials functionalized with antimicrobial peptides to prevent surgical site infections

Surgical site infections (SSI) often occur after invasive surgery, which is as a serious health problem, making it important to develop new biomaterials to prevent infections. Spider silk is a natural biomaterial with excellent biocompatibility, low immunogenicity and controllable biodegradability. Through recombinant DNA technology, spider silk-based materials can be bioengineered and functionalized with antimicrobial (AM) peptides 1. The aim of this study is to develop new materials by combining spider silk chimeric proteins with AM properties and silk fibroin extracted from Bombyx mori cocoons to prevent microbial infection. Here, spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6 mer and 15 mer) were fused with the AM peptides Hepcidin and Human Neutrophil peptide 1 (HNP1). The spider silk domain maintained its self-assembly features allowing the formation of beta-sheets to lock in structures without any chemical cross-linking. The AM properties of the developed chimeric proteins showed that 6 mer + HNP1 protein had a broad microbicidal activity against pathogens. The 6 mer + HNP-1 protein was then assembled with different percentages of silk fibroin into multifunctional films. In vitro cell studies with a human fibroblasts cell line (MRC5) showed nontoxic and cytocompatible behavior of the films. The positive cellular response, together with structural properties, suggests that this new fusion protein plus silk fibroin may be good candidates as multifunctional materials to prevent SSI.

Clonació del gen de l'envolta de vih en el plàsmid pnl Δenv-Ren per la obtenció de recombinants

Estudi elaborat a partir d’una estada al Laboratori de Inmunopatología del SIDA del Dr Alcamí a l’Instituto de Salud Carlos III-Centro Nacional de Microbiologia, entre finals de desembre de 2006 i març de 2007. L’objectiu ha estat millorar la caracterització de l’envolta del VIH-1 mitjançant l’obtenció de virus recombinants, ja que això permet estudiar l’envolta viral tant genètica com fenotípicament. En aquest cas, s’ha estudiat l'envolta viral dels pacients sotmesos a vacunació terapèutica amb cèl•lules dendrítiques polsades amb virus autòlegs. Durant aquesta estada es realitza un aprenentatge profund de les tècniques adequades per a l'amplificació i clonatge del gen complet de l'envolta del VIH-1 (env), així com de l’obtenció de virus recombinants amb l’envolta del pacient i els corresponents assaigs de tropisme viral i neutralització sèrica. Aquesta metodologia empra el virus quimèric pNL4.3 delta_env Renilla, construït a partir del virus de referència NL4.3 i que té dues característiques importants: la primera és que conté un gen marcador Renilla, que a l’interior de les cèl•lules infectades té activitat luciferasa. La utilització del virus pNL4.3 delta_env Renilla en assaigs de neutralització presenta diversos avantatges front altres assaigs més convencionals, tant a nivell de sensibilitat i especificitat com d’estalvi de temps.

Cultured human fetal astrocytes can be induced by interferon-gamma to express HLA-DR.

Interferon-gamma (IFN-gamma) modulates the expression of Class II major histocompatibility antigens (MHC), thus providing a potential regulatory mechanism for local immune reactivity in the context of MHC-restricted antigen presentation. Within the central nervous system (CNS), the expression of MHC Class II antigens has been demonstrated on human reactive astrocytes and glioma cells. In order to investigate the modulation of HLA-DR on normal astrocytes, two cell lines were grown from a 20-week-old fetal brain. In situ none of the fetal brain cells expressed HLA-DR as determined by immunohistology on frozen tissue sections. The two cell lines, FB I and FB II, expressed GFAP indicating their astrocytic origin. FB I was HLA-DR negative at the first tissue culture passages, but could be induced to express HLA-DR when treated with 500 U/ml IFN-gamma. FB II was spontaneously HLA-DR positive in the early passages, lost the expression of this antigen after 11 passages and could also be induced to express HLA-DR by IFN-gamma. The induction of HLA-DR expression was demonstrated both by a binding RIA and by immunoprecipitation using a monoclonal antibody (MAB) directed against a monomorphic determinant of HLA-DR. The HLA-DR alloantigens were determined on FB II cells after IFN-gamma treatment, by immunofluorescence and by cytotoxicity assays, and were shown to be DR4, DR6, Drw52, DRw53 and DQwl. These results show that human fetal astrocytes can be induced to express HLA-DR by IFN-gamma in vitro and support the concept that astrocytes may function as antigen-presenting cells.

Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages.

Organs developing as appendages of the ectoderm are initiated from epithelial thickenings called placodes. Their formation is regulated by interactions between the ectoderm and underlying mesenchyme, and several signalling molecules have been implicated as activators or inhibitors of placode formation. Ectodysplasin (Eda) is a unique signalling molecule in the tumour necrosis factor family that, together with its receptor Edar, is necessary for normal development of ectodermal organs both in humans and mice. We have shown previously that overexpression of the Eda-A1 isoform in transgenic mice stimulates the formation of several ectodermal organs. In the present study, we have analysed the formation and morphology of placodes using in vivo and in vitro models in which both the timing and amount of Eda-A1 applied could be varied. The hair and tooth placodes of K14-Eda-A1 transgenic embryos were enlarged, and extra placodes developed from the dental lamina and mammary line. Exposure of embryonic skin to Eda-A1 recombinant protein in vitro stimulated the growth and fusion of placodes. However, it did not accelerate the initiation of the first wave of hair follicles giving rise to the guard hairs. Hence, the function of Eda-A1 appears to be downstream of the primary inductive signal required for placode initiation during skin patterning. Analysis of BrdU incorporation indicated that the formation of the epithelial thickening in early placodes does not involve increased cell proliferation and also that the positive effect of Eda-A1 on placode expansion is not a result of increased cell proliferation. Taken together, our results suggest that Eda-A1 signalling promotes placodal cell fate during early development of ectodermal organs.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study.

BACKGROUND & AIMS: Hepatitis C virus (HCV) induces chronic infection in 50% to 80% of infected persons; approximately 50% of these do not respond to therapy. We performed a genome-wide association study to screen for host genetic determinants of HCV persistence and response to therapy. METHODS: The analysis included 1362 individuals: 1015 with chronic hepatitis C and 347 who spontaneously cleared the virus (448 were coinfected with human immunodeficiency virus [HIV]). Responses to pegylated interferon alfa and ribavirin were assessed in 465 individuals. Associations between more than 500,000 single nucleotide polymorphisms (SNPs) and outcomes were assessed by multivariate logistic regression. RESULTS: Chronic hepatitis C was associated with SNPs in the IL28B locus, which encodes the antiviral cytokine interferon lambda. The rs8099917 minor allele was associated with progression to chronic HCV infection (odds ratio [OR], 2.31; 95% confidence interval [CI], 1.74-3.06; P = 6.07 x 10(-9)). The association was observed in HCV mono-infected (OR, 2.49; 95% CI, 1.64-3.79; P = 1.96 x 10(-5)) and HCV/HIV coinfected individuals (OR, 2.16; 95% CI, 1.47-3.18; P = 8.24 x 10(-5)). rs8099917 was also associated with failure to respond to therapy (OR, 5.19; 95% CI, 2.90-9.30; P = 3.11 x 10(-8)), with the strongest effects in patients with HCV genotype 1 or 4. This risk allele was identified in 24% of individuals with spontaneous HCV clearance, 32% of chronically infected patients who responded to therapy, and 58% who did not respond (P = 3.2 x 10(-10)). Resequencing of IL28B identified distinct haplotypes that were associated with the clinical phenotype. CONCLUSIONS: The association of the IL28B locus with natural and treatment-associated control of HCV indicates the importance of innate immunity and interferon lambda in the pathogenesis of HCV infection.