BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

Abstract Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major EGFP or L. major EGFP-LUC ) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel. Disease progression was followed by footpad swelling measurements and parasite burden in draining lymph nodes using microtitration assay and real-time PCR, and immune responses were also evaluated in spleen. EGFP-expressing parasites generated larger swellings in comparison with wild-type (L. major) while mice immunized with rEGFP and challenged with wild-type parasite were quite comparable in footpad swelling with control group without significant difference. However, both conventional and molecular approaches revealed no significant difference in parasite load between different groups. More importantly, no significant inflammatory responses were detected in groups with higher swelling size measured by interferon-γ (IFN-γ), interleukin (IL)-10, IL-5, and nitric oxide against frozen and thawed lysate of parasite as stimulator. Altogether, these results clearly revealed that EGFP protein expressed in prokaryotic and eukaryotic hosts is not an immunological reactive molecule and acts as a neutral protein without any side effects in mice. So, EGFP expressing Leishmania could be a safe and reliable substitution for wildtypes that simplifies in situ follow-up and eliminates the animal scarification wherever needed during the study

Prevalence, years lived with disability, and trends in anaemia burden by severity and cause, 1990-2021: findings from the Global Burden of Disease Study 2021

Background Anaemia is a major health problem worldwide. Global estimates of anaemia burden are crucial for developing appropriate interventions to meet current international targets for disease mitigation. We describe the prevalence, years lived with disability, and trends of anaemia and its underlying causes in 204 countries and territories. Methods We estimated population-level distributions of haemoglobin concentration by age and sex for each location from 1990 to 2021. We then calculated anaemia burden by severity and associated years lived with disability (YLDs). With data on prevalence of the causes of anaemia and associated cause-specific shifts in haemoglobin concentrations, we modelled the proportion of anaemia attributed to 37 underlying causes for all locations, years, and demographics in the Global Burden of Disease Study 2021. Findings In 2021, the global prevalence of anaemia across all ages was 24·3% (95% uncertainty interval [UI] 23·9–24·7), corresponding to 1·92 billion (1·89–1·95) prevalent cases, compared with a prevalence of 28·2% (27·8–28·5) and 1·50 billion (1·48–1·52) prevalent cases in 1990. Large variations were observed in anaemia burden by age, sex, and geography, with children younger than 5 years, women, and countries in sub-Saharan Africa and south Asia being particularly affected. Anaemia caused 52·0 million (35·1–75·1) YLDs in 2021, and the YLD rate due to anaemia declined with increasing Socio-demographic Index. The most common causes of anaemia YLDs in 2021 were dietary iron deficiency (cause-specific anaemia YLD rate per 100 000 population: 422·4 [95% UI 286·1–612·9]), haemoglobinopathies and haemolytic anaemias (89·0 [58·2–123·7]), and other neglected tropical diseases (36·3 [24·4–52·8]), collectively accounting for 84·7% (84·1–85·2) of anaemia YLDs. Interpretation Anaemia remains a substantial global health challenge, with persistent disparities according to age, sex, and geography. Estimates of cause-specific anaemia burden can be used to design locally relevant health interventions aimed at improving anaemia management and prevention. Funding Bill & Melinda Gates Foundation

Antibiotic-Free Nanoplasmids as Promising Alternatives for Conventional DNA Vectors

DNA vaccines with their extraordinary properties are the best choice as vectors for subunit vaccines but are not in compliance with safety regulations, mainly because of the antibiotic resistance genes on their backbone. New generations of plasmids with minimum bacterial backbones are now developed as promising alternatives to pass the safety rules and be replaced for conventional plasmids. Here we have compared the nanoplasmid (with RNA-out selection system and professional HTLV-1 containing promoter) and the conventionally used pcDNA plasmid, as regards the transfection efficiency. The EGFP gene was cloned in both pcDNA-3.1+ and NTC9385R-MSC and transfected into COS-7 cells for expression evaluation by flow cytometry. Meanwhile, qPCR was used to analyze the EGFP mRNA copy numbers. It was concluded that the nanoplasmid, with its extraordinary properties, can be a tempting alternative to conventional pcDNA in equal or equimolar concentrations for vaccine design. These promising results can put DNA vaccines back into focus, especially regarding diseases controlled by robust cellular immune responses

Assessment of genetic variation and evolutionary history of Caucasian Grouse (Lyrurus mlokosiewiczi)

Caucasian Grouse (Lyrurus mlokosiewiczi) is an endemic species found in the Caucasus whose population is declining. Initial assessment of genetic variation and phylogenetic status of the species confirmed the monophyly of L. mlokosiewiczias and indicated a sister relationship between L. mlokosiewiczi and L. tetrix (Black grouse). Further the Caucasian grouse from Georgia, Caucasus and Iran created three genetic groups with no shared haplotype. This separation could be the result of different evolutionary events or geographic distances between them. Four different haplotypes were identified in north-western Iran, distributed inside and outside Arasbaran protected area (APA), suggesting the expansion of APA to include Caucasian grouse habitats in the Kalibar Mountains (western APA) and enhance the protection of the species in the region

Isolation, characterization, and functional study of extracellular vesicles derived from Leishmania tarentolae

Leishmania (L.) species are protozoan parasites with a complex life cycle consisting of a number of developmental forms that alternate between the sand fly vector and their host. The non-pathogenic species L. tarentolae is not able to induce an active infection in a human host. It has been observed that, in pathogenic species, extracellular vesicles (EVs) could exacerbate the infection. However, so far, there is no report on the identification, isolation, and characterization of L. tarentolae EVs. In this study, we have isolated and characterized EVs from L. tarentolaeGFP+ (tEVs) along with L. majorGFP+ as a reference and positive control. The EVs secreted by these two species demonstrated similar particle size distribution (approximately 200 nm) in scanning electron microscopy and nanoparticle tracking analysis. Moreover, the said EVs showed similar protein content, and GFP and GP63 proteins were detected in both using dot blot analysis. Furthermore, we could detect Leishmania-derived GP63 protein in THP-1 cells treated with tEVs. Interestingly, we observed a significant increase in the production of IFN-γ, TNF-α, and IL-1β, while there were no significant differences in IL-6 levels in THP-1 cells treated with tEVs following an infection with L. major compared with another group of macrophages that were treated with L. major EVs prior to the infection. Another exciting observation of this study was a significant decrease in parasite load in tEV-treated Leishmania-infected macrophages. In addition, in comparison with another group of Leishmania-infected macrophages which was not exposed to any EVs, tEV managed to increase IFN-γ and decrease IL-6 and the parasite burden. In conclusion, we report for the first time that L. tarentolae can release EVs and provide evidence that tEVs are able to control the infection in human macrophages, making them a great potential platform for drug delivery, at least for parasitic infections.ISSN:2235-298

Schematic presentation of sampling lesions with tape strip discs, DNA isolation and further identification of <i>Leishmania</i> species.

<p>1) patient´s lesion 2) disc 3) disc applied to patient´s lesion 4) even pressure applied on lesion using a plunger 5) sterile removal of disc from the lesion 6) DNA isolation from disc 7) confirmation of <i>Leishmania</i> by kDNA1 8) species-specific identification (PCR ITS-RFLP).</p

Sensitive areas for sampling.

<p>A) eyelid B) around the eye C) ear D) on the lips.</p

Diagnostic accuracy of parasite culture from biopsies and tape strip disc.

<p>Diagnostic accuracy of parasite culture from biopsies and tape strip disc.</p

Disease duration, gender distribution and infecting species of <i>Leishmania</i> in CL patients with acute, chronic or healed disease.

<p>Disease duration, gender distribution and infecting species of <i>Leishmania</i> in CL patients with acute, chronic or healed disease.</p

Clinical profiles of 119 cutaneous leishmaniasis patients.

<p>Clinical profiles of 119 cutaneous leishmaniasis patients.</p