Statistical Experimental Designs for cLTB-Syn Vaccine Production Using Daucus carota Cell Suspension Cultures.

The carrot-made LTB-Syn antigen (cLTB-Syn) is a vaccine candidate against synucleinopathies based on carrot cells expressing the target antigen LTB and syn epitopes. Therefore, the development of an efficient production process is required with media culture optimization to increase the production yields as the main goal. In this study, the effect of two nitrogen sources (urea and glutamate) on callus cultures producing cLTB-Syn was studied, observing that the addition of 17 mM urea to MS medium favored the biomass yield. To optimize the MS media composition, the influence of seven medium components on biomass and cLTB-Syn production was first evaluated by a Plackett-Burman design (PBD). Then, three factors were further analyzed using a central composite design (CCD) and response surface methodology (RSM). The results showed a 1.2-fold improvement in biomass, and a 4.5-fold improvement in cLTB-Syn production was achieved at the shake-flask scale. At the bioreactor scale, there was a 1.5-fold increase in biomass and a 2.8-fold increase in cLTB-Syn yield compared with the standard MS medium. Moreover, the cLTB-Syn vaccine induced humoral responses in BALB/c mice subjected to either oral or subcutaneous immunization. Therefore, cLTB-Syn is a promising vaccine candidate that will aid in developing immunotherapeutic strategies to combat PD and other neurodegenerative diseases without the need for cold storage, making it a financially viable option for massive immunization.

Congenital Juvenile Xanthogranuloma in the Perioral Region: A Case Image.

Juvenile xanthogranuloma (JXG) is the most common form of non-Langerhans cell histiocytosis in childhood. It often presents with cutaneous involvement and exhibits a predilection for the head and neck region. This article illustrates a case of congenital JXG in a 5-month-old boy, characterized by a solitary, well-circumscribed nodule above the left upper lip. Histopathologically, the lesion exhibited histiocytes with eosinophilic cytoplasm and Touton giant cells. Immunohistochemistry revealed histiocytes positive for CD68 and Factor XIIIa, while negative for S-100 protein. Clinicians should become familiar with the broad clinical spectrum of cutaneous JXG, particularly its congenital presentation, in order to ensure timely and accurate management.

Production and Immunogenicity Assessment of LTp50: An Escherichia coli-Made Chimeric Antigen Targeting S1- and S2-Epitopes from the SARS-CoV-2/BA.5 Spike Protein.

Subunit vaccines stand as a leading approach to expanding the current portfolio of vaccines to fight against COVID-19, seeking not only to lower costs but to achieve long-term immunity against variants of concern and have the main attributes that could overcome the limitations of the current vaccines. Herein a chimeric protein targeting S1 and S2 epitopes, called LTp50, was designed as a convenient approach to induce humoral responses against SARS-CoV-2. LTp50 was produced in recombinant Escherichia coli using a conventional pET vector, recovering the expected antigen in the insoluble fraction. LTp50 was purified by chromatography (purity > 90%). The solubilization and refolding stages helped to obtain a stable protein amenable for vaccine formulation. LTp50 was adsorbed onto alum, resulting in a stable formulation whose immunogenic properties were assessed in BALB/c mice. Significant humoral responses against the S protein (BA.5 variant) were detected in mice subjected to three subcutaneous doses (10 µg) of the LTp50/alum formulation. This study opens the path for the vaccine formulation optimization using additional adjuvants to advance in the development of a highly effective anti-COVID-19 vaccine directed against the antigenic regions of the S protein, which are less prone to mutations.

Establishment of the Daucus carota SMC-1 Cell Suspension Line for Poliovirus Vaccine Development.

The development of virus-free, oral vaccines against poliovirus capable of inducing mucosal protective immunity is needed to safely combat this pathogen. In the present study, a carrot cell line expressing the poliovirus VP2 antigen was established at the level of callus and cell suspensions, exploring the effects of culture media (MS and B5), supplementation with urea, phytoregulators (2,4-D : KIN), and light conditions (continuous light, photoperiod, and total darkness). The best callus growth was obtained on B5 medium supplemented with 2 mg/L of 2,4-D + 2 mg/L kinetin and 0.0136 g/L of urea and in continuous light conditions. Suspension cultures of the SMC-1 line in 250 mL Erlenmeyer flasks had a maximum growth of 16.07 ± 0.03 g/L DW on day 12 with a growth rate of µ=0.3/d and a doubling time of 2.3 days. In a 2 L airlift bioreactor, the biomass yield achieved was 25.6 ± 0.05 g/L DW at day 10 with a growth rate of µ= 0.58/d and doubling time of 1.38 d. Cell growth was 1.5 times higher in bioreactors than in shake flasks, highlighting that both systems resulted in the accumulation of VP2 throughout the time in culture. The maximum VP2 yield in flasks was 387.8 µg/g DW at day 21, while in the reactor it was 550.2 µg/g DW at day 18. In conclusion, bioreactor-based production of the VP2 protein by the SMC-1 suspension cell line offers a higher productivity when compared to flask cultures, offering a key perspective to produce low-cost vaccines against poliomyelitis.

Frailty syndrome and end-stage kidney disease outcomes at a Latin American dialysis center

Introduction. Frailty syndrome generates a high risk of adverse outcomes and mortality, and its prevalence is elevated in patients with end-stage kidney disease. Few studies have reported the prevalence and outcomes of frailty in populations from less developed countries. Objective. To identify the clinical outcomes and factors associated with the frailty syndrome in patients with stage five chronic kidney disease who started renal replacement therapy - both hemodialysis and peritoneal dialysis- in a dialysis center in Bucaramanga, Colombia. Materials and methods. This was a prospective study of patients with end-stage kidney disease who initiated dialysis at a center in Colombia and had a twelve-month follow-up. Results. The overall frailty prevalence was 50.47% and two out of three patients older than 65 years had the syndrome. We found significantly higher followup mortality among patients with frailty: odds ratio of 2.95 (CI: 1.07- 8.13; p=0.036) in unadjusted analysis. Conclusions. Literature shows that compared to developed nations, Latin American adults are facing a higher prevalence of chronic diseases, and frailty syndrome is increasing. In this study, according to the FRAIL scale, having a frailty syndrome predicts a higher mortality; hypoalbuminemia and low creatinine levels at the beginning of dialysis could act as predictors of its diagnosis.

Introducción. El síndrome de fragilidad implica un alto riesgo de desenlaces adversos y mortalidad, y tiene una prevalencia elevada en pacientes con enfermedad renal en etapa terminal. Hay pocos estudios que investiguen la prevalencia y los desenlaces de este síndrome de fragilidad en las poblaciones de los países en desarrollo. Objetivo. Identificar los desenlaces clínicos y los factores asociados al síndrome de fragilidad en los pacientes con enfermedad renal crónica en estadio cinco que inician terapia de reemplazo renal -con hemodiálisis o diálisis peritoneal- en un centro de diálisis de Bucaramanga, Colombia. Materiales y métodos. Se trató de un estudio prospectivo de pacientes con enfermedad renal en etapa terminal que iniciaron diálisis en un centro de Colombia y a quienes se les hizo seguimiento durante doce meses. Resultados. La prevalencia global del síndrome de fragilidad fue del 50,47 % y dos de cada tres pacientes mayores de 65 años lo presentaban. Se encontró una mortalidad significativamente mayor entre los pacientes con síndrome de fragilidad: razón de probabilidad de 2,95 (IC:1,07-8,13; p=0,036) en el análisis no ajustado. Conclusiones. La literatura muestra que, en comparación con los países desarrollados, los adultos latinoamericanos presentan una mayor prevalencia de enfermedades crónicas y un aumento progresivo del síndrome de fragilidad. En este estudio, la fragilidad -según la escala FRAIL- predijo una mayor mortalidad. Además, la hipoalbuminemia y los niveles bajos de creatinina al inicio de la diálisis podrían actuar como elementos predictores de su diagnóstico.

Rationale, formative research, and protocol for Calma, Conversa, y Cría: A pilot mindful parenting intervention with Latina women.

Background: Latina mothers' stress is associated with their children's health behaviors and risk for obesity; however, existing pediatric health promotion programs have not focused on maternal stress reduction. Methods: Herein we describe a study design that will examine the acceptability and feasibility of Calma, Conversa, y Cría (CCC) a 6-week mindful parenting intervention designed to reduce stress. We present the results of qualitative research with Latina mothers and experts in Latinx health and mindfulness who provided culturally-relevant feedback on existing mindful parenting strategies to inform the development of CCC. Fifty Latina mothers of children ages 3-11 years will be randomly assigned to CCC or an enhanced usual care health education intervention. Acceptability will be assessed through participant satisfaction surveys and exit interviews. Feasibility will be determined through detailed tracking of recruitment, retention, and attendance rates. A signal regarding any group differences in maternal stress, health-related parenting practices, child diet, child physical activity, and child quality of life will be explored. Discussion: The development of interventions that can reduce maternal stress and risk for obesity in Latinx children is critical to significantly reduce negative health impacts in this underserved population. Our approach includes the identification of effective cultural adaptations that should improve the feasibility and acceptability of mindful parenting strategies in Latinx families, ideally reducing maternal stress and improving parenting behaviors related to child health. If successful, CCC will be examined in a larger efficacy trial involving the measurement of objective biomarkers of children's chronic disease risk.

Frailty syndrome and end-stage kidney disease outcomes at a Latin American dialysis center. / Desenlaces de enfermedad renal crónica y síndrome de fragilidad en un centro de diálisis de Latinoamérica

INTRODUCTION: Frailty syndrome generates a high risk of adverse outcomes and mortality, and its prevalence is elevated in patients with end-stage kidney disease. Few studies have reported the prevalence and outcomes of frailty in populations from less developed countries. OBJECTIVE: To identify the clinical outcomes and factors associated with the frailty syndrome in patients with stage five chronic kidney disease who started renal replacement therapy - both hemodialysis and peritoneal dialysis- in a dialysis center in Bucaramanga, Colombia. MATERIALS AND METHODS: This was a prospective study of patients with end-stage kidney disease who initiated dialysis at a center in Colombia and had a twelve-month follow-up. RESULTS: The overall frailty prevalence was 50.47% and two out of three patients older than 65 years had the syndrome. We found significantly higher followup mortality among patients with frailty: odds ratio of 2.95 (CI: 1.07- 8.13; p=0.036) in unadjusted analysis. CONCLUSIONS: Literature shows that compared to developed nations, Latin American adults are facing a higher prevalence of chronic diseases, and frailty syndrome is raising. In this study, according to the FRAIL scale, having a frailty syndrome predicts a higher mortality; hypoalbuminemia and low creatinine levels at the beginning of dialysis could act as predictors of its diagnosis.

Introduction. Frailty syndrome generates a high risk of adverse outcomes and mortality, and its prevalence is elevated in patients with end-stage kidney disease. Few studies have reported the prevalence and outcomes of frailty in populations from less developed countries.Objective. To identify the clinical outcomes and factors associated with the frailty syndrome in patients with stage five chronic kidney disease who started renal replacement therapy ­both hemodialysis and peritoneal dialysis­ in a dialysis center in Bucaramanga, Colombia.Materials and methods. This was a prospective study of patients with end-stage kidney disease who initiated dialysis at a center in Colombia and had a twelve-month follow-up. Results. The overall frailty prevalence was 50.47% and two out of three patients older than 65 years had the syndrome. We found significantly higher followup mortality among patients with frailty: odds ratio of 2.95 (CI: 1.07- 8.13; p=0.036) in unadjusted analysis.Conclusions. Literature shows that compared to developed nations, Latin American adults are facing a higher prevalence of chronic diseases, and frailty syndrome is increasing. In this study, according to the FRAIL scale, having a frailty syndrome predicts a higher mortality; hypoalbuminemia and low creatinine levels at the beginning of dialysis could act as predictors of its diagnosis.

Adolescent attendance at transcultural psychotherapy: a retrospective cohort study.

Migrant adolescents in therapy for psychological problems are at risk of poor attendance or even of dropping out. Transcultural psychotherapy has been developed in France to take cultural diversity into account in psychological treatment and to deal with the specific difficulties encountered in the psychotherapeutic treatment of this population. This study aims to assess adolescents' attendance rates to this form of psychotherapy and to explore the association of these rates with demographic, cultural, and clinical variables. We conducted a retrospective clinical cohort study of 148 adolescents aged from 11 to 20 years treated between 2008 and 2018 at two transcultural psychotherapy centers in Paris. Statistical analyses tested demographic, cultural, and clinical hypotheses. The main result was the high attendance rate at transcultural psychotherapy sessions among adolescents (77.8%). Attendance rates were not associated with age, gender, family size, generation of migration, or cultural area of origin, but were significantly linked to support in therapy, specifically, the presence at the first transcultural psychotherapy session of the first-line therapist, an interpreter, or both. Transcultural psychotherapy appears to be an effective method for addressing the complex symptoms experienced by migrant adolescents. Better attendance at sessions is statistically significantly associated with factors favoring a therapeutic alliance, specifically, the presence of the first-line therapist or an interpreter in TPT sessions and the existence of support from a social worker. The holistic approach of transcultural psychotherapy to adolescent care may explain the high attendance rates observed.

Transgenic plants expressing a Clostridium difficile spore antigen as an approach to develop low-cost oral vaccines.

Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a low-grade fever, watery stools, and minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon. Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant-based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB-CdeM), carrying the CdeM antigen, fused to the carboxi-terminus of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB-CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB-CdeM antigen in the range of 52-90 µg/g dry weight leaf tissues. The antigenicity of the plant-made LTB-CdeM antigen was evidenced by GM1-ELISA and immunogenicity assessment performed in test mice revealed that the LTB-CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant-based vaccines against C. difficile infection.

Expression and immunogenicity assessment of a plant-made immunogen targeting the cytotoxic T-lymphocyte associated antigen-4: a possible approach for cancer immunotherapy.

Cancer immunotherapy is a promising intervention to fight against this global health problem. In particular targeting immune checkpoints, such as cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed-death protein 1 (PD-1), by specific monoclonal antibodies is a current treatment for many malignances. A possible innovation in this field is based on the induction of humoral responses in the host by suppressing the effects of such immune checkpoints and as consequence favoring the activation of cellular immunity against the tumor cells. In this study, chimeric protein comprising the B subunit of Escherichia coli heat-labile enterotoxin as carrier and the extracellular domain of CTLA-4 (LTB-CTLA4) was produced in Nicotiana benthamiana by transient expression. The recombinant protein was accumulated up to 1.29 µg/g of leaves fresh weight on 4 day-post-infiltration. The integrity of the plant-made LTB-CTLA4 antigen was confirmed by western blot analysis and ELISA. Immunogenicity of the plant-made LTB-CTLA4 was assessed in BALB/c mice and the results showed that humoral responses were induced against both the LTB and CTLA-4 moieties. The plant-made LTB-CTLA4 stands as a promising candidate for the design of advanced protection studies against cancer in murine models.

Acoustic analysis of cry in with cleft palate patients / Análisis acústico del llanto en pacientes con hendidura palatina

INTRODUCTION: Crying in newborns and infants is a functional expression of biological interest. Through, acoustic analysis of crying, results of a primary palatoplasty can be evaluated in patients with cleft palate before development of speech, is objective, non-invasive, quick and simple tool for vocal exploration. The objective is to determine Fundamental Frequency (F0) of spontaneous crying in patients with pre and postoperative cleft palate, and compare with healthy control group through PRAAT software, in period between 2017 and 2019. MATERIALS AND METHODS: A sample of 16 patients from 18 to 30 months of age with cleft palate was obtained. Recordings were made of spontaneous pre and postoperative crying, and healthy control group; later were digitally recorded, and analyzed through PRAAT Software. RESULTS: 56.2 % were female and 43.7 % were male, witn average age of 25.8 months. Primary palatoplasty was performed using Veau Wadrill technique and Furlow technique. F0 of crying was Pitch average value 349.125 Hz preoperative and Pitch average postoperative control 369.775 Hz. More acute crying emissions were observed in postoperative period (p < 0.000), presenting similar values with control group (p < 0.000). CONCLUSION: Fundamental frequency (F0) was determined in patients with cleft palate, they presented a significant increase of 15,5 % in postoperative controls of primary palatoplasty, similar to healthy control group

INTRODUCCIÓN: El llanto de los recién nacidos y lactantes es una expresión funcional de interés biológico, por lo cual, a través del análisis acústico del llanto se pueden evaluar los resultados de una palatoplastia primaria en pacientes con hendidura palatina antes del desarrollo del habla, por ser una herramienta objetiva, no invasiva, rápida y sencilla de exploración vocal. El objetivo es determinar la frecuencia fundamental (F0) del llanto espontáneo en los pacientes con hendidura palatina pre y posoperatorio, y comparar con el grupo control sano a través del software PRAAT, en el periodo entre el año 2017 y 2019. MATERIALES Y MÉTODOS: Se obtuvo una muestra de 16 pacientes de 18 a 30 meses de edad con hendidura palatina. Se realizaron grabaciones del llanto espontáneo pre y posoperatorio, y del grupo control sano; posteriormente fueron grabadas digitalmente, y analizadas a través del software PRAAT. RESULTADOS: El 56,2 % fue del género femenino y el 43,7 % del género masculino, con edad promedio de 25,8 meses. Se realizó palatoplastia primaria mediante técnica de Veau Wadrill y técnica Furlow. La F0 del llanto fue de valor promedio Pitch 349.125 Hz preoperatorio y un control posoperatorio promedio Pitch 369.775 Hz. Se evidenció emisiones del llanto más agudas en el posoperatorio (p < 0,000), presentando valores similares con el grupo control (p < 0,000). CONCLUSIÓN: Se determinó frecuencia fundamental (F0) en los pacientes con hendidura palatina, presentaron un incremento significativo de 15,5 % en los controles posoperatorio de palatoplastia primaria, similares al grupo control sano

Chlamydomonas reinhardtii chloroplasts express an orally immunogenic protein targeting the p210 epitope implicated in atherosclerosis immunotherapies.

KEY MESSAGE: An algae-based vaccine model against atherosclerosis was developed with positive findings in terms of antigen yield and immunogenicity in mouse. Several immunotherapies against atherosclerosis have been evaluated at the preclinical level thus far, with some of them currently under evaluation in clinical trials. In particular, the p210 epitope from ApoB100 is known to elicit atheroprotective responses. Considering that Chlamydomonas reinhardtii is an attractive host for the production and delivery of subunit vaccines, in this study a chimeric protein consisting of the B subunit of the cholera toxin and the p210 epitope from ApoB100 (CTB:p210) has been expressed in C. reinhardtii chloroplast as an attempt to establish an oral vaccine candidate against atherosclerosis. The Chlamydomonas-made CTB:p210 protein was successfully expressed at levels of up to 60 µg per g of fresh weight biomass. The antigenic activity of the CTB and the p210 moiety was preserved in the CTB:p210 chimera. Moreover the algae-made CTB:p210 showed an immunogenic activity, when orally administered to BALB/c mice, as evidenced the presence of anti-p210 serum antibodies in mice treated with the algae-derived CTB:p210. The antibody response lasts for at least 80 days after the last boost. This experimental model is proposed as a convenient tool in the development of low cost atherosclerosis vaccines of easy compliance and friendly delivery. Further studies will determine the therapeutic potential of this algae-made vaccine in atherosclerosis animal models.

Towards the development of an oral vaccine against porcine cysticercosis: expression of the protective HP6/TSOL18 antigen in transgenic carrots cells.

MAIN CONCLUSION: The Taenia solium HP6/TSOL18 antigen was produced in carrot cells, yielding an immunogenic protein that induced significant protection in an experimental murine model against T. crassiceps cysticercosis when orally administered. This result supports the potential of HP6/TSOL18-carrot as a low-cost anti-cysticercosis vaccine candidate. Cysticercosis is a zoonosis caused by Taenia solium that can be prevented by interrupting the parasite life cycle through pig vaccination. Several injectable vaccine candidates have been reported, but the logistic difficulties and costs for its application limited its use in nationwide control programs. Oral plant-based vaccines can deal with this limitation, because of their easy administration and low cost. A stable expression of the HP6/TSOL18 anti-T. solium cysticercosis protective antigen in carrot calli transformed with an optimized transgene is herein reported. An antigen accumulation up to 14 µg g(-1) of dry-weight biomass was achieved in the generated carrot lines. Mouse immunization with one of the transformed calli induced both specific IgG and IgA anti-HP6/TSOL18 antibodies. A statistically significant reduction in the expected number of T. crassiceps cysticerci was observed in mice orally immunized with carrot-made HP6/TSOL18, in a similar extent to that obtained by subcutaneous immunization with recombinant HP6/TSOL18 protein. In this study, a new oral plant-made version of the HP6/TSOL18 anti-cysticercosis vaccine is reported. The vaccine candidate should be further tested against porcine cysticercosis.

The Corn Smut ('Huitlacoche') as a New Platform for Oral Vaccines.

The development of new alternative platforms for subunit vaccine production is a priority in the biomedical field. In this study, Ustilago maydis, the causal agent of common corn smut or 'huitlacoche'has been genetically engineered to assess expression and immunogenicity of the B subunit of the cholera toxin (CTB), a relevant immunomodulatory agent in vaccinology. An oligomeric CTB recombinant protein was expressed in corn smut galls at levels of up to 1.3 mg g-1 dry weight (0.8% of the total soluble protein). Mice orally immunized with 'huitlacoche'-derived CTB showed significant humoral responses that were well-correlated with protection against challenge with the cholera toxin (CT). These findings demonstrate the feasibility of using edible corn smut as a safe, effective, and low-cost platform for production and delivery of a subunit oral vaccine. The implications of this platform in the area of molecular pharming are discussed.

A Plant-Derived Multi-HIV Antigen Induces Broad Immune Responses in Orally Immunized Mice.

Multi-HIV, a multiepitopic protein derived from both gp120 and gp41 envelope proteins of the human immunodeficiency virus (HIV), has been proposed as a vaccine prototype capable of inducing broad immune responses, as it carries various B and T cell epitopes from several HIV strains. In this study, the immunogenic properties of a Multi-HIV expressed in tobacco chloroplasts are evaluated in test mice. BALB/c mice orally immunized with tobacco-derived Multi-HIV have elicited antibody responses, including both the V3 loop of gp120 and the ELDKWA epitope of gp41. Based on splenocyte proliferation assays, stimulation with epitopes of the C4, V3 domain of gp120, and the ELDKWA domain of gp41 elicits positive cellular responses. Furthermore, specific interferon gamma production is observed in both CD4+ and CD8+ T cells stimulated with HIV peptides. These results demonstrate that plant-derived Multi-HIV induces T helper-specific responses. Altogether, these findings illustrate the immunogenic potential of plant-derived Multi-HIV in an oral immunization scheme. The potential of this low-cost immunization approach and its implications on HIV/AIDS vaccine development are discussed.

An Env-derived multi-epitope HIV chimeric protein produced in the moss Physcomitrella patens is immunogenic in mice.

KEY MESSAGE: The first report on the recombinant production of a candidate vaccine in the moss system. The need for economical and efficient platforms for vaccine production demands the exploration of emerging host organisms. In this study, the production of an antigenic protein is reported employing the moss Physcomitrella patens as an expression host. A multi-epitope protein from the Human Immunodeficiency Virus (HIV) based on epitopes from gp120 and gp41 was designed as a candidate subunit vaccine and named poly-HIV. Transgenic moss plants were generated carrying the corresponding poly-HIV transgene under a novel moss promoter and subsequently seven positive lines were confirmed by PCR. The poly-HIV protein accumulated up to 3.7 µg g(-1) fresh weight in protonema cultures. Antigenic and immunogenic properties of the moss-produced recombinant poly-HIV are evidenced by Western blots and by mice immunization assays. The elicitation of specific antibodies in mice was observed, reflecting the immunogenic potential of this moss-derived HIV antigen. This is the first report on the production of a potential vaccine in the moss system and opens the avenue for glycoengineering approaches for the production of HIV human-like glycosylated antigens as well as other vaccine prototypes under GMP conditions in moss bioreactors.

Production of a plant-derived immunogenic protein targeting ApoB100 and CETP: toward a plant-based atherosclerosis vaccine.

In an effort to initiate the development of a plant-based vaccination model against atherosclerosis, a cholera toxin B subunit (CTB)-based chimeric protein was designed to target both ApoB100 and CETP epitopes associated with immunotherapeutic effects in atherosclerosis. Epitopes were fused at the C-terminus of CTB to yield a protein called CTB:p210:CETPe. A synthetic gene coding for CTB:p210:CETPe was successfully transferred to tobacco plants with no phenotypic alterations. Plant-derived CTB:p210:CETPe was expressed and assembled in the pentameric form. This protein retained the target antigenic determinants, as revealed by GM1-ELISA and Western blot analyses. Higher expresser lines reached recombinant protein accumulation levels up to 10 µg/g fresh weight in leaf tissues and these lines carry a single insertion of the transgene as determined by qPCR. Moreover, when subcutaneously administered, the biomass from these CTB:p210:CETPe-producing plants was able to elicit humoral responses in mice against both ApoB100 and CETP epitopes and human serum proteins. These findings evidenced for the first time that atherosclerosis-related epitopes can be expressed in plants retaining immunogenicity, which opens a new path in the molecular farming field for the development of vaccines against atherosclerosis.

The potential of Physcomitrella patens as a platform for the production of plant-based vaccines.

The moss Physcomitrella patens has a number of advantages for the production of biopharmaceuticals, including: i) availability of standardized conditions for cultivation in bioreactors; ii) not being part of the food chain; iii) high biosafety; iv) availability of highly efficient transformation methods; v) a haploid, fully sequenced genome providing genetic stability and uniform expression; vi) efficient gene targeting at the nuclear level allows for the generation of mutants with specific post-translational modifications (e.g., glycosylation patterns); and vii) oral formulations are a viable approach as no toxic effects are attributed to ingestion of this moss. In the light of this panorama, this opinion paper analyzes the possibilities of using P. patens for the production of oral vaccines and presents some specific cases where its use may represent significant progress in the field of plant-based vaccine development. The advantages represented by putative adjuvant effects of endogenous secondary metabolites and producing specific glycosylation patterns are highlighted.

Modification of the expression of two NPR1 suppressors, SNC1 and SNI1, in soybean confers partial resistance to the soybean cyst nematode, Heterodera glycines.

Systemic acquired resistance (SAR) is an enhanced defence response triggered when plants detect a pathogen. The response is extended to uninfected organs to protect against future attack. NPR1 is a nuclear leucine-rich repeat protein with a key role in SAR. It binds specifically to salicylic acid, and acts as a transcriptional coregulator of SAR activators and an inhibitor of transcriptional repressors. The proteins encoded by Suppressor of NPR1, Constitutive (SNC1) and Suppressor of NPR1, Inducible (SNI1) interact with NPR1 to regulate the expression of pathogenesis-related genes. The Arabidopsis thaliana (L.) Heynh. snc1 mutant exhibits a constitutive resistance response, but in the sni1 mutant, the SNI1 protein is rendered incapable of suppressing pathogen resistance genes. To study the influence of SNC1 and SNI1 on resistance to the soybean cyst nematode (Heterodera glycines), soybean (Glycine max (L.) Merr.) roots were separately transformed with four constructs designed to: (i) overexpress GmSNC1, the soybean orthologue of AtSNC1; (ii) overexpress AtSNI1; (iii) silence GmSNC1 and (iv) silence GmSNI1. A significant reduction of the female nematode population was observed in Treatments (i) and (iv). The expression of SAR marker genes was analysed in these treatments. The unusual pattern of expression of pathogen resistance genes shows there are differences in the effect resistance genes have on soybean and A. thaliana. Although NPR1 is involved in the cross-talk between the salicylic acid, jasmonic acid and ethylene pathways, understanding the nematode resistance mechanism in plants is still imprecise. These results provide further insights into the soybean defence response.

Should they play outside? Cardiorespiratory fitness and air pollution among schoolchildren in Bogotá / ¿Deberían los niños jugar al aire libre? Acondicionamiento cardiorespiratorio y contaminación del aire en Bogotá

Objective: This study was aimed at comparing cardiorespiratory fitness (CF), measured as VO2 max, amongst school children exposed to varying levels of particulate matter (PM10), and air pollution in Bogotá, Colombia. Methods: This was a cross-sectional study; it involved 1,045 children aged 7-12 attending four public schools served by different public transit routes and systems. Three schools were classified as being highly polluted (HP) and one slightly polluted (SP). The children and their parents were surveyed to collect data regarding their socio-demographic characteristics, physical activity habits and respiratory disease background. Objective measurements of weight and height were used to calculate the body mass index. VO2max was estimated using the 20-meter shuttle-run test, previously validated for Bogotá. Spirometry was performed on 435 children. Results: After adjustment for covariates, no difference was found in VO2max between children attending SP or HP schools (girls SP 45.8 ml/kg/min vs HP 44.6 ml/kg/min, p=0.11; boys SP 47.2 ml/kg/min cf HP 48.2 ml/kg/min, p=0.41). Conclusions: VO2max levels did not differ amongst children attending schools exposed to high compared to low levels of air pollution and PM. A longitudinal study assessing children’s VO2max levels in relation to exposure to highly-polluted areas is warranted.

Objetivo: Comparar el acondicionamiento cardiorespiratorio medido como VO2 máximo en niños escolarizados expuestos a diferentes niveles de PM10 en Bogotá. Métodos: Estudio de corte transversal. Se incluyeron 1045 niños de 7-12 años de 4 colegios públicos con diferentes corredores viales y sistemas de transporte público. Tres colegios tenían alta contaminación (AC) y uno baja contaminación (BC). Se aplicó una encuesta a niños y padres sobre características sociodemográficas, actividad física, antecedentes de enfermedades respiratorias y salud. Se midió objetivamente peso y talla para calcular el índice masa corporal. El VO2 máximo se estimó con la prueba de Leger validada para Bogotá. Se realizaron espirometrias en 435 niños. Resultados: Después de ajustar por covariables, no se encontraron diferencias significativas en el VO2 máximo de los niños en colegios con BC ò AC. (Niñas BC: 45,8ml/kg/min vs. AC: 44,6ml/kg/min; p=0,11, niños BC: 47,2ml/kg/min vs. AC: 48,2ml/kg/min; p=0,41). Conclusión: No se encontraron diferencias en el VO2 máximo de los niños que asistían a los colegios de AC ó BC. Se recomienda un estudio longitudinal que evalúe los niveles de VO2 máximo en los niños expuestos a áreas altamente contaminadas.