Transient production of receptor-binding domain of SARS-CoV-2 in Nicotiana benthamiana plants induces specific antibodies in immunized mice.

BACKGROUND: The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has currently affected millions of people around the world. To combat the rapid spread of COVID-19 there is an urgent need to implement technological platforms for the production of vaccines, drugs and diagnostic systems by the scientific community and pharmaceutical companies. The SARS-CoV-2 virus enters the cells by the interaction between the receptor-binding domain (RBD) present in the viral surface spike protein and its human receptor ACE2. The RBD protein is therefore considered as the target for potential subunit-based vaccines. METHODS AND RESULTS: We evaluate the use of Nicotiana benthamiana plants as the host to transiently-producing recombinant RBD (RBDr) protein. The identity of the plant-produced RBDr was confirmed by immune assays and mass spectrometry. Immunogenicity was confirmed through the specific antibodies generated in all of the immunized mice compared to the PBS treated group. CONCLUSIONS: In conclusions, the immunogenicity of the RBDr produced in N. benthamiana was confirmed. These findings support the use of plants as an antigen expression system for the rapid development of vaccine candidates.

The intestinal regionalization of acute norovirus infection is regulated by the microbiota via bile acid-mediated priming of type III interferon.

Evidence has accumulated to demonstrate that the intestinal microbiota enhances mammalian enteric virus infections1. For example, we and others previously reported that commensal bacteria stimulate acute and persistent murine norovirus infections2-4. However, in apparent contradiction of these results, the virulence of murine norovirus infection was unaffected by antibiotic treatment. This prompted us to perform a detailed investigation of murine norovirus infection in microbially deplete mice, revealing a more complex picture in which commensal bacteria inhibit viral infection of the proximal small intestine while simultaneously stimulating the infection of distal regions of the gut. Thus, commensal bacteria can regulate viral regionalization along the intestinal tract. We further show that the mechanism underlying bacteria-dependent inhibition of norovirus infection in the proximal gut involves bile acid priming of type III interferon. Finally, the regional effects of the microbiota on norovirus infection may result from distinct regional expression profiles of key bile acid receptors that regulate the type III interferon response. Overall, these findings reveal that the biotransformation of host metabolites by the intestinal microbiota directly and regionally impacts infection by a pathogenic enteric virus.

The major targets of acute norovirus infection are immune cells in the gut-associated lymphoid tissue.

Noroviruses are the leading cause of food-borne gastroenteritis outbreaks and childhood diarrhoea globally, estimated to be responsible for 200,000 deaths in children each year 1-4 . Thus, reducing norovirus-associated disease is a critical priority. Development of vaccines and therapeutics has been hindered by the limited understanding of basic norovirus pathogenesis and cell tropism. While macrophages, dendritic cells, B cells and stem-cell-derived enteroids can all support infection of certain noroviruses in vitro 5-7 , efforts to define in vivo norovirus cell tropism have generated conflicting results. Some studies detected infected intestinal immune cells 8-12 , other studies detected epithelial cells 13 , and still others detected immune and epithelial cells 14-16 . Major limitations of these studies are that they were performed on tissue sections from immunocompromised or germ-free hosts, chronically infected hosts where the timing of infection was unknown, or following non-biologically relevant inoculation routes. Here, we report that the dominant cellular targets of a murine norovirus inoculated orally into immunocompetent mice are macrophages, dendritic cells, B cells and T cells in the gut-associated lymphoid tissue. Importantly, we also demonstrate that a norovirus can infect T cells, a previously unrecognized target, in vitro. These findings represent the most extensive analyses to date of in vivo norovirus cell tropism in orally inoculated, immunocompetent hosts at the peak of acute infection and thus they significantly advance our basic understanding of norovirus pathogenesis.

High accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1) influenza.

Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.

Comparative in vitro and experimental in vivo studies of the anti-epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants.

A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.

[Association of insulin resistance to different anthropometric measures and cardiovascular risk factors in a non-diabetic population]. / Relación de la resistencia a la insulina con diferentes medidas antropométricas y factores de riesgo cardiovascular en una población no diabética.

BACKGROUND AND OBJECTIVE: Insulin resistance (IR) has been directly related to obesity, particularly central obesity, and to other cardiovascular risk factors (CVRFs). Direct IR quantification is difficult in clinical practice, and indirect methods such as HOMA (homeostasis model assessment) have therefore been developed. The aim of this study was to assess the association of IR, as measured by HOMA, with different anthropometric measures and some CVRFs. MATERIALS AND METHODS: A cross-sectional, observational study was carried out in a general population sample older than 18 years in the province of Albacete, Spain. Sample size was 678 subjects. Participants completed a survey and underwent physical examinations and laboratory tests. Obesity measures included body mass index, waist perimeter, and sagittal abdominal diameter. Data analysis was performed using SPSS 15.0 software. RESULTS: Mean values of obesity measures were higher in males as compared to females and increased with age. IR prevalence was 39.8%. All assessed anthropometric measures, decreased HDL (high density lipoprotein) cholesterol and increased non-HDL cholesterol were independently associated to the risk of IR. CONCLUSIONS: A clear association exists between different anthropometrical measures and IR in the general population. There is also an association between lipid profile cahnges and the risk of experiencing IR.

Biodistribución y farmacocinética de 99mTc-ciprofloxacina obtenida de una formulación modificada / Biokinetics performance of 99mTc-ciprofloxacin obtained from a new formulation

El propósito de la presente investigación fue evaluar el comportamiento biocinético de la 99mTc-ciprofloxacina obtenida de una nueva formulación. Un ensayo in vitro y un modelo de infección experimental demostraron su afinidad por bacterias vivas. La vida media en sangre fue de 5,89 +/- 0,85 horas. La biodistribución mostró alta acumulación en músculos infectados y baja en tejidos sanos.

The aim of the present investigation was to evaluate the biokinetics performance of 99mTc-ciprofloxacin obtained from a new formulation. Both in vitro assays and experimental infection models demonstrated its affinity for viable bacteria.Half life in blood was 5.89 +/- 0.85 hours. The biodistribution showed high accumulation on infected muscles and low on healthy tissues.

Phase I single-dose study of intracavitary-administered Nimotuzumab labeled with 188 Re in adult recurrent high-grade glioma.

Radioimmunotherapy (RIT) may improve the management of malignant gliomas. A Phase I clinical trial was performed to evaluate, for the first time, the toxicity and clinical effect of an intracavitary administration of a single dose of Nimotuzumab (h-R3) labeled wit (188)Re. Nimotuzumab is a humanized monoclonal antibody directed against epidermal growth factor receptors. Three patients with anaplastic astrocytoma (AA) and 8 with glioblastoma multiforme (GBM) were intended to be treated with 3 mg of mAb labelled with 10 or 15 mCi of (188)Re. In patients treated with 10 mCi (n=6) transitory worsening of pre-existing neurological symptoms were observed. Two patients treated with 15 mCi (n=4) developed early severe neurological symptoms and one also developed late severe toxicity (radionecrosis). In the group treated with 10 mCi, 1 GBM patient died in progression 6 months after the treatment, 2 patients (1 GBM and 1 AA) developed stable disease during 3 months. One GBM patient had partial response for more than 1 year and 2 patients (1 GBM and 1 AA) were asymptomatic and in complete response after 3 years of treatment. Maximal tolerated dose of the radioimmunoconjugate (188)Re-Nimotuzumab was 3 mg of the h-R3 labelled with 10 mCi of (188)Re. The radioimmunoconjugate showed a high retention in the surgical created resection cavity and the brain adjacent tissues with a mean value of 85.5 % of the injected dose one hour post-administration. This radioimmunoconjugate may be relatively safe and a promising therapeutic approach for treating high grade gliomas.

[Large uterine leiomyoma in a young patient]. / Leiomiomatosis uterina de grandes elementos en una paciente joven.

BACKGROUND: Great elements uterine leiomyomas are the most common tumours in the reproductive life affecting up to 30 % of the women in the United States. Its aetiology remains uncertain; cytogenic studies suggest that 40 to 50% present chromosomic abnormalities. Clinical manifestations are: hypermenorrhea and abdominal pain. In the great element cases, patients suffered intestinal constipation and rectal tenesm. The treatment includes analogous of human chorionic gonadotrophin liberation hormone (GnRH), progesterone, surgical treatment, myomectomy and hysterectomy, uterine artery embolization, high frequency ultrasound, laser, cryotherapy and thermoablation. CLINICAL CASE: An 18 year-old female, menarquia at 12, periods 28/4, nubil. 6 months previous: intestinal constipation, tumour-like mass in hypogastrium, of about 8 cm in diameter, which increased gradually up to 18 cm, smooth, movile and irregular. The pelvic ultrasound showed a tumour of 140 mm dependent of uterus. Myomectomy was performed. The histopathologic report was a 19.9 cm uterine leiomyoma, weighing 949 g and with hyaline degeneration. The patient was asymptomatic and without relapse a year later. CONCLUSIONS: Myomectomy is the choice treatment for the large uterine myomatosis when the patient's fertility is to be preserved.

Biodistribution and internal dosimetry of the 188Re-labelled humanized monoclonal antibody anti-epidemal growth factor receptor, nimotuzumab, in the locoregional treatment of malignant gliomas.

OBJECTIVE: To evaluate the biodistribution, internal radiation dosimetry and safety of the 188Re-labelled humanized monoclonal antibody nimotuzumab in the locoregional treatment of malignant gliomas. METHODS: Single doses of 370 or 555 MBq of 188Re-labelled nimotuzumab were locoregionally administered to nine patients with recurrent high-grade gliomas, according to an approved dose-escalation study. SPECT, planar scintigraphy and magnetic resonance images were combined for dosimetric and pharmacokinetic studies. Blood and urine samples were collected to evaluate clinical laboratory parameters and for absorbed doses calculations. Biodistribution, internal dosimetry, human anti-mouse antibody response and toxicity were evaluated and reported. RESULTS: The 188Re-nimotuzumab showed a high retention in the surgically created resection cavity with a mean value of 85.5+/-10.3%ID 1 h post-injection. It produced mean absorbed doses in the tumour region of approximately 24.1+/-2.9 Gy in group I (patients receiving 370 MBq) and 31.1+/-6.4 Gy in group II (patients receiving 555 MBq); the normal organs receiving the highest absorbed doses were the kidneys, liver and urinary bladder. About 6.2+/-0.8%ID was excreted by the urinary pathway. The maximum tolerated dose was 370 MBq because two patients showed severe adverse effects after they received 555 MBq of 188Re-nimotuzumab. No patient developed human anti-mouse antibody response. CONCLUSIONS: A locoregional single dose of 188Re-labelled nimotuzumab of approximately 370 MBq could be used safely in the routine treatment of patients suffering with high-grade gliomas. The efficacy of this therapy needs to be evaluated in a phase II clinical trial.

Phase I/II clinical trial of the humanized anti-EGF-r monoclonal antibody h-R3 labelled with 99mTc in patients with tumour of epithelial origin.

AIM: To evaluate the biodistribution, internal radiation dosimetry and toxicity of the humanized MAb h-R3 labelled with Tc in humans. METHODS: Twenty-five patients with suspected epithelial-derived tumours were included in this study and divided into two groups: group I consisted of 10 patients who received 3 mg/1110 MBq (3 mg/30 mCi); and group II consisted of 15 patients who received 6 mg/2220 MBq (6 mg/60 mCi). Single photon emission computed tomography (SPECT) and planar images, and multiple blood and urine samples were collected up to 24 h after injection. Haematological parameters and adverse effects were classified according to the WHO criteria. Biodistribution, human anti-mouse antibody (HAMA) response and absorbed doses were estimated and reported. RESULTS: Liver, spleen, kidneys and heart were identified as source organs. Their higher uptakes were 53.3+/-6.4%ID, 2.0+/-1.4%ID, 9.8+/-4.3%ID and 2.8+/-0.9%ID, respectively. The urinary bladder and large intestine also had a significant uptake. The mean urinary excretion was around 22%ID. The liver received the highest absorbed doses followed by the kidneys and the urinary bladder wall. There were no haematological or biochemical abnormalities with clinical significance related to the product. No patient developed HAMA response. Preliminary analysis of clinical results showed a sensitivity of 76.5% and a specificity of 100%. CONCLUSIONS: The results of this study suggest that Tc-h-R3 could be used in patients in a safe and effective way, for the diagnosis of epithelial-derived tumours at the two evaluated dose levels.

Evaluación de un método práctico para estabilizar el d, l-HMPAO / Evaluation of a practical method for stabilization of d, l-HMPAO

El objetivo de este estudio fue evaluar la estabilidad del d,l-HMPAO reconstituido y almacenado a -20º C. El kit se reconstituyó con un mililitro de solución salina. Fracciones de 0.333 mililitros fueron almacenadas a -20°C. El marcaje y el control de calidad se realizaron entre los 0-300 minutos posteriores. Fueron realizados estudios de SPECT cerebral y de cuerpo entero a 5 pacientes, con el kit intacto y con más de 30, 60 y 90 minutos de reconstituidos y almacenados. La calidad de las imágenes fue valorada por un observador experimentado. La integridad química se conservó por un tiempo de 2 horas (>80 por ciento de marcaje). Se acumuló en el cerebro entre 3,5-7 por ciento del 99mTc-d,l-HMPAO utilizando el producto hasta 90 minutos después de reconstituido. Las imágenes obtenidas fueron de adecuada utilidad clínica con un significativo ahorro de un 33,4 por ciento por cada dosis.

Línea básica de susceptibilidad a insecticidas de Aedes aegypti de Venezuela / Susceptibility base-line to insecticide for Aedes aegypti of Venezuela

En los años 1982-84 el Servicio de Evaluación Biológica de Plaguicidas determino la línea básica de susceptibilidad de Aedes aegypti a Malathion y Fenitrothion con cepas de varios estados costeros de Venezuela y del estado Aragua. Siguiendo las normas de la OMS se confesionaron cuadros y gráficos donde se aprecian tiempos letales 50 y 99% para las cepas trabajadas y dosis diagnósticas para cada producto. La clasificación de cepas en susceptible, indeterminada o resistente obedece al oficio AL-2037-81, difundiendo instrucciones de la OMS al respecto. Los resultados fueron los siguientes: a Malathion: susceptible la cepa Carabobo resistente la de Miranda; a Sumithion: susceptibles las cepas de Bioterio, Zulia y Nueva Esparta; a Fenthion: suceptibles las cepas Aragua y Carabobo, resistente de la Miranda; todas las demás fueron catalogadas de indeterminadas

Nueva linea basica de susceptibilidade a insecticidas de Rhodnius prolixus / New basic line of susceptibility to insecticides of Rhodnius prolixus

En 1975, el Servicio de Evaluación Biologica de Plaguicidas del Ministerio de Sanidad y Asistencia Social de Venezuela se establecio la linea basica de susceptibilidad a insecticidas de cepas de bioterio de Rhodnius prolixus, transmisor de la enfermedad de Chagas, empleando la tecnica de exposiciónes continuadas a papeles de filtro impregnados de soluciones insecticidas. Se confecciono una tabla donde se establecieron los tiempos de lectura en que se registraban el 50 y el 100 por ciento de mortalidad; esta tabla se utilizo durante 5 anos en Venezuela y en otros países de América Latina como patron para clasificar diversas especies de triatomineos recolectados en el medio natural. En 1982 se modifico la tecnica para establecer los tiempos de lectura en que se registraban el 50 y el 95 por ciento de mortalidad utilizando ninfas de bioterio en el quinto estadio. Se consideraron tres períodos de exposición a una sola concentracion de insecticida que se aumentaba para producir alta mortalidad a las 24 horas, tiempo en que se realizó una lectura unica. Con los resultados obtenidos se confecciono una tabla que se utiliza en la actualidad en Venezuela para determinar la susceptibilidad, tolerancia o resistencia de especies de triatomineos recolectados en el medio natural.