Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo.

BACKGROUND: Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. RESULTS: Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. CONCLUSIONS: Our results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule.

Chimeric VLPs Bearing VP60 from Two Serotypes of Rabbit Haemorrhagic Disease Virus Are Protective against Both Viruses.

The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.

Chrysalises as natural production units for recombinant subunit vaccines.

The baculovirus vector expression system (BEVS) combines cultured insect cells and genetically modified Autographa californica nuclear polyhedrosis virus (AcMNPV)-derived baculovirus vectors. This expression system has been widely used for the expression of hundred of proteins for more than 30 years, existing commercial products manufactured at large scale by this methodology, mainly subunit vaccines. At an industrial scale, insect cells, as any other cultured cells, require artificial media and a strict control of environmental sterile conditions in the complex and expensive bioreactors. Here we describe an efficient alternative to produce recombinant biologics using the versatile and productive baculovirus vectors. It consists in natural biocapsules (pupae from Trichoplusia ni (Hübner) Lepidoptera), containing millions of insect cells in perfect physiological conditions, ready to be programmed by a genetically modified AcMNPV-derived baculovirus vector to produce large quantities of any recombinant protein. This technology, denominated CrisBio, has been tested to produce dozens of proteins, reaching productivities on the range of milligrams per infected pupa, that can be translated into dozens of vaccine doses, for example. The biologics production by CrisBio was industrialized with the design of both insect rearing and pupae storage single-use plastic devices, compatible with machines specifically designed for the automation of pupae manipulation and inoculation. These devices and machines reduce manual operations, increase batches consistency and facilitate the scaled production of any recombinant protein. As a mode of examples, the productivity in CrisBio technology platform of two virus-like particle (VLP) vaccine antigens is described in this work.

Effect of growth hormone treatment on lymphocyte functions in old male rats.

INTRODUCTION: Age-related changes in the communication between the neuroendocrine and the immune system have been scarcely studied. Aging in mammals is associated with an impairment of the immune response, especially regarding lymphocyte functions. Furthermore, the endocrine system is also affected by aging, one of the most significant changes being the decrease in the secretion of several hormones such as growth hormone (GH). OBJECTIVE: The aim of the present work was to study whether GH replacement therapy in old male rats could improve several lymphocyte functions. METHODS: Spleen and axillary node lymphocytes from old (24 months of age) male Wistar rats were used in the present study to investigate the effect of GH (2 mg/kg daily during 10 weeks) on chemotaxis, lymphoproliferative response to the mitogen concanavalin A, interleukin 2 release and natural killer cell activity. RESULTS: We have found that the administration of GH can reduce or even reverse the age-related changes observed in these key immune function parameters. Moreover, we have observed that the recovery of such immune functions is able to reach similar values as those exhibited by young control animals of 6 months of age. CONCLUSION: Considering that the immune system is a marker of health and a predictor of longevity, hormone replacement therapies with GH, by increasing the immune function and thus delaying or slowing down some aspects of the aging process, could facilitate successful aging.

Improvement of immune cell functions in aged mice treated for five weeks with soybean isoflavones.

Aging is associated with an impaired immune system as well as with a decline of several hormones, such as estrogens. Dietary phytoestrogens have been proposed as an alternative to hormone replacement therapy. The beneficial action of soybean compounds is probably due to isoflavones, whose role as immunomodulators has been investigated with increasing interest. The aim of the present work was to study whether a 5-week administration of a diet enriched in soybean isoflavones and green tea could influence the immune function of mice that were sham-operated or ovariectomized. Natural killer activity (NK) and lymphoproliferation in response to the mitogens concanavaline A (Con A) and lipopolysaccharide (LPS), were studied in peritoneal leukocytes. Both treatments with soybean or green tea plus soybean were highly effective in improving these immune parameters with respect to control groups. Since the immune system is a marker of health and a predictor of longevity, an adequate treatment with isoflavones could be useful in slowing down the effects of the aging process through an improvement in the two relevant immune functions studied.

Oxidative stress in leukocytes from young prematurely aging mice is reversed by supplementation with biscuits rich in antioxidants.

Aging is associated with a progressive dysregulation of immune responses as a result of increased oxidative stress. Therefore, we have assessed the oxidative stress status of peritoneal leukocytes from young prematurely aging mice (PAM) as compared with non-prematurely aging mice (NPAM), as well as the effects on this oxidative stress of a dietary supplementation with biscuits rich in antioxidants (vitamin C, vitamin E, beta-carotenes, zinc and selenium). We found that, in the peritoneal leukocytes, the levels of several parameters of oxidation such as extracellular superoxide anion (O(2)(-)), Prostaglandin E(2) (PGE(2)), nitric oxide, oxidized glutathione (GSSG) and lipid peroxidation (malondialdehyde, MDA) were higher in PAM as compared with NPAM, whereas the antioxidant defences such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities, as well as reduced glutathione (GSH) levels, were decreased. Consequently, young PAM showed an oxidative stress in their leukocytes, which is characteristic of mice of an older chronological age. Antioxidant diet supplementation was able to restore redox homeostasis, increasing the antioxidant and decreasing the oxidant levels. Accordingly, supplementation with adequate levels of antioxidants, from an early age, could be useful to preserve health, especially in prematurely aging populations.

Improvement of leukocyte functions in prematurely aging mice after five weeks of diet supplementation with polyphenol-rich cereals.

OBJECTIVE: We investigated the beneficial effects of diet supplementation with two types of cereals naturally rich in polyphenolic compounds on several functions of peritoneal leukocytes from prematurely aging mice (PAM). METHODS: Two-hundred sixty healthy mice, 8 wk of age, were recruited and their behavioral responses were tested in a simple T-maze to identify PAM. Then the mice were fed a diet supplemented with 20% (wt/wt) of two different cereal fractions, named B (wheat germ) and C (buckwheat flour), rich in polyphenols (gallic acid, catechin, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, sinapic acid, ferulic acid, quercetin, and rutin), or a standard diet (controls) for 5 wk. Several parameters of innate (adherence to tissues, chemotaxis, phagocytosis, microbicidal capacity, and natural killer activity) and acquired immune (lymphoproliferation and interleukin-2 release) responses were measured. RESULTS: The PAM control group showed worse immune functions (P < 0.001 to 0.05) compared with the non-PAM control group. The PAM group that received cereal B showed increases in phagocytosis (P < 0.01), microbicidal activity (P < 0.001 to 0.01), natural killer activity (P < 0.001) and lymphoproliferation in response to lipopolysaccharide (P < 0.01) and interleukin-2 release (P < 0.001), and the PAM group that received cereal C showed a similar pattern, with increases in macrophage chemotaxis (P < 0.01), phagocytosis (P < 0.01), microbicidal activity (P < 0.001 to 0.01), natural killer activity (P < 0.01), lymphoproliferative response to concanavalin A and lipopolysaccharide (P < 0.001), and interleukin-2 release (P < 0.001). CONCLUSIONS: Dietary supplementation with polyphenol-rich cereals appears to have a protective effect on immune cell functions in mice with premature senescence. Thus, regular intake of these compounds could delay normal aging and improve quality of life.

Dietary supplementation with antioxidants improves functions and decreases oxidative stress of leukocytes from prematurely aging mice.

OBJECTIVES: Aging is accompanied by chronic inflammation and oxidative stress, which lead to a marked impairment of immune function and therefore increased mortality. This study assessed the effect of dietary supplementation, for 15 wk, with 5% and 20% (w/w) of biscuits enriched with nutritional doses of vitamins C and E, zinc, selenium, and beta-carotenes on function and oxidative stress parameters of peritoneal leukocytes from middle-aged, prematurely aging mice (PAM) and non-prematurely aging mice (NPAM). METHODS: After supplementation we measured leukocyte functions (adherence, chemotaxis, phagocytosis, intracellular reactive oxygen species levels, lymphoproliferation, natural killer activity, and interleukin-2 release), antioxidant defenses (superoxide dismutase, glutathione peroxidase, and reduced glutathione), oxidant compounds (extracellular O(2)(-), glutathione disulfide, glutathione disulfide/reduced glutathione ratio, tumor necrosis factor-alpha, nitric oxide, and prostaglandin E(2)), and lipid and DNA oxidative damage, measured by malondialdehyde and 8-oxo,7,8-dihydro-2'-deoxyguanosine levels, respectively. RESULTS: In general, leukocyte functions were improved and redox homeostasis was restored after intake of antioxidants. In consequence, malondialdehyde and 8-oxo,7,8-dihydro-2'-deoxyguanosine in PAM and NPAM were strikingly decreased after 5% and 20% supplementation (malondialdehyde, P < 0.001 in PAM; P < 0.01 in NPAM after both treatments; 8-oxo,7,8-dihydro-2'-deoxyguanosine, P < 0.01 after 5% supplementation and P < 0.001 after 20% supplementation in PAM and NPAM). Moreover, the effect of the antioxidants was stronger in PAM than in NPAM, and 20% supplementation was more effective than 5%. CONCLUSION: Our data suggest that improvement of leukocyte function and restoration of redox balance after consumption of adequate levels of antioxidants from adulthood may be useful to attain healthy aging, especially in animals with premature aging.

Effect of PVC and iron materials on Mn(II) deposition in drinking water distribution systems.

Polyvinyl chloride (PVC) and iron pipe materials differentially impacted manganese deposition within a drinking water distribution system that experiences black water problems because it receives soluble manganese from a surface water reservoir that undergoes biogeochemical cycling of manganese. The water quality study was conducted in a section of the distribution system of Tegucigalpa, Honduras and evaluated the influence of iron and PVC pipe materials on the concentrations of soluble and particulate iron and manganese, and determined the composition of scales formed on PVC and iron pipes. As expected, total Fe concentrations were highest in water from iron pipes. Water samples obtained from PVC pipes showed higher total Mn concentrations and more black color than that obtained from iron pipes. Scanning electron microscopy demonstrated that manganese was incorporated into the iron tubercles and thus not readily dislodged from the pipes by water flow. The PVC pipes contained a thin surface scale consisting of white and brown layers of different chemical composition; the brown layer was in contact with the water and contained 6% manganese by weight. Mn composed a greater percentage by weight of the PVC scale than the iron pipe scale; the PVC scale was easily dislodged by flowing water. This research demonstrates that interactions between water and the infrastructure used for its supply affect the quality of the final drinking water.

Diet supplementation for 5 weeks with polyphenol-rich cereals improves several functions and the redox state of mouse leucocytes.

BACKGROUND: Cereals naturally contain a great variety of polyphenols, which exert a wide range of physiological effects both in vitro and in vivo. Many of their protective effects, including an improvement of the function and redox state of immune cells in unhealthy or aged subjects come from their properties as powerful antioxidant compounds. However, whether cereal-based dietary supplementation positively affects the immune function and cellular redox state of healthy subjects remains unclear. AIM OF THE STUDY: To investigate the effects of supplementation (20% wt/wt) for 5 weeks with four different cereal fractions on healthy mice. METHODS: Several parameters of function and redox state of peritoneal leukocytes were measured. The cereals, named B (wheat germ), C (buckwheat flour), D (fine rice bran) and E (wheat middlings) contained different amounts of gallic acid, p-hydroxybenzoic acid, vanillic acid, sinapic acid, p-coumaric acid, ferulic acid, quercetin, catechin, rutin and oryzanol as major polyphenols. RESULTS: In general, all cereal fractions caused an improvement of the leukocyte parameters studied such as chemotaxis capacity, microbicidal activity, lymphoproliferative response to mitogens, interleukin-2 (IL-2) and tumor necrosis factor (TNFalpha) release, as well as oxidized glutathione (GSSG), GSSG/GSH ratio, catalase (CAT) activity and lipid oxidative damage. We observed similar effects among the cereal fractions. CONCLUSIONS: The results suggest that some of these effects may due, at least partially, to the antioxidant activity of the polyphenols naturally present in cereals. Since an appropriate function of the leukocytes has been proposed as marker of the health state, a short-term intake of cereals seems to be sufficient to exert a benefit in the health of the general population. However, further studies are needed to assess the optimal doses and to find out which active polyphenols are able to mediate the observed physiological effects before recommending their regular consumption.

Factores de estrés laboral en el personal de enfermería en Consulta Externa Hospital Emergencias Grau 2014 / Factors of occupational stress in the personnel of nursing in the external consultation at the Emergency Hospital Grau 2014

El Objetivo fue determinar los factores de estrés laboral en el personal de Enfermería en Consulta Externa. Hospital de Emergencias Grau. Material y Método: El estudio es de nivel aplicativo, tipo cuantitativo, método descriptivo de corte transversal. La población estuvo conformada por 50. La técnica fue la encuesta y el instrumento un formulario tipo cuestionario aplicado previo consentimiento informado. Resultados: Los factores de estrés laboral, del 100 por ciento (50), 54 por ciento (27) refieren que están presentes y 46 por ciento (23) ausentes. En la dimensión personal 54 por ciento (27) manifiestan que están presentes y 46 por ciento (23) ausente; en la dimensión organizacional 68 por ciento (34) expresan que están presentes y 32 por ciento (16) ausente; y en la dimensión ambiental 72 por ciento (38) presente y 28 por ciento (16) ausente. Conclusiones: Los factores de estrés laboral en el personal de enfermería de consulta externa, el mayor porcentaje expresan que están presentes el estado civil, la disponibilidad de los recursos materiales en su servido, la facilidad para cambiar los turnos, el clima laboral, los ambientes donde realizan la atención están libres de contaminación, están alejados de ruidos y condiciones físicas externas; seguido de un porcentaje considerable que refieren que están ausentes el número de hijos, los turnos que realiza y trabajan en otra institución...

The Objective was to determine the factors of occupational stress in nurses working in external consultation, Emergency Hospital Grau. Material and Method: The study is application level, quantitative, descriptive cross-sectional method. The population was formed by 50. The technique was the survey and the instrument a form type questionnaire applied prior informed consent. Results: Factors of occupational stress, of 100 per cent (50), 54 per cent (27) refer that they are present and 46 per cent (23) absent. In the dimension personal 54 per cent (27) manifest are present and 46 per cent (23) away; in the dimension organizational 68 per cent (34) expressed that they are present and 32 per cent (16) away; and in the environmental dimension 72 per cent (38) present and 28 per cent (16) away. Conclusions: Factors of occupational stress in nurses of outpatient, the highest percentage expressed that marital status, availability of material resources at your service, the ease of changing shifts, work climate are present, environments where do care are free from contamination, are far from noise and external physical conditions; followed by a considerable percentage refer to the number of children, shifts that are absent and working at another institution...

Parálisis de Bell: evaluación del tratamiento esteroidal / Bell's palsy: evaluation of steroidal treatment

Se realiza un estudio prospectivo, estratificado y randomizado en 120 pacientes portadores de parálisis facial periférica idiopática comparando la evolución de los casos con y sin tratamiento esteroidal. El tratamiento esteroidal se inició antes de 10- día de evolución con prednisona en dosis de 1 mg por kilógramo de peso y por día. El grupo control no recibió tratamiento. La motilidad facial fue evaluada según la escala de Adour modificada. A los 120 días la recuperación fue satisfactoria en el 91,1% de los casos del grupo control y en el 97,6% de los tratados con corticoides, lo que no tiene significación estadística. Sin embargo, según nuestros resultados el uso de corticoides favoreció la recuperación precoz (28 días) en las parálisis faciales periféricas idiopáticas completas si se las compara con el grupo control. Esta diferencia es estadísticamente significativa (63,6 versus 26,6% respectivamente y con p < 0.04). Como consecuencia del trabajo se concluye que: 1) No hay diferencia significativa en la evolución final de las parálisis faciales periféricas idiopáticas con y sin tratamiento esteroidal y 2) El uso de corticoides podría, sin embargo, favorecer la recuperación precoz de las parálisis completas. Esta afirmación requiere del estudio de una casuística más numerosa que permita llegar a conclusiones definitivas

Parálisis de Bell: evolución natural / Bell's palsy: its natural clinical course

Se realiza un estudio descriptivo y prospectivo de la evolución natural de 115 pacientes con parálisis facial periférica idiopática sin tratamiento ni kinesiterapia específica, reunidos entre abril de 1986 y mayo de 1987. Se analizan los factores pronósticos, en base al protocolo de Adour modificado. Se efectuaron 762 controles. La recuperación fue satisfactoria en un 91,6%. No hubo diferencia en la evolución según edad, sexo, lado afectado y patología agregada. Analizando la evolución del compromiso de los músculos considerados por separado, se observa un gran sincronismo en su recuperación final. Sin embargo, los pacientes que tuvieron una parálisis facial incompleta lograron una mejoría precoz dentro del primer mes (p<0.001). Se concluye que en el material estudiado, la evolución natural fue altamente satisfactoria; que el tipo de parálisis incompleta o completa es un indicador pronóstico del tiempo necesario para obtener la recuperación de la motilidad facial y que el control de la recuperación funcional de un solo músculo puede ser suficiente indicador evolutivo de la parálisis facial periférica

Primer año de vida en hijos de adolescentes / First year of life in the children born of adolescents

Los hallazgos más importantes en el grupo de madres adolescentes y de sus hijos fueron: 1. Frecuencia significativamente mayor de soltería (53,9% contra sólo 19,1%). 2. Porcentaje significativamente mayor de casos con peso de nacimiento bajo (9% contra 0,6%). 3. Mayor proporción de niños con cumplimiento de todos los controles de salud (69% contra 46%). 4. Calendario de vacunaciones completo en el 79,4%. 5. Ausencia de morbilidad durante el primer año de vida en el 19%. Más de 6 episodios de morbilidad en el 29% lo que es significativamente superior a los niños controles (13,7%). 6 Necesidad de hospitalización en el 15% de los caso, porcentaje que es similar al de los controles. 7. Calificación de alto riesgo en el 91% de los casos y sólo en el 48% de los controles, diferencia que es significativa

Características materno-neonatales del retardo de crecimiento intrauterino en la gestación de término según curva nacional de crecimiento intrauterino / Maternal and neonatal characteristics of fetal growth retardation in terminal gestation according to national ponderal intrauterine index

Se estudian 100 recién nacidos de término, pequeños para edad gestacional, según curva de crecimiento intrauterina nacional (Juez y Ventura) y se comparan con 130 niños de término, adecuados para edad gestacional, nacidos en el Hospital San Borja-Arriarán, entre 1989 y 1991. Se analiza las características clínicas y morbilidad neonatal para ambos grupos. Un 91% de los niños pequeños, fueron considerados con retardo de tipo simétrico, según índice ponderal. Simultáneamente se estudió en las 230 madres, factores favorecedores de retardo de crecimiento intrauterino. Se apreció diferencias estadísticamente significativas, en ambos grupos de madres, especialmente en relación a factores nutricionales, talla materna, actividad laboral y presencia de oligoamnios. En los recién nacidos PEG, no se evidenciaron las alteraciones clásicas descritas en el período neonatal, en relación al grupo control. Atribuimos esta ausencia de morbilidad, al hecho que el percentil 10, de la curva nacional, al considerar niños con mayores pesos de nacimiento, incluye entre los niños considerados PEG, un número importante que son constitucionalmente pequeños, y por lo tanto no corresponden a retardo de crecimiento intrauterino. Sugerimos que para aumentar la sensibilidad debería considerarse el percentil 5 de la curva nacional