Performance of Marmoset Monkeys as Embryo Donors Is Reflected by Different Stress-Related Parameters.

Non-human primates (NHPs) serve as embryo donors for embryo collection in order to mimic genetic diseases in humans by genetic modification. Reproductive health of the embryo donors is crucial, and chronic distress needs to be avoided. Embryo retrieval rates (ERR), anti-Müllerian hormone (AMH) concentrations, cortisol levels, and body weight fluctuations were assessed as markers for fertility and distress. With regard to successful embryo retrievals (total n = 667), the animals were either used for extended periods (long-term group; LTG) or only for short periods (short-term group; STG). Retrospective evaluation expectedly showed that animals in the LTG had a higher ERR than animals in the STG (p < 0.0001). Importantly, ERR in the LTG remained stable throughout the experimental period, and high embryo rates were already encountered during the first year of experimental use (p = 0.0002). High ERR were associated with high AMH and low cortisol levels, and minimal body weight fluctuations following anesthesia, indicating a superior ability of the LTG animals to handle distress. We conclude that the long-term experimental use of marmosets does not impair their fertility or health status per se, supporting the view that animal reuse can be in accordance with the 3R-principle, implying reduction, replacement, and refinement in animal experimentation.

Generation and Breeding of EGFP-Transgenic Marmoset Monkeys: Cell Chimerism and Implications for Disease Modeling.

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.

Risco nutricional de pacientes críticos utilizando o NUTRIC Score / Nutritional risk of critically ill patients using the NUTRIC Score

Introdução: Na avaliação nutricional do paciente crítico devem-se utilizar instrumentos validados, considerar a gravidade da doença e a função gastrointestinal. Esses fatores devem ser verificados, pois servem para identificar pacientes que podem se beneficiar de uma terapia nutricional especializada. Por isso, a triagem nutricional é o primeiro passo para a estratificação de pacientes com risco nutricional para a instituição precoce de uma terapia nutricional segura e planejada. Método: Estudo exploratório, prospectivo realizado com pacientes internados no Centro de Terapia Intensiva (CTI) do Hospital São Vicente de Paulo, Passo Fundo-RS, no período de agosto a setembro de 2016. Foram incluídos pacientes admitidos na CTI, nas primeiras 24 horas de internação. Os dados foram coletados utilizando a ferramenta de risco nutricional denominada NUTRIC Score, bem como variáveis demográficas: sexo e idade, variáveis clínicas: data de internação, data de admissão e alta da CTI e número de comorbidades. A heterogeneidade dos pacientes foi analisada, a fim de entender a influência dos dados demográficos nos resultados, pelo teste t de Student, correlação linear simples e análise variância (ANOVA). Resultados: Foram incluídos no estudo 28 pacientes críticos, prevalecendo o gênero feminino, com média de idade de 55,75±15,47 anos. Os principais motivos de admissão na unidade de terapia intensiva foram os traumas e politraumas (35,7%), sendo que 64,3% dos pacientes apresentaram duas ou mais comorbidades associadas. Na pontuação final do NUTRIC Score, prevaleceu a pontuação alta (67,9%), indicando risco nutricional. Dentre os pacientes avaliados, 73,7% (14) dos pacientes com pontuação alta no NUTRIC Score, classificados em risco nutricional, foram a óbito (p=0,025).Conclusões: Observou-se elevada prevalência de risco nutricional entre os pacientes críticos estudados, confirmando a importância do acompanhamento nutricional adequado. O NUTRIC Score se mostrou uma ferramenta de fácil aplicação para triagem de pacientes críticos em unidade de terapia intensiva.

Natural Killer Group 2D Ligand Depletion Reconstitutes Natural Killer Cell Immunosurveillance of Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and aggressive tumor originating from the epithelial lining of the upper aero-digestive tract accounting for 300,000 annual deaths worldwide due to failure of current therapies. The natural killer group 2D (NKG2D) receptors on natural killer (NK) cells and several T cell subsets play an important role for immunosurveillance of HNSCC and are thus targeted by tumor immune evasion strategies in particular by shedding of various NKG2D ligands (NKG2DLs). Based on plasma and tumor samples of 44 HNSCC patients, we found that despite compositional heterogeneity the total plasma level of NKG2DLs correlates with NK cell inhibition and disease progression. Strikingly, based on tumor spheroids and primary tumors of HNSCC patients, we found that NK cells failed to infiltrate HNSCC tumors in the presence of high levels of NKG2DLs, demonstrating a novel mechanism of NKG2DL-dependent tumor immune escape. Therefore, the diagnostic acquisition of the plasma level of all NKG2DLs might be instrumental for prognosis and to decipher a patient cohort, which could benefit from restoration of NKG2D-dependent tumor immunosurveillance. Along these lines, we could show that removal of shed NKG2DLs (sNKG2DLs) from HNSCC patients' plasma restored NK cell function in vitro and in individual patients following surgical removal of the primary tumor. In order to translate these findings into a therapeutic setting, we performed a proof-of-concept study to test the efficacy of adsorption apheresis of sNKG2DLs from plasma after infusion of human MICA in rhesus monkeys. Complete removal of MICA was achieved after three plasma volume exchanges. Therefore, we propose adsorption apheresis of sNKG2DLs as a future preconditioning strategy to improve the efficacy of autologous and adoptively transferred immune cells in cellular cancer immunotherapy.

Direito humano à alimentação adequada: percepções e práticas de nutricionistas a partir do ambiente escolar / Human right to adequate food: perceptions and practices of nutritionists in a school environment / Derecho humano a la alimentación adecuada: percepciones y prácticas de nutricionistas en ambiente escolar

Resumo A incorporação da perspectiva dos direitos e da cidadania às políticas públicas de alimentação e nutrição passa pela prática social dos atores envolvidos com a concretização dessas políticas. O objetivo do estudo foi compreender percepções e práticas de nutricionistas sobre o direito humano à alimentação adequada no âmbito de sua atuação profissional no Programa Nacional de Alimentação Escolar. O estudo, desenvolvido em 2014, foi qualitativo, e a coleta de dados, realizada por grupo focal. Participaram 11 nutricionistas de municípios da Mesorregião Oeste Catarinense. As falas foram gravadas, transcritas e analisadas segundo a técnica de análise de conteúdo temática. Os resultados indicaram que as participantes tinham percepção abrangente sobre o direito humano à alimentação adequada, em diferentes dimensões e relações com os aspectos inerentes a ela. As nutricionistas destacaram a importância e o desafio da atuação intersetorial, articulada à comunidade. Contudo, pareceu haver limitação ao trabalho em equipe e à interlocução com as famílias, possivelmente por fragilidade de suporte teórico-metodológico e político para intervenção nesse campo. As percepções abrangentes das nutricionistas, mas pouco fundamentadas teórica e politicamente sobre o direito humano à alimentação adequada, evidenciaram o desafio a se enfrentar na formação profissional, tanto na graduação como na educação permanente.

Abstract Incorporating the perspective of rights and citizenship to public policies on food and nutrition goes through the social practice of the players involved in the implementation of these policies. The aim of the study was to understand the nutritionists' perceptions and practices on the human right to adequate food in the context of their professional activities in the National Program for School Feeding. The study was qualitative, developed in 2014, and data were collected by means of a focus group. Eleven nutritionists from municipalities located in the West Santa Catarina Mesoregion (Brazil) participated. The statements were recorded, transcribed, and analyzed according to the thematic content analysis technique. The results showed that the participants had a comprehensive perception of the human right to adequate food, in different dimensions and relations with the aspects related to it. The nutritionists emphasized the importance and the challenge of intersectoral action linked to the community. However, there appeared to be a limitation in teamwork and in dialog with the families, possibly due to fragile theoretical-methodological and political support for intervention in this field. The nutritionists' comprehensive perceptions on the human right to adequate food, although poorly substantiated from the theoretical and political viewpoint, showed the challenge to be faced in vocational training, both in undergraduate and in permanent education.

Resumen La incorporación de la perspectiva de los derechos y la ciudadanía a las políticas públicas de alimentación y nutrición pasa por la práctica social de los actores involucrados con la concreción de estas políticas. El objetivo del estudio aquí presentado fue comprender las percepciones y las prácticas de nutricionistas sobre el derecho humano a la alimentación adecuada en el ámbito de su actuación profesional en el Programa Nacional de Alimentación Escolar. El estudio, realizado en 2014, fue de naturaleza cualitativa, y la recolección de datos, realizada por un grupo focal. Participaron 11 nutricionistas de municipios de la Mesoregión Oeste Catarinense (Brasil). Las respuestas se grabaron, se transcribieron y se analizaron según la técnica de análisis de contenido temático. Los resultados indicaron que los participantes tenían una percepción abarcativa sobre el derecho humano a la alimentación adecuada, en diferentes dimensiones y relaciones con los aspectos inherentes a la misma. Las nutricionistas destacaron la importancia y el desafío de la actuación intersectorial, articulada a la comunidad. Sin embargo, pareció haber una limitación al trabajo en equipo y a la interlocución con las familias, posiblemente por la fragilidad del soporte teórico-metodológico y político para intervenir en ese campo. Las percepciones abarcativas de las nutricionistas, pero poco fundamentadas teórica y políticamente sobre el derecho humano a la alimentación adecuada, pusieron de manifiesto el desafío a ser enfrentado en la formación profesional, tanto en el pregrado como en la educación permanente.

Suspect Guillain-Barré syndrome in a male rhesus macaque (Macaca mulatta).

Guillain-Barré syndrome (GBS) is a rare, mainly acute inflammatory polyneuropathy in humans. It is frequently post-infectious with auto antibodies being formed against myelin sheaths, resulting in a progressive and more-or-less severe paralysis of the motor neuron and cranial nerves. Mortality is low and 60 % of the patients recover completely from the disease after intensive treatment. In animals, there are a few diseases that closely resemble GBS, but cases of GBS in monkeys seem to be scarce. In this case report, the clinical course of a progressive tetraplegia in a male rhesus macaque is described. Clinical, cerebrospinal fluid (CSF), electroneurography (ENG) and electromyography (EMG), and pathological findings revealed symptoms very similar to human GBS.

Unique case of granulomatous arteritis in a grey mouse lemur (Microcebus murinus) - first case description.

Overall, diseases of the vascular system are rarely observed entities among nonhuman primates that are commonly associated with systemic infections, septicemia or bacteremia. Rhesus monkeys infected with simian immunodeficiency virus (SIV) may develop a chronic occlusive arteriopathy of unknown etiology in late stages of the disease. This SIV associated arteriopathy is the only well-known specific vascular entity described in nonhuman primates. We herein report a unique case of granulomatous arteritis in a grey mouse lemur affecting multiple organs, which is not comparable to other disease entities formerly described in nonhuman primates. The features of the entity most closely resemble disseminated visceral giant cell arteritis in humans. A concise description of the disease is given, and the differential diagnoses are discussed. An idiopathic pathogenesis is suspected.

Virus safety of islet cell transplantation from transgenic pigs to marmosets.

Transplantation of pig islet cells for the treatment of diabetes may be a more effective approach compared with the application of insulin. However, before introduction into the clinic, efficacy and safety of this treatment have to be shown. Non-human primate models may be used for this, despite the fact that they are characterised by several limitations. Here we investigate the prevalence of porcine endogenous retroviruses (PERVs), which are present in the genome of all pigs and which may infect human cells, as well as of porcine herpes viruses in donor pigs and their potential transmission to non-human primate recipients. Despite the fact that all three subtypes of PERV were present in all and porcine cytomegalovirus (PCMV) was found in some of the pigs, neither PERVs nor PCMV were found in the recipient animals under the experimental conditions applied. Porcine lymphotropic herpes viruses (PLHV) were not found in the donor pigs, hepatitis E virus (HEV) was not found in the recipients.

Airway hyper-responsiveness in lipopolysaccharide-challenged common marmosets (Callithrix jacchus).

Animal models with a high predictive value for human trials are needed to develop novel human-specific therapeutics for respiratory diseases. The aim of the present study was to examine lung-function parameters in marmoset monkeys (Callithrix jacchus) that can be used to detect pharmacologically or provocation-induced AHR (airway hyper-responsiveness). Therefore a custom-made lung-function device that allows application of defined aerosol doses during measurement was developed. It was hypothesized that LPS (lipopolysaccharide)-challenged marmosets show AHR compared with non-challenged healthy subjects. Invasive plethysmography was performed in 12 anaesthetized orotracheally intubated and spontaneously breathing marmosets. Pulmonary data of R(L) (lung resistance), C(dyn) (dynamic compliance), EF50 (mid-expiratory flow), P(oes) (oesophageal pressure), MV (minute volume), respiratory frequency (f) and V(T) (tidal volume) were collected. Measurements were conducted under baseline conditions and under MCh (methacholine)-induced bronchoconstriction. The measurement was repeated with the same group of animals after induction of an acute lung inflammation by intratracheal application of LPS. PDs (provocative doses) of MCh to achieve a certain increase in RL were significantly lower after LPS administration. AHR was demonstrated in the LPS treated compared with the naïve animals. The recorded lung-function data provide ground for pre-clinical efficacy and safety testing of anti-inflammatory substances in the common marmoset, a new translational NHP (non-human primate) model for LPS-induced lung inflammation.

Phytoestrogens: endocrine disrupters or replacement for hormone replacement therapy?

OBJECTIVES: This review presents findings with clear statements from the literature as well as own results of effects of soy, red clover and their isoflavones as well as of the Cimicifuga racemosa extract BNO 1055. Experimental and clinical effects on climacteric complaints, osteoprotective effects, activity in the urogenital tract, and risks concerning cardiovascular diseases and mammary and endometrial tissue will be compared, also in comparison to classical hormone preparations. The question whether soy and red clover products and/or Cimicifuga racemosa (CR) preparations are endocrine disrupters or may fulfill the criteria of the so-called phyto-SERMs will be discussed. METHODS: Review of selected publications since 1980 and summary of unpublished own results of the authors. RESULTS: Experimental and clinical evidences suggest that soy/red clover and their isoflavones do not fulfill the criteria of an ideal SERM. They appear to have mild osteoprotective effects but do not improve climacteric complaints. Furthermore, they seem to stimulate uterine growth and mammary epithelial proliferation. In ovariectomized rats, the CR extract BNO 1055 showed many of the beneficial effects of 17beta-estradiol, including effects in the brain/hypothalamus to reduce serum LH levels, effects in the bone to prevent osteoporosis and estrogenic effects in the urinary bladder. The CR extract BNO 1055 had no uterotrophic effect. CONCLUSION: If clinical studies confirm these results, the Cimicifuga racemosa preparation BNO 1055 would appear as an ideal SERM and may therefore be an alternative to hormone replacement therapy.

Genomics and diversity of the common marmoset monkey NK complex.

The common marmoset monkey (Callithrix jacchus) is a New World primate that is increasingly used in biomedical research as a model organism. Due to the occurrence of natural bone marrow chimerism, it represents a particularly useful primate model in immunological research. In this study, we describe the genomic organization of the CD94, NKG2, and LY49L genes in the NK complex (NKC) of the common marmoset based on complete sequencing of a bacterial artificial chromosome clonal contig. This region of the marmoset NKC is 1.5 times smaller than its human counterpart, but the genes are colinear and orthologous. One exception is the activating NKG2CE gene, which is probably an ancestral form of the NKG2C- and NKG2E-activating receptor genes of humans and great apes. The two completely sequenced marmoset bacterial artificial chromosome clones are derived from distinct haplotypes, which differ by 200 sites in the overlapping sequence. Analyses of NKC genes in nine additional marmoset individuals revealed a moderate degree of polymorphism of the CD94, NKG2A, NKG2CE, and NKG2D genes. Furthermore, expression analyses identified several alternatively spliced transcripts, particularly of the CD94 gene. Several products of alternative splicing of NKC genes are highly conserved among primates. Alternative transcriptional start sites were found, but these probably do not lead to a change of the translational start site or result in longer or shorter cytoplasmic regions of these type II membrane receptors.

GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs.

We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43 kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [d-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor-like antigen.

Evidence for selective estrogen receptor modulator activity in a black cohosh (Cimicifuga racemosa) extract: comparison with estradiol-17beta.

OBJECTIVE: Some phytoestrogens are believed to have selective estrogen receptor modulator (SERM) activity with no action in the uterus but beneficial effects in the hypothalamo/pituitary unit and in the bone and are presently the focus of clinical interest. In the present experiments, the effects of the clinically used Cimicifuga racemosa (CR) extract BNO 1055 in the uterus, in the bone and on serum luteinizing hormone (LH) were compared with the effects of estradiol-17beta (E(2)) under acute and chronic conditions in ovariectomized rats. METHODS: Ovariectomized rats were treated either acutely (6 h) or chronically (3 Months) with E(2) or the CR extract. Gene expression of some estrogen-regulated genes in the metaphysis of the tibia and the uterus was determined. Furthermore, bone mineral density was measured by quantitative computer tomography. RESULTS: When given acutely, both E(2) and the CR extract inhibited LH secretion and slightly stimulated gene expression of IGF-I, collagen-1alpha1, osteoprotegerin and osteocalcin (all osteoblast products), and of tartrate-resistant acid phosphatase (TRAP, an osteoclast product) in the metaphysis of the femur. While E(2) stimulated uterine weight and expression of progesterone receptor (PR), the complement protein (C3) and IGF-I genes, and inhibited gene expression of the estrogen receptor beta (ERbeta) in the uterus, no such effect was observed under acute CR treatment. After chronic application with pelleted food over 3 Months E(2) had profound effects in the uterus on weight and gene expression (ERbeta, PR, C3 and IGF-I) which were not seen in the CR-treated animals. Within 3 Months after ovariectomy, control rats had lost more than 50% of the metaphyseal bone mass of the tibia, an effect prevented by E(2) and partially by CR supplementation. CONCLUSIONS: These data confirm the concept that the CR extract BNO 1055 contains as yet unidentified substances with SERM properties which act in the hypothalamo/pituitary unit and in the bone but not in the uterus.

Phytoestrogens: endocrine disrupters or replacement for hormone replacement therapy?

OBJECTIVES: This review presents findings with clear statements from the literature as well as own results of effects of soy, red clover and their isoflavones as well as of the Cimicifuga racemosa extract BNO 1055. Experimental and clinical effects on climacteric complaints, osteoprotective effects, activity in the urogenital tract, and risks concerning cardiovascular diseases and mammary and endometrial tissue will be compared, also in comparison to classical hormone preparations. The question whether soy and red clover products and/or Cimicifuga racemosa (CR) preparations are endocrine disrupters or may fulfill the criteria of the so-called phyto-SERMs will be discussed. METHODS: Review of selected publications since 1980 and summary of unpublished own results of the authors. RESULTS: Experimental and clinical evidences suggest that soy/red clover and their isoflavones do not fulfill the criteria of an ideal SERM. They appear to have mild osteoprotective effects but do not improve climacteric complaints. Furthermore, they seem to stimulate uterine growth and mammary epithelial proliferation. In ovariectomized rats, the CR extract BNO 1055 showed many of the beneficial effects of 17beta-estradiol, including effects in the brain/hypothalamus to reduce serum LH levels, effects in the bone to prevent osteoporosis and estrogenic effects in the urinary bladder. The CR extract BNO 1055 had no uterotrophic effect. CONCLUSION: If clinical studies confirm these results, the Cimicifuga racemosa preparation BNO 1055 would appear as an ideal SERM and may therefore be an alternative to hormone replacement therapy.