Advances in epigenetic regulation of Chinese hamster ovary cells / 生物工程学报

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.

A Synergistic Nephroprotective Activity of A Miracle Drink Ayurvedic Proprietary Medicine Renal Support A Novel Therapy for Chronic Kidney Disease in Human

Miracle drink “Renal Support (S-5)” an Ayurvedic formulation in conjugation with other cardiovascular support (S3), Sugar Care (S10), and liver health support (S4) was scientifically evaluated on 12 humans subjects for its therapeutic potential in treating chronic kidney diseases caused due to: a) Induce of pain killers medication, and other medications, b) Chronic diabetes, c) Blood pressure. Patients suffering from renal failure due to over medications, pain killer medication and BP were advised to take 15ml of Renal Support and S3 twice a day morning and evening before food, and 15ml of S4 trice a day. As the main biomarker of kidney disease, creatinine was monitored every month till three months of treatment whereas; blood urea and hemoglobin were screened at month end. Cytotoxicity and nephroprotective activity of Renal Support were evaluated on Baby Hamster Kidney Fibroblast cells (BHK-21). Radical decline in serum creatinine content was observed from 6.31mg/dl to 1.80mg/dl (68%), 1.20mg/dl (79%), and 0.84mg/dl (82%) on 30, 60, and 90 days of treatment respectively and in 90 days of treatment most of the patients showed 50 to 83% creatinine reduction. A significant decrease in the blood urea from 91mg/dl to 30mg/dl(67%) and hemoglobin content from 7.27 to 11.77g% was observed in 30days of treatment and the majority of patients showed >50% of blood urea reduction. No toxicity of Renal Support towards BHK-21 was noticed and showed 40.92% and 47.54% nephroprotective activity. A novel, natural-based, and safe Ayurveda formulation with significant nephroprotective potential for CKD treatment was proposed in the present study.

A comprehensive morphometric study of visceral and subcutaneous adipose tissue depots in mice, hamsters and rats / Un estudio morfométrico completo de los depósitos de tejido adiposo visceral y subcutáneo en ratones, hámsters y ratas

SUMMARY: Adipose tissue morphology of different fat tissue depots can be described using the number of adipocytes and cell surface of adipocytes. This study deals with characteristics and morphometric analysis of white and brown adipose tissue depots in healthy adult laboratory mice, hamsters and rats of both sexes. The number of unilocular adipocytes in white adipose tissue differs from one adipose tissue depot to another, with the largest number of adipocytes in mice and a similar number in hamsters and rats. The smallest surface area and the largest percentage of small unilocular adipocytes were found in mice. White adipose tissue in hamsters and rats was predominantly made out of a larger percentage of medium-sized adipocytes and a smaller percentage of small and medium-sized adipocytes. Uncoupling protein 1 positive multilocular adipocytes were found in classic brown adipose tissue depots with larger percentages in mice (93.20 %) and hamsters (91.30 %), while rats had a smaller percentage (78.10 %). In white and brown adipose tissue, significant differences between species and both sexes within the same species were found, indicating the influence of sexual dimorphism. The presented morphometric results could serve as a basis for further studies concerning experimental animal models of metabolic disorders and obesity.

RESUMEN: La morfología del tejido adiposo de diferentes depósitos de tejido graso se puede describir utilizando el número de adipocitos y la superficie celular de los adipocitos. Este estudio analiza las características y el análisis morfométrico de los depósitos de tejido adiposo blanco y marrón en ratones, hamsters y ratas de laboratorio, adultos sanos de ambos sexos. El número de adipocitos uniloculares en el tejido adiposo blanco difiere de un depósito de tejido adiposo a otro, con el mayor número de adipocitos en ratones y un número similar en hámsteres y ratas. La superficie más pequeña y el mayor porcentaje de adipocitos uniloculares pequeños se encontraron en ratones. El tejido adiposo blanco en hámsteres y ratas estaba compuesto predominantemente por un mayor porcentaje de adipocitos de tamaño mediano y un porcentaje menor de adipocitos de tamaño pequeño y mediano. Los adipocitos multiloculares positivos para la proteína desacopladora 1 se encontraron en depósitos de tejido adiposo marrón clásico con mayores porcentajes en ratones (93,20 %) y hámsters (91,30 %), mientras que las ratas tenían un porcentaje menor (78,10 %). En el tejido adiposo blanco y pardo se encontraron diferencias significativas entre especies y entre ambos sexos dentro de una misma especie, lo que indica la influencia del dimorfismo sexual. Los resultados morfométricos presentados podrían servir como base para futuros estudios sobre modelos animales experimentales de trastornos metabólicos y obesidad.

Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

Site-specific integration and stable expression of exogenous protein at a novel site on CHO cell chromosome / 中国药科大学学报

@#Finding stable expression sites on the chromosomes of Chinese hamster ovary (CHO) cells is an effective method to solve the problem of unstable expression of CHO cells in long-term culture. Our group used lentiviral transfection to integrate the tracer gene (Zsgreen1) into the chromosome of CHO cells and found multiple potential stable expression sites. This study verified the ability of one of the sites located in the 148052-148157 bp region on chromosome NW_003614241.1 to stably express exogenous proteins.The expression of Zsgreen1 gene was first observed, and CRISPR/Cas9 technology was then used to integrate the enhanced green fluorescent protein (EGFP) gene into this site. Three strains of EGFP gene integrated cells were obtained. After 60 generations of suspension culture, the fluorescence intensity of the cells had no significant changes, which proved that this site can stably express the EGFP gene. The same method was used to construct recombinant CHO cell lines expressing the human serum albumin (HSA) gene, and was verified by Western blot that this site could express and secrete HSA. It shows that the above-mentioned sites can be integrated and can stably express exogenous proteins.

The hamster egg penetration test may decrease intracytoplasmic sperm injection utilization while maintaining high conventional fertilization rates / 亚洲男科学杂志(英文版)

This was a cohort study of in vitro fertilization (IVF) subjects at the University of Utah, Salt Lake City (UT, USA) utilizing partner sperm. Cycles where both the hamster egg penetration test (HEPT) and semen analysis were performed within 2 years prior to IVF cycles were stratified into four groups based on a normal or an abnormal HEPT and morphology. The mean conventional and intracytoplasmic sperm injection (ICSI) fertilization rates were calculated in each group. We performed a univariate analysis on the primary outcome comparing clinically interesting subjects. We performed a cost-effectiveness analysis of a policy of HEPT versus universal ICSI in couples with an abnormal morphology. Among patients with a normal HEPT, there was no difference in the mean conventional fertilization rates between those with a normal and an abnormal morphology. There was no difference in the mean conventional fertilization rates between subjects with a normal morphology without a hamster test and those with a normal HEPT without a morphology assessment. In 1000 simulated cycles with an abnormal morphology, a policy of HEPT was cost saving compared to universal ICSI, yet produced similar fertilization rates. The HEPT is similar to the World Health Organization edition 5 (WHO-5) morphology in predicting successful conventional fertilization while allowing decreased utilization of ICSI. A policy of HEPT for males with abnormal morphology saves cost in selecting couples for a fertilization method.

Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS / 药物分析学报

Ensuring the removal of host cell proteins (HCPs) during downstream processing of recombinant pro-teins such as monoclonal antibodies (mAbs) remains a challenge.Since residual HCPs might affect product stability or safety,constant monitoring is required to demonstrate their removal to be below the regulatory accepted level of 100 ng/mg.The current standard analytical approach for this procedure is based on ELISA;however,this approach only measures the overall HCP content.Therefore,the use of orthogonal methods,such as liquid chromatography-mass spectrometry (LC-MS),has been established,as it facilitates the quantitation of total HCPs as well as the identification and quantitation of the indi-vidual HCPs present.In the present study,a workflow for HCP detection and quantitation using an automated magnetic bead-based sample preparation,in combination with a data-independent acquisi-tion (DIA) LC-MS analysis,was established.Employing the same instrumental setup commonly used for peptide mapping analysis of mAbs allows for its quick and easy implementation into pre-existing workflows,avoiding the need for dedicated instrumentation or personnel.Thereby,quantitation of HCPs over a broad dynamic range was enabled to allow monitoring of problematic HCPs or to track changes upon altered bioprocessing conditions.

Identification and sequence analysis of prolactin receptor and its differential expression profile at various developmental stages in striped hamsters

Prolactin (PRL) plays critical roles in regulation of biological functions with the binding of specific prolactin receptor (PRLR). Revealing the expression patterns of PRLR at different developmental stages is beneficial to better understand the role of PRL and its mechanism of action in striped hamsters. In this study, the cDNA sequence of PRLR (2866-base-pairs) was harvested from the pituitary of mature female striped hamsters (Cricetulus barabensis) that contains an 834-base-pair 5′-untranslated region (1-834 bp), a 1848-base-pair open reading frame (835-2682 bp), and a 184-base-pair 3′-untranslated region (2683-2866). The 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids. In the mature PRLR, two prolactin-binding motifs, 12 cysteines, and five potential Asn-linked glycosylation sites were detected. Our results showed that the PRLR mRNA quantity in the hypothalamus, pituitary, ovaries, or testis was developmental-stage-dependent, with the highest level at sub-adult stage and the lowest level at old stage. We also found that PRLR mRNAs were highest in pituitary, medium level in hypothalamus, and lowest in ovaries or testis. PRLR mRNAs were significantly higher in males than in females, except in the hypothalamus and pituitary from 7-week-old striped hamsters. Moreover, the PRLR mRNAs in the hypothalamus, pituitary, and ovaries or testis were positively correlated with the expression levels of GnRH in the hypothalamus. These results indicated that the PRLR has conserved domain in striped hamster, but also possesses specific character. PRLR has multiple biological functions including positively regulating reproduction in the striped hamster.

Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates

BACKGROUND: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. RESULTS: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa). CONCLUSIONS: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.

Effect of topical 2% eucalyptus extract on 5-fu-induced oral mucositis in male golden hamsters

Abstract Oral mucositis is a common inflammatory complication among patients with cancer. This study evaluated the histopathological, stereological, and antioxidant markers of 2% eucalyptus extract in induced oral mucositis in male golden hamsters. In this animal study, oral mucositis was induced in 30 male golden hamsters by 5-FU (60 mg/kg) on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle once daily on days 3 and 4. On days 14-17, 2% eucalyptus hydroalcoholic extract gel and Calendula officinalis extract gel groups were treated and then compared with a non-treated control group. The histopathological and stereological scores and the pouch content of malondialdehyde, as well as the activities of glutathione and myeloperoxidase in the pouch tissue, were evaluated. Histopathologic scores of oral mucositis were lower in the eucalyptus gel group than those of the calendula and control groups (p<0.05). Also, a lower malondialdehyde level and higher myeloperoxidase and glutathione activities were detected in the eucalyptus group in comparison to the calendula and control groups (p<0.001). The thickness of the mucosa and submucosa increased in the eucalyptus group. The numerical density of the fibroblast and the volume density of the collagen significantly increased in the eucalyptus group. In conclusion, the use of eucalyptus hydroalcoholic extract may be associated with reduced intensity of oral mucositis, diminished concentration of malondialdehyde, increased activity of myeloperoxidase and glutathione, increased volume of mucosa and submucosa, increased fibroblast and collagen in the induced oral mucositis in golden hamsters undergoing 5-FU consumption.

Resumo A mucosite oral é uma complicação inflamatória comum em pacientes com câncer. Este estudo avaliou os marcadores histopatológicos, estereológicos e antioxidantes de Eucalyptus 2% na mucosite oral induzida em hamsters dourados machos. Neste estudo em animais, a mucosite oral foi induzida em 30 hamsters golden masculinos por 5-FU (60 mg / kg) nos dias 0, 5 e 10 do estudo. A bolsa da bochecha foi arranhada com uma agulha estéril uma vez ao dia nos dias 3 e 4. Nos dias 14 a 17, os grupos de gel de eucalipto a 2% e curativos à base de gel foram tratados e comparados com um grupo controle. Foram avaliados os escores histopatológicos e estereológicos e o conteúdo de malondialdeído na bolsa, bem como as atividades de glutationa e mieloperoxidase no tecido da bolsa. Os escores histopatológicos de mucosite foram menores no grupo de gel de eucalipto a 2% do que os do gel e do grupo controle (p <0,05). Além disso, um nível mais baixo de malondialdeído e maiores atividades de mieloperoxidase e glutationa foram detectadas no grupo tratado com eucalipto em comparação aos grupos à base de gel e controle (p <0,001). A espessura da mucosa e submucosa aumentou no grupo Eucalyptus. A densidade numérica do fibroblasto e a densidade do volume do colágeno aumentaram significativamente nos grupos tratados com eucalipto. Em conclusão, o uso do extrato hidroalcoólico de Eucalyptus pode estar associado a menor intensidade de mucosite oral, diminuição da concentração de malondialdeído, aumento da atividade de mieloperoxidase e glutationa, aumento do volume de mucosa e submucosa, aumento de fibroblastos e colágeno na mucosite oral induzida em hamsters dourados em consumo de 5 UF.

2,6-and 3,5-dimethylaniline-induced mutagenesis in Chinese hamster ovary cells expressing human cytochrome P450 1A2 and sulfotransferase

Aim: The aim of this study was to test the hypothesis that human cytochrome P450 1A2 (CYP1A2) and sulfotransferase (SULT) contribute to the phase I and II bioactivation of 2,6-dimethylaniline (2,6-DMA) and 3,5-dimethylaniline (3,5-DMA) in affecting the incidence of genotoxicity.Methodology: 5P3H1 cells carrying cytochrome P450 1A2 (CYP1A2) and SULT cells were treated with various concentrations of 2,6-and 3,5-DMA for 48 hr or their N-hydroxyl and aminophenol metabolites for 1 hr in the absence or presence of 2,6-Dichloro-4-nitrophenol (DCNP). Cell lethality was assayed by trypan blue exclusion and induced mutagenesis of adenine phosphoribosyl transferase (aprt) gene was also evaluated. Results: A significant dose-dependent increase in cytotoxicity and mutant fraction was observed after treatment with 2,6- and 3,5-DMA, and their metabolites; N-hydroxy and aminophenol metabolites are more potent than the parent compounds. Addition of sulfotransferase inhibitor DCNP decreased the cytotoxic and mutagenic effects of 2,6- and 3,5-DMA, and their metabolites in a dose-dependent manner. Interpretation: This research indicate that 2,6 and 3,5-DMA are mutagenic, and their toxicity in model systems depend on metabolic activation. This activation is mediated by CYP1A2 and SULT enzymes

Increasing transgenic expression in recombinant Chinese hamster ovary cells using introns in different directions / 生物工程学报

The aim of this study is to investigate the effect of the chimeric intron in different directions on the expression of the nerve growth factor (NGF) in recombinant Chinese hamster ovary (CHO) cells. The chimeric intron that contained the splice sequence of the first intron of the human β-globin and the human immunoglobulin heavy chain variable region intron was used. NGF gene was cloned into the expression vectors containing the chimeric intron in the forward or reverse direction, followed by transfecting into CHO cells, and screened under G418 to produce the stable transfected CHO cells. Fluorescence quantitative PCR, ELISA, and Western blotting were performed to detect the recombinant NGF gene expression in CHO cells. The results showed that the chimeric introns could significantly enhance the expression of NGF in recombinant CHO cells. Moreover, the enhancing effect on NGF expression level by the intron in the forward direction showed stronger than that of the reverse direction both at mRNA and protein level. In conclusion, the chimeric intron could increase NGF expression in stably transfected CHO cells and the effect is associated with the direction of the intron insertion.

The treatment effect of novel hGHRH homodimer to male infertility hamster

Extra-hypothalamic growth hormone-releasing hormone (GHRH) plays an important role in reproduction. To study the treatment effect of Grin (a novel hGHRH homodimer), the infertility models of 85 male Chinese hamsters were established by intraperitoneally injecting 20 mg/kg of cyclophosphamide once in a week for 5 weeks and the treatment with Grin or human menopausal gonadotropin (hMG) as positive control was evaluated by performing a 3-week mating experiment. 2–8 mg/kg of Grin and 200 U/kg of hMG showed similar effect and different pathological characteristics. Compared to the single cyclophosphamide group (0%), the pregnancy rates (H-, M-, L-Grin 26.7, 30.8, 31.3%, and hMG 31.3%) showed significant difference, but there was no difference between the hMG and Grin groups. The single cyclophosphamide group presented loose tubules with pathologic vacuoles and significant TUNEL positive cells. Grin induced less weight of body or testis, compactly aligned tubules with little intra-lumens, whereas hMG caused more weight of body or testis, enlarging tubules with annular clearance. Grin presented a dose-dependent manner or cell differentiation-dependentincrease in testicular GHRH receptor, and did not impact the levels of blood and testicular GH, testosterone. Grin promotes fertility by proliferating and differentiating primitive cells through up-regulating testicular GHRH receptor without triggering GH secretion, which might solve the etiology of oligoasthenozoospermia.

Stable co-expression of human kappa opioid receptor and PKAcat-EGFP in CHO cells and functional identification / 中国药理学通报

Aim To establish a cell model which stably co-ex-press human kappa opioid receptor (hKOR) and enhanced green fluorescent protein ( EGFP) labeled catalytic domain of cAMP-dependent protein kinase A(PKAcat) fusion protein (PKAcat-EGFP) in Chinese hamster ovary(CHO) cells, laying the foun-dation for the high-throughput screening of hKOR drugs and drug molecular mechanisms in vitro. Methods Hygromycin B resist-ant hKOR recombinant plasmid [ pcDNA3.1/Hygro ( + ) -hKOR] was transfected into CHO cells stably expressing PKA-cat-EGFP by a lipofectin based method. Transfected cells were selected in culture medium containing hygromycin B. The posi-tive clones were selected by PKA redistribution assay. Z’ factor was used for evaluation and validation the reliability of the cell model. PKA redistribution assay and LANCE cAMP 384 Kit were used to test the function of the receptors in selected clone. Results CHO-PKAcat-EGFP/hKOR-13 cell model exhibited stable response in PKA redistribution assay and LANCE cAMP 384 Kit. Treated with 100 nmol·L-1U-50488 for 30 min, the average value of Z’ factor was 0.596, proving the reliability of the cell model. The hKOR expression in cell model remained stable after a few generations. Conclusion The CHO-PKAcat-EGFP/hKOR-13 cell model with stable co-expression of hKOR and PKAcat-EGFP has been successfully established.

Advances in development and application of Chinese hamster resource / 中国比较医学杂志

Chinese hamsters are a kind of valuable laboratory animal resources and play an important role in medical,genetics and pharmaceutical research. More and more biological characteristics of Chinese hamsters have been discovered with in-depth research,and many Chinese hamster models have been established so far. This paper is a brief overview of the development and research progress of Chinese hamsters and their application in taxonomy and medical research.

Quality control of chromosome preparation in the chromosome aberration test in vitro / 中国比较医学杂志

Objective To study the influencing factors and quality control of chromosome preparation during chromosome aberration test in vitro,and to summarize and analyze the method and key points of successful preparation of chromosome specimens in vitro. Methods Chinese hamster lung cells(CHL)were used for cell culture and chromosome preparation. Mitomycin and cyclophosphamide were used as positive mutagens. After routine hypotonic treatment,fixation, and squash preparation, finally, to read the film under the microscope. Results The CHL chromosome aberration test showed that both the chromosome aberration rates of mitomycin- and cyclophosphamide-treated cells were significantly increased(>20%),while the aberration rates in the negative control group were less than 5%, either with and without metabolic activation. The success rate was high and the prepared chromosomes were well dispersed with a moderate length. Conclusions Many factors can affect the specimen preparation in chromosome aberration test. Every step is very important,and it should be strictly following the operating procedures. It is of importance to grasp the principle of each step and to operate carefully,patiently and scientifically in order to prepare good specimens.

A pilot study of the modulation of sirtuins on arylamine -acetyltransferase 1 and 2 enzymatic activity

Arylamine -acetyltransferase (NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide, which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2, SIRT1 and SIRT6 in peripheral blood mononuclear cells (PBMC) from healthy subjects were examined by flow cytometry and Western blot. The activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist (resveratrol) and inhibitor (nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1+ cells compared to SIRT6 in lymphocytes. NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence of resveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

Comparison of urinary proteomes among rats, guinea pigs and golden hamsters / 生物工程学报

Biomarkers are the detectable changes associated with physiological or pathological changes. Urine as excreta of the body, without the mechanisms to maintain a homeostatic internal environment, can reflect a variety of changes in the body. Using animal models can simulate human disease processes, monitor disease changes, and provide clues to early diagnosis. Rats as commonly used model animals are not the dominant models for all disease, thus comparing the urinary proteins of rats with other animals to provide clues to the selection of other animal dominant models. In this study, urinary proteins were digested and profiled by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The urinary proteins of rats, guinea pigs and golden hamsters were compared. The results showed that the number of urine proteins in the three different animals was different, and also different in every system of the body. This provides a basis for selecting the best animal models for different diseases.

Maternal infection by Leishmania braziliensis in hamster does not influence the course of disease in progeny / A infecção materna por Leishmania braziliensis em hamster não influencia o curso da doença na prole

Introduction: Immunoglobulins, soluble antigens, cells, cytokines and other immune system products can be transferred from infected mother to her offspring, leading to suppression or stimulation of immune response. Objective: To evaluate the influence of gender and maternal infection with Leishmania braziliensis in the course of the disease in the offspring of hamsters. Methods: Offspring born from infected mother (IMO) or non-infected mother (NIMO) by Leishmania braziliensis, both sexes, was infected with the same strain of the mother after 30 days of life and followed for 18 weeks. We evaluated the thickness of the lesion, parasite load and histology of the lesions. Results: The number of parasite in both lesions and lymph node of IMO offspring showed a significant reduction in the 5th week post-infection compared to the NIMO offspring; however, this did not correspond to clinical symptoms. Histopathological analysis revealed that in the IMO offspring, the inflammatory process was more prominent. In relation to gender, it was observed that the male offspring showed lesion thickness and higher parasite burden than females. Conclusion: Maternal infection by L. braziliensis in hamsters does not appear to influence the course of the disease in the homologous offspring infection, as well as the male offspring presented augmented susceptibility to L. braziliensis infection regardless of whether they were born from IMO or NIMO. Also, the reduction of the granuloma index in the IMO offspring, together with the higher inflammatory response, suggests a less effective cellular response in the chronic phase of the disease in these animals. (AU)

Introdução: Imunoglobulinas, antígenos solúveis, células, citocinas e outros produtos do sistema imune podem ser transferidos de mãe infectada para a sua prole, levando à supressão ou estimulação da resposta imune. Objetivo: Avaliar a influência do gênero e a infecção materna por Leishmania braziliensis no curso da doença na prole de hamsters. Métodos: Filhotes nascidos de mãe infectada (MI) e mãe não infectada (MNI) por L. braziliensis, ambos os sexos, foram infectados com a mesma cepa da mãe após 30 dias de vida e acompanhados por 18 semanas. Avaliou-se a espessura da lesão, a carga parasitária e os aspectos histopatológicos das lesões. Resultados: A carga parasitária (lesões e linfonodo de drenagem das lesões) da prole nascida de MI mostrou diminuição significativa na 5a semana pós-infecção, comparada àquela nascida de MNI, no entanto, esta diminuição não correspondeu aos sintomas clínicos. A análise histopatológica revelou que na prole nascida de MI, o processo inflamatório mostrou-se mais proeminente. Em relação ao gênero observou-se que os filhotes machos apresentaram espessura das lesões e carga parasitária maiores do que as fêmeas. Conclusão: A infecção materna por L. braziliensis parece não influenciar o curso da doença na infecção homóloga da prole, bem como os filhotes machos apresentaram aumentada susceptibilidade à infecção por L. braziliensis, independente se eles nasceram de MI ou MNI. Além disso, a redução no index de granulomas na prole nascida de MI, em conjunto com a maior resposta inflamatória, sugere uma resposta celular menos efetiva na fase crônica da doença nestes animais. (AU)

Impact of sodium butyrate and mild hypothermia on metabolic and physiological behaviour of CHO TF 70R cells

Background: To reduce costs associated with productivity of recombinant proteins in the biopharmaceutical industry, research has been focused on regulatory principals of growth and survival during the production phases of the cell culture. The main strategies involve the regulation of cell proliferation by the modulation of cell cycle control points (G1/S or G2/M) with mild hypothermia and the addition of sodium butyrate (NaBu). In this study, batch culture strategies were evaluated using CHO TF 70R cells producing the recombinant human tissue plasminogen activator (rh-tPA), to observe their individual and combined effect on the cellular physiological state and relevant kinetic parameters. Results: NaBu addition has a negative effect on the mitochondrial membrane potential (ΔΨm), the values of which are remarkably diminished in cultures exposed to this cytotoxic compound. This effect was not reflected in a loss of cell viability. NaBu and mild hypothermic conditions increased the doubling time in the cell cultures, suggesting that these strategies triggered a general slowing of each cell cycle phase in a different way. Finally, the individual and combined effect of NaBu and mild hypothermia produced an increase in the specific rh-tPA productivity in comparison to the control at 37°C without NaBu. Nevertheless, both strategies did not have a synergistic effect on the specific productivity. Conclusions: The combination of NaBu addition and mild hypothermic condition causes an impact on physiological and metabolic state of CHO TF 70R cells, decreasing cell growth rate and improving glucose consumption efficiency. These results therefore provide a promising strategy to increase specific productivity of rh-tPA.