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Considering the lack of consensus related to the impact of selective IL-6 receptor inhibition on bone remodeling and the scarcity of reports, especially on large bone defects, this study proposed to evaluate the biological impact of the selective inhibitor of interleukin-6 receptor (tocilizumab) in an experimental model of critical calvarial defect in rats. In this preclinical and in vivo study, 24 male Wistar rats were randomly divided into two groups (n=12/group): defect treated with collagen sponge (CG) and defect treated with collagen sponge associated with 2 mg/kg tocilizumab (TCZ). The defect in the parietal bone was created using an 8-mm diameter trephine drill. After 90 days, the animals were euthanized, and tissue samples (skull caps) were evaluated through micro-CT, histological, immunohistochemistry, cytokines, and RT-qPCR analyses. Tocilizumab reduced mononuclear inflammatory infiltration (P<0.05) and tumor necrosis factor (TNF)-α levels (P<0.01) and down-regulated tissue gene expression of BMP-2 (P<0.001), RUNX-2 (P<0.05), and interleukin (IL)-6 (P<0.05). Moreover, it promoted a stronger immunostaining of cathepsin and RANKL (P<0.05). Micro-CT and histological analyses revealed no impact on general bone formation (P>0.05). The bone cells (osteoblasts, osteoclasts, and osteocytes) in the defect area were similar in both groups (P>0.05). Tocilizumab reduced inflammatory cytokines, decreased osteogenic protein, and increased proteases in a critical bone defect in rats. Ninety days after the local application of tocilizumab in the cranial defect, we did not find a significant formation of bone tissue compared with a collagen sponge.
Assuntos
Citocinas , Modelos Animais de Doenças , Ratos Wistar , Receptores de Interleucina-6 , Crânio , Animais , Masculino , Citocinas/metabolismo , Receptores de Interleucina-6/antagonistas & inibidores , Crânio/efeitos dos fármacos , Ratos , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Microtomografia por Raio-X , Peptídeo Hidrolases/metabolismo , Imuno-Histoquímica , Distribuição AleatóriaRESUMO
Advanced therapy medicinal products (ATMP) are medicines for human use that are based on genes, cells or tissues. Over the past years, an increasing number of ATMP entered the market for treatment of cancer, genetic disorders, skeletal defects and metabolic diseases. However, the ATMP production methods often change from the initial concept to commercialization. This change is needed to improve the manufacturing feasibility for scaling up or scaling out. Moreover, the production must adhere to current good manufacturing practices (GMP), and needs to follow a risk-based approach, which often is challenging to implement due to the novelty of the products. Since most of the early ATMP development is done in academia, an environment that is not familiar with regulatory requirements for ATMP production in GMP, the initial manufacturing choice for pre-clinical studies is usually very different from what is required for clinical use. This leads to a lengthy production process optimization, unnecessary repetition of experiments and ultimately waste of funding. This consideration prompted us to provide an intermediate step between early ATMP production in research settings to GMP manufacturing. We built a dedicated facility, and we called this environment 'pre-GMP' to highlight that it is a step toward preparation to GMP manufacturing. This environment supports process development and provides a manufacturing fitness room before transferring to GMP suites. This paper addresses the relevance of pre-GMP, underlining the advantages and the possible disadvantages of this additional framework that may be key in accelerating the pace of ATMP toward clinic.
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BACKGROUND: Obesity is associated with numerous cardiac complications, including arrhythmias, cardiac fibrosis, remodeling and heart failure. Here we evaluated the therapeutic potential of mesenchymal stromal cells (MSCs) and their conditioned medium (CM) to treat cardiac complications in a mouse model of high-fat diet (HFD)-induced obesity. METHODS: After obesity induction and HFD withdrawal, obese mice were treated with MSCs, CM or vehicle. Cardiac function was assessed using electrocardiography, echocardiography and treadmill test. Body weight and biochemical parameters were evaluated. Cardiac tissue was used for real time (RT)-polymerase chain reaction (PCR) and histopathologic analysis. RESULTS/DISCUSSION: Characterization of CM by protein array showed the presence of different cytokines and growth factors, including chemokines, osteopontin, cystatin C, Serpin E1 and Gas 6. HFD-fed mice presented cardiac arrhythmias, altered cardiac gene expression and fibrosis reflected in physical exercise incapacity associated with obesity and diabetes. Administration of MSCs or CM improved arrhythmias and exercise capacity. This functional improvement correlated with normalization of GATA4 gene expression in the hearts of MSC- or CM-treated mice. The gene expression of connexin 43, troponin I, adiponectin, transforming growth factor (TGF) ß, peroxisome proliferator activated receptor gamma (PPARγ), insulin-like growth factor 1 (IGF-1), matrix metalloproteinase-9 (MMP9) and tissue inhibitor of metalloproteinases 1 (TIMP1) were significantly reduced in MSCs, but not in CM-treated mice. Moreover, MSC or CM administration reduced the intensity of cardiac fibrosis. CONCLUSION: Our results suggest that MSCs and CM have a recovery effect on cardiac disturbances due to obesity and corroborate to the paracrine action of MSCs in heart disease models.
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Meios de Cultivo Condicionados/farmacologia , Dieta Hiperlipídica/efeitos adversos , Coração/fisiopatologia , Transplante de Células-Tronco Mesenquimais/métodos , Obesidade/fisiopatologia , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/terapia , Citocinas/metabolismo , Fibrose/genética , Fibrose/patologia , Fator de Transcrição GATA4/genética , Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos Endogâmicos BALB C , Miocárdio/patologia , Obesidade/etiologiaRESUMO
The aim of this experiment was to determine effects of treating peripartum dairy cows with body condition score ≥3.75 with recombinant bovine somatotropin (rbST) on immune, inflammatory, and metabolic responses. Holstein cows (253±1d of gestation) were assigned randomly to 1 of 3 treatments: untreated control (n=53), rbST87.5 (n=56; 87.5mg of rbST), and rbST125 (n=57; 125mg of rbST). Cows in the rbST87.5 and rbST125 treatments received rbST weekly from -21 to 28d relative to calving. Growth hormone, insulin-like growth factor 1, haptoglobin, tumor necrosis factor α, nonesterified fatty acids, ß-hydroxybutyrate, glucose, and cortisol concentrations were determined weekly from -21 to 21d relative to calving. Blood sampled weekly from -14 to 21d relative to calving was used for hemogram and polymorphonuclear leukocyte (PMNL) expression of adhesion molecules, phagocytosis, and oxidative burst. Cows were vaccinated with ovalbumin at -21, -7, and 7d relative to calving, and blood was collected weekly from -21 to 21d relative to calving to determine IgG anti-ovalbumin concentrations. A subsample of cows had liver biopsied -21, -7, and 7d relative to calving to determine total lipids, triglycerides, and glycogen content. Growth hormone concentrations prepartum (control=11.0±1.2, rbST87.5=14.1±1.2, rbST125=15.1±1.3ng/mL) and postpartum (control=14.4±1.1, rbST87.5=17.8±1.2, rbST125=21.8±1.1ng/mL) were highest for rbST125 cows. Cows treated with rbST had higher insulin-like growth factor 1 concentrations than control cows (control=110.5±4.5, rbST87.5=126.2±4.5, rbST125=127.2±4.5ng/mL) only prepartum. Intensity of L-selectin expression was higher for rbST125 than for control and rbST87.5 cows [control=3,590±270, rbST87.5=3,279±271, rbST125=4,371±279 geometric mean fluorescence intensity (GMFI)] in the prepartum period. The PMNL intensities of phagocytosis (control=3,131±130, rbST87.5=3,391±133, rbST125=3,673±137 GMFI) and oxidative burst (control=9,588±746, rbST87.5=11,238±761, rbST125=12,724±781 GMFI) were higher for rbST125 cows than for control cows during the prepartum period. Concentrations of serum IgG anti-ovalbumin tended to be higher for rbST125 cows than for control cows (control=0.75±0.11, rbST87.5=0.94±0.10, rbST125=1.11±0.11 optical density) in the prepartum period. Haptoglobin concentration was significantly reduced 7d postpartum for rbST125 treatment compared with control and rbST87.5 treatments (control=2.74±0.28, rbST87.5=2.81±0.28, rbST125=1.87±0.28 optical density). Although treatment tended to affect postpartum ß-hydroxybutyrate (control=747.5±40.2, rbST87.5=753.2±40.1, rbST125=648.8±39.7 µmol/L), it did not affect liver contents of total lipids, triglycerides, or glycogen. Incidence of metritis among rbST125 cows was reduced compared with that in control cows (control=23.1, rbST87.5=18.0, rbST125=7.8%). Treatment of dairy cows with 125mg of rbST improved innate immune responses and IgG concentration, with limited effects on metabolism.
Assuntos
Doenças dos Bovinos/fisiopatologia , Hormônio do Crescimento/administração & dosagem , Inflamação/veterinária , Período Periparto , Período Pós-Parto , Ácido 3-Hidroxibutírico/sangue , Imunidade Adaptativa/imunologia , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/metabolismo , Ácidos Graxos não Esterificados/sangue , Feminino , Hormônio do Crescimento/sangue , Haptoglobinas/análise , Inflamação/imunologia , Inflamação/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Selectina L/metabolismo , Neutrófilos/imunologia , Ovalbumina/imunologia , Período Periparto/fisiologia , Fagocitose/imunologia , Período Pós-Parto/imunologia , Explosão Respiratória/imunologiaRESUMO
Eighty-one Holstein and Holstein-cross dairy calves fed calf milk replacer (CMR) were used to determine response to increasing amounts of supplemental fat during mild cold stress. Calves (n=27) were randomly assigned to 1 of 3 treatments: (1) low fat [LF; 28% crude protein:15% fat milk replacer (28:15 MR)]; (2) medium fat [MF; 28:15 MR+113 g/d of commercial fat supplement (FS)]; (3) high fat (HF; 28:15 MR+227 g/d of FS). The MF and HF calves received FS from d 2 to 21, and all calves were fed LF from d 22 to 49. The CMR was fed at 1.4% of birth body weight (BBW) from d 1 to 10, at 1.8% of BBW from d 11 to 42, and at 0.9% of BBW from d 43 to 49. Calves were weaned on d 49 and remained in hutches until d 56. The CMR was reconstituted to 13% solids. Calves were fed a commercial starter grain (19.2% crude protein on a dry matter basis) ad libitum and offered warm water after CMR feeding. Calves were fed CMR twice daily at 0630 and 1730 h in hutches bedded with straw. Starter intake, CMR intake, and ambient temperature were measured daily, and body weight (BW), hip height, and body length were measured weekly. Data were analyzed using PROC MIXED in SAS (SAS Institute Inc., Cary, NC) as a randomized design with linear and quadratic contrasts. Calf BBW averaged 42.0 ± 1.0 kg, total serum protein averaged 5.8 ± 0.1mg/dL, and birth ambient temperature averaged 5.0 ± 1.1°C. Feeding FS increased metabolizable energy intake (MEI) over maintenance but decreased efficiency of conversion of BW gain:MEI. Starter intake by LF calves was greatest until the beginning of weaning, after which starter intake was similar among treatments. Because of higher starter intake, total MEI was similar among treatments. Feed efficiency through d 49 was greater for calves fed MF and HF. Average daily gain during fat supplementation was greater for MF and HF than for LF. Lack of increase in BW gain and feed efficiency between MF and HF treatments indicated that HF did not result in advantages over MF. Supplementing fat to preweaned calves fed CMR increased BW gain and decreased starter intake through d 21 which had carryover effects on starter intake on d 49 and reduced hip height and tended to reduced withers height and body length by d 56. The addition of supplemental fat to LF, during mild cold stress, may result in a suboptimal ratio of crude protein to metabolizable energy in the CMR.
Assuntos
Ração Animal/análise , Bovinos , Temperatura Baixa , Gorduras na Dieta/farmacologia , Metabolismo Energético/fisiologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Peso Corporal/fisiologia , Dieta/veterinária , Gorduras na Dieta/administração & dosagem , Suplementos Nutricionais , Infecções Respiratórias , Estresse Fisiológico , Desmame , Aumento de Peso/fisiologiaRESUMO
This study examined the effects of a forage source [wheat straw (WS) versus grass hay (GH)] prepartum and supplemental carbohydrate source [corn (dry feed; DF) versus molasses (liquid feed; LF)] on pre- and postpartum intake, digestibility, selective particle consumption, milk yield, and lipid metabolism. The objectives were to determine if forage or pre- and postpartum supplement alters periparturient intake, energy balance, and milk yield. Sixty (n=15) multiparous dairy cows were used in a randomized complete block design with a 2 × 2 factorial arrangement of treatments to compare WS versus GH diets supplemented with either DF or LF. Dietary treatments were (1) WS prepartum + DF pre- and postpartum (WSDF), 2) WS prepartum + LF pre- and postpartum (WSLF), (3) GH prepartum + DF pre- and postpartum (GHDF), and (4) GH prepartum + LF pre- and postpartum (GHLF). Treatments began at dry-off, × before expected calving. During the prepartum phase, cows maintained dry matter intake (DMI) at 2.0% of body weight and prepartum energy balance remained positive for all treatments until calving. Prepartum GH diets had a more positive energy balance compared with WS diets. On week -5, energy balance was more positive for GHDF than for WSDF or GHLF. Energy balance for WSLF, however, was lower on week -3 and -1 than GHDF. Liquid feed decreased dry matter digestibility and increased prepartum liver triglyceride, serum nonesterified fatty acids (NEFA), and tended to increase ß-hydroxybutyrate. After calving, LF decreased DMI and energy balance, but not yield of milk or 3.5% fat-corrected milk, resulting in greater feed efficiency compared with DF. Forage did not affect postpartum DMI, but milk yield tended to be higher for WS versus GH. The DMI expressed as percentage of body weight was not affected by supplement or prepartum forage type. Cows fed WS had lower serum NEFA, higher liver glycogen, and tended to have a lower triglyceride to glycogen ratio postpartum than GH. Serum NEFA peaked on d 14 for all treatments and then declined thereafter. In postpartum diets, more particles were retained on the top screen for LF (>19.0mm) of the Penn State Particle Separator, which also tended to have more particles in the second screen (particles 19.0-8.0mm). Supplement had minimal effect on postpartum selective particle consumption. In conclusion, feeding diets containing WS resulted in lower postpartum serum NEFA, higher liver glycogen, and a tendency for greater milk production and lower liver triglyceride to glycogen than those containing GH. Liquid feed reduced postpartum DMI but not yield of milk yield or 3.5% fat-corrected milk yield, resulting in an improvement in feed efficiency. Future research should continue to investigate the use of single dry cow diet feeding strategies as they affect pre- and postpartum animal responses.
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Dieta/veterinária , Lactação/fisiologia , Metabolismo dos Lipídeos/fisiologia , Período Periparto/fisiologia , Ração Animal/análise , Animais , Bovinos/fisiologia , Suplementos Nutricionais , Ingestão de Energia/fisiologia , Metabolismo Energético/fisiologia , Feminino , Fígado/metabolismo , Melaço , Período Periparto/efeitos dos fármacos , Poaceae , Triticum , Zea maysRESUMO
The aims of this study were to determine if feeding frequency (FF) of milk replacer (MR; meals/d) alters starter intake, growth, and efficiency of growth in nursery calves fed a conventional or accelerated MR. We hypothesized that smaller and more frequent MR meals would increase starter intake and growth when greater amounts of MR nutrients are fed to nursery calves. Forty-eight Holstein and Holstein-cross heifer and bull calves were assigned to treatments in a 2 × 2 factorial arrangement of MR and FF. Treatments included (1) standard 20% CP and 20% fat MR fed at 1.5% of body weight (BW; 2 meals/d; STD2), (2) standard 20% CP and 20% fat MR fed at 1.5% of BW (4 meals/d; STD4), (3) modified 26% CP and 18% fat MR fed at 2.0% of BW (2 meals/d; MOD2), and (4) modified 26% CP and 18% fat MR fed at 2.0% of BW (4 meals/d; MOD4; n=12). All calves were fed at 0600 and 1700 h and STD4 and MOD4 calves were fed 2 additional meals at 1100 and 1400 h, resulting in the same amount of MR offered for the 2 × and 4 × treatments. Treatments were fed from d 2 to 42 of age and all MR feeding rates were adjusted weekly to maintain MR solids intakes at 1.5 or 2.0% of BW for STD and MOD diets, respectively, and were reconstituted to 15% total solids. Milk replacer FF was reduced by 50% on d 36 and calves were weaned on d 42. Calves were housed in hutches bedded with straw and offered water and a texturized 18% CP starter ad libitum. Calf body weight and body structure were measured weekly and starter intake and fecal scores were measured daily. Through both 42 and 56 d, calves fed MOD had greater CP and fat intake, resulting in increased average daily gain, heart girth, circulating nonesterified fatty acids (NEFA), and muscle total lipid compared with calves fed STD diets. Calves fed MOD diets consumed less starter grain than STD calves but total dry matter intake was similar among treatments. Increased FF for STD calves resulted in greater starter intake at weaning but increasing FF of MOD calves did not have this effect. Due to differences in starter intake, total dry matter by calves through d 56 was similar across treatments. Additionally, increased FF tended to increase serum NEFA concentrations. Serum NEFA concentration was negatively correlated with starter intake. The BW of calves fed STD2 and STD4 treatments almost doubled and the BW of calves on the MOD2 and MOD4 treatments doubled by d 56. Increased FF of the conventional MR program but not accelerated MR program increased starter intake. Increased FF did not affect growth, starter intake, or gain:feed ratio.
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Ração Animal , Bovinos/crescimento & desenvolvimento , Ingestão de Alimentos/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/fisiologia , Bovinos/fisiologia , Indústria de Laticínios/métodos , Dieta/veterinária , Proteínas Alimentares/metabolismo , Feminino , Masculino , Valor Nutritivo/fisiologia , Aumento de Peso/fisiologiaRESUMO
Nuclear and radiological accidents have demonstrated the need for prior planning for exposure assessment as well as guidelines for the implementation of protection and remediation measures of contaminated areas. Typically, the description of the efficiency of the measures in the literature is associated with the reduction of the concentration of the environmental media where they are applied. In order to verify the efficiency related to the reduction in doses, some basic scenarios were established, taking into account aspects of a typical tropical climate, such as building materials (urban areas) and types of crops and farming practices, considering the seasonality and soil type typical of the southeastern region of Brazil. The Integrated System for Emergency (SIEM) program was used to perform the simulations. The results indicate that decision-making processes must be made in accordance with the actual conditions of contamination and use of the affected area. For rural areas, the effectiveness of measures depends on many factors specific to each site, such as seasonality, produced crops, diet habits and degree of subsistence on the items in the diet, which make it unfeasible to develop generic predefined scenarios. The criteria for classification of measurements were defined as: (i) the efficiency in reducing the doses in the first year, in which largest dose rates are observed; (ii) the efficiency in reducing the long-term dose, considering 50 y for adults and (iii) the effect of delay in implementation of the measures on the reduction of doses.
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Poluentes do Solo , Solo , Agricultura , Brasil , Exposição AmbientalRESUMO
The reproductive cycles of lizards, including Tropidurus species, have been widely studied. However, few studies describe in detail the ultrastructure and the epithelial changes in the epididymis. Using histology and trasmission electron microscopy, we show the seasonal changes in the testis and epididymis of the lizard Tropidurus itambere, during its annual reproductive cycle. The reproductive cycle of T. itambere was analysed from June 1988 to June 1989 and from June 2001 to June 2002. While the frequency of reproductive males in the population varied throughout the year, there were reproductive males in most months except for February through April. During this nonreproductive period, there is a reduction in the mean seminiferous tubule volume and few sperm were found in both the testis and the epididymis.
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Epididimo/anatomia & histologia , Lagartos/anatomia & histologia , Estações do Ano , Espermatogênese/fisiologia , Testículo/anatomia & histologia , Animais , Epididimo/ultraestrutura , Lagartos/fisiologia , Masculino , Microscopia Eletrônica de Transmissão , Reprodução/fisiologia , Testículo/ultraestruturaRESUMO
The reproductive cycles of lizards, including Tropidurus species, have been widely studied. However, few studies describe in detail the ultrastructure and the epithelial changes in the epididymis. Using histology and trasmission electron microscopy, we show the seasonal changes in the testis and epididymis of the lizard Tropidurus itambere, during its annual reproductive cycle. The reproductive cycle of T. itambere was analysed from June 1988 to June 1989 and from June 2001 to June 2002. While the frequency of reproductive males in the population varied throughout the year, there were reproductive males in most months except for February through April. During this nonreproductive period, there is a reduction in the mean seminiferous tubule volume and few sperm were found in both the testis and the epididymis.
O ciclo reprodutivo de alguns lagartos já foi extensamente estudado, incluindo algumas espécies do gênero Tropidurus. Entretanto, poucos estudos mostram os aspectos morfológicos do testículo e epidídimo, durante o ciclo reprodutivo anual, e não existe nenhuma descrição ultraestrutural das variações epiteliais no epidídimo. O presente estudo foi feito para mostrar, através da histologia e microscopia eletrônica de transmissão, detalhes das mudanças sazonais no testículo e epidídimo durante o ciclo reprodutivo anual. Acompanhamos o ciclo de Tropidurus itambere, na floresta atlântica brasileira, durante o período de junho de 2001 a junho de 2002. Os machos variam em sua atividade reprodutiva, mas foram considerados potencialmente reprodutivos na maioria dos meses, à exceção dos meses de fevereiro a abril. Durante este período, ocorreu uma redução no volume médio dos túbulos seminíferos e uma menor quantidade de espermatozóides no testículo e no epidídimo.
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Animais , Masculino , Epididimo/anatomia & histologia , Lagartos/anatomia & histologia , Estações do Ano , Espermatogênese/fisiologia , Testículo/anatomia & histologia , Epididimo/ultraestrutura , Lagartos/fisiologia , Microscopia Eletrônica de Transmissão , Reprodução/fisiologia , Testículo/ultraestruturaRESUMO
In the present work, three commercial acid (pH 3.5-4) pyrogallol-containing hair gels, SunSet Alizador Negro (two formulations) and Embelleze Henê Gel, were tested for mutagenicity using two well-established assays. In the Salmonella mutagenicity assay using 648-5000 microg/plate of cosmetic samples, none of the samples reached a 2-fold increase in revertants relative to the controls. Both in the absence and in the presence of S9, the dose-response relation in strains TA98, TA100, TA102, TA1535, and TA1537 was not significant (p>0.01). In the mouse bone marrow micronucleus assay, 10 Swiss male mice were orally administered 2000 mg/kg of sample per body weight/day. The ratio between polychromatic and normochromatic erythrocytes as well as the presence of micronuclei in bone marrow cells were determined. Equal numbers of micronucleated polychromatic erythrocytes were detected between the cells of each treated group and the negative control, using ANOVA and chi-square analyses. Thus, none of the products induced mutagenesis in either assay. Previous studies have shown pyrogallol is mutagenic in various test systems, including Salmonella. However studies have also shown that acidic conditions may repress the reactive-oxygen species (ROS) produced by pyrogallol, and ROS is considered the primary mechanism for the mutagenicity of pyrogallol. Consistent with this are our results, which show that acidic, commercially available pyrogallol-containing hair gels are neither mutagenic in Salmonella nor induce micronuclei in mouse bone marrow in vivo.