Calbindin-D9k mRNA expression in the rat uterus following exposure to methoxychlor: a comparison of oral and subcutaneous exposure

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein that is induced by estrogenic compounds possibly through estrogen receptors. We compared CaBP-9k mRNA expression in the uterus with uterotrophic response in immature rats exposed to methoxychlor (MC), an environmental chemical with estrogenic activity. MC was orally or subcutaneously administered to 3-week-old female Sprague-Dawley rats for 3 days. The weights of the uterus and vagina significantly increased in the oral treatment group at a dose of 50, 100 and 200 mg/kg, but those of the subcutaneous (SC) treatment group only increased at 200 mg/kg. Northern blot analysis showed that CaBP-9k mRNA expression was significantly induced in a dose-dependent manner at doses of 50, 100 and 200 mg/kg/day in the oral treatment group. SC administration of MC induced significant expression at only a dose of 200 mg/kg/day; this was similar to the uterotrophic response. MC has an estrogenic effect on the uterus as shown by the increase in weight and induction of CaBP-9k mRNA expression, which were much greater following exposure via oral gavage than via the SC route. The strong correlation between the results of in vivo uterotrophic assay and CaBP-9k mRNA expression suggests that CaBP-9k mRNA expression in the rat uterus may be used as an early gene marker for detection of the estrogenic effects of putative environmental chemicals.     

Maternal exposure to bisphenol a during late pregnancy resulted in an increase of Calbindin-D9k mRNA and protein in maternal and postnatal rat uteri

It has been reported that Calbindin-D9k (CaBP-9k) is rapidly and strongly induced by environmental estrogenic compounds, possibly through estrogen receptors (ERalpha) in the uterus of mammals. CaBP-9k can be evaluated as an early gene marker for assaying estrogenic effects of putative environmental chemicals in the rat uterus. This study was undertaken to investigate CaBP-9k mRNA and protein expression in the postnatal rat uterus following maternal exposure to 17beta-estradiol (E2) and bisphenol A (BPA) during the neonatal period. Treatment with a high dose of BPA (600 mg/kg body weight (BW) per day) resulted in a 3-fold increase in CaBP-9k mRNA expression for 3 days, while a single dose of E2 (40 microg/kg BW per day) induced 2-fold increase of this gene in the maternal uterus. In an agreement with maternal CaBP-9k mRNA, postnatal CaBP-9k mRNA in the uterus increased 4-fold when treated with BPA (600 mg/kg BW per day). In addition, treatment with increasing concentrations of BPA resulted in significant increases in CaBP-9k protein in the maternal rat uterus. It is of interest that increasing doses of BPA induced a significant ERalpha mRNA increase in the postnatal uterus. Furthermore, immunohistochemistry revealed that treatment with BPA induced CaBP-9k protein in the maternal uterus. We demonstrated that maternal exposure to BPA during late pregnancy induced CaBP-9k mRNA and protein in maternal and postnatal rat uteri. These results suggest that rapid absorption and distribution of environmental estrogenic compounds occurs in maternal and neonatal rat uteri and these chemicals can easily pass though the placenta during pregnancy to affect postnatal reproductive functions.     

Adipogenesis: usefulness of in vitro and in vivo experimental models

The use of experimental models is the foundation of experimental biology, so it is important to know how much the models can tell us about actual animals. Inconsistent or contradictory results from in vitro models are often associated with the perception that a particular model or results are somehow wrong and therefore cannot tell us anything important about how an animal works. In fact, in vitro conditions do not create new biology. Differences between in vitro and in vivo behavior can only result from the actual cellular repertoire, which provides a powerful tool to uncover new information. Adipose tissue research provides a useful context for examining this issue because the regulation of adipose growth and metabolism has important economic implications for livestock production. Examples are discussed in which either excess skepticism or narrow interpretation of results slowed progress toward our current understanding of adipose biology. Similarly, contemporary examples using genomics are used to suggest that large inconsistencies are still apparent with in vitro methods. Careful consideration of these inconsistencies may provide new insights.     

Avirulence of viable but non-culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 degrees C and Scott A strain at 4 degrees C. No culturable bacteria were detected in the VBNC state, although 104 active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state.     

In vitro and in vivo binding of phenylbutazone and related drugs to equine feeds and digesta

In vitro and in vivo studies of phenylbutazone binding to equine ingesta and digesta were undertaken. In vitro binding to chopped hay and powdered pony nuts in buffer solutions at 37 degrees C was found to be time-, concentration- and pH-dependent. Percentage binding generally increased with time, decreased with concentration and varied with buffer pH in an unpredictable manner. Other non-steroidal anti-inflammatory drugs (NSAIDs) also bound to hay, the degree of binding being less for meclofenamate and least for flunixin in comparison with phenylbutazone. Phenylbutazone became bound to digesta collected from eight regions of the gastrointestinal tract when they were spiked with a concentration of 1 mg.10 g-1 digesta, the amounts ranging from 80.0 per cent (duodenum) to 99.6 per cent (stomach). Binding also occurred to equine digesta following the oral administration of phenylbutazone (4.4 mg.kg-1) to three ponies. It was concluded that drug uptake by and release from equine ingesta and digesta were probably adsorptive and desorptive processes. The clinical significance of the findings for the use of NSAIDs in equine medicine was considered.     

桉叶注射剂体内外抗菌作用研究

考察桉叶注射液体内外抗菌作用。桉叶注射剂体外对金黄色葡萄球菌和链球菌的最低抑菌浓度(M IC)分别为0.004 g/mL和0.008 g/mL,最低杀菌浓度(MBC)分别为0.008 g/mL和0.015 g/mL,对金黄色葡萄球菌的作用明显强于对链球菌。体内抑菌试验中,小白鼠共分4组,均注射1个MLD菌液,1-3组分别为感染前12 h给药、感染同时给药、感染后12 h给药,4组为细菌对照组,结果表明,在感染同时给药效果最佳,且对两种菌人工感染的治疗效果相当。     

纳米蜂胶黄酮对猪细小病毒的体内外抗病毒作用

采用体内外试验研究纳米蜂胶黄酮抗猪细小病毒(PPV)作用。体外试验是以普通蜂胶黄酮为对照,分3种加药方式(先加蜂胶、后加蜂胶、蜂胶和病毒同时加入)加至PK-15细胞中,用MTT法检测细胞活性,实时荧光PCR检测PK-15中PPV含量,观察蜂胶黄酮对PK-15抗PPV及清除PPV能力的影响。体内试验是给豚鼠注射PPV后灌胃纳米蜂胶黄酮和普通蜂胶黄酮,检测肺脏、肾脏、肝脏、性腺及血液中PPV含量、血清中HI含量。结果显示:(1)与普通蜂胶黄酮相比,纳米蜂胶黄酮可显著抑制PPV在PK-15上生长,高浓度效果优于低浓度,3种加药方式中先加药物的效果最好;(2)高中剂量的纳米蜂胶黄酮可以显著抑制PPV在血液中复制、减轻PPV对豚鼠体重的影响、提高血清HI效价。结果表明:蜂胶黄酮经纳米化处理后,可以显著提高其抗PPV活性。     

Enrofloxacin is able to control Toxoplasma gondii infection in both in vitro and in vivo experimental models

Currently, toxoplasmosis is treated with sulfadiazine and pyrimethamine. However, this treatment presents several adverse side effects; thus, there is a critical need for the development and evaluation of new drugs, which do not present the same problems of the standard therapy. Enrofloxacin is a fluoroquinolone antibiotic known to control infection against several bacteria in veterinary medicine. Recently, this drug has demonstrated protective effects against protozoan parasites such as Neospora caninum. The present study aimed to determine the effect of enrofloxacin in the control of Toxoplasma gondii infection. For this purpose, human foreskin fibroblast (HFF) cells were infected with T. gondii RH strain and treated with sulfadiazine, penicillin/streptomycin, pyrimethamine, or enrofloxacin. Following treatment, we analyzed the infection index, parasite intracellular proliferation and the number of plaques. Additionally, tissue parasitism and histological changes were investigated in the brain of Calomys callosus that were infected with T. gondii (ME49 strain) and treated with either sulfadiazine or enrofloxacin. Enrofloxacin was able to reduce the infection index, intracellular proliferation and the number of plaques in HFF cells infected by T. gondii in comparison with untreated or penicillin/streptomycin-treated ones. Enrofloxacin was more protective against T. gondii in HFF infected cells than sulfadiazine treatment (P<0.001). In addition, pyrimethamine, enrofloxacin or the associations of sulfadiazine plus pyrimethamine, enrofloxacin plus sulfadiazine or enrofloxacin plus pyrimethamine-treatments were able to reduce the plaque numbers in HFF cells infected by T. gondii when compared to medium, penicillin/streptomycin or sulfadiazine alone. In vivo experiments demonstrated that enrofloxacin diminished significantly the tissue parasitism as well as the inflammatory alterations in the brain of C. callosus infected with T. gondii when compared with untreated animals. Based on our findings, it can be concluded that enrofloxacin is a potential alternative drug for the treatment of toxoplasmosis.     

In vitro and in vivo anthelmintic activity of Euphorbia helioscopia L

The anthelmintic potentials of the aqueous and methanol extracts of Euphorbia helioscopia were investigated. In folklore medicine, E. helioscopia (Euphorbiaceae) is used in the treatment of various gastrointestinal ailments and diseases. A worm motility inhibition (WMI) assay and egg hatch assay (EHA) were used for in vitro assessment, and a faecal egg count reduction (FECR) assay was used for an in vivo study. The in vitro study revealed anthelmintic effects of crude methanolic extracts of E. helioscopia on live Haemonchus contortus worms as evident from their paralysis and/or death at 8h after exposure, different concentrations (12.5mgml(-1), 25mgml(-1) and 50mgml(-1)) of aqueous and methanolic extracts were used against H. contortus which exhibited dose-dependent anthelmintic effects on H. contortus. Aqueous and methanolic extracts of aerial parts (stem, leaves and flowers) of E. helioscopia were found to have very low percent inhibitory egg hatching as compared to levamisole. It is concluded that the entire plant of E. helioscopia possesses significant anthelmintic activity and could be a potential alternative for treating cases of helminth infections in ruminants.     

In vitro and in vivo acaricidal activity of a herbal extract

The anti-tick efficacy of combined aqueous herbal extracts of Azadirachta indica leaves, Nicotiana tabacum leaves, Calotropis procera flowers and Trachyspermum ammi seeds was evaluated using adult immersion test, larval packet test and ear bag method. The extract exhibited lethal effects on egg laying (index of egg laying=0.371404±0.00435), hatching (22.35%) and total larval mortality at 50 mg ml(-1) and reduced tick intensity on the infested calves (18 detached out of 35 at 45% (w/w) suspension, topically applied). The herbal extract exerted dose- and time-dependent response against all the developmental stages of Rhipicephalus (Boophilus) microplus considered in this study, thus justified their use in the traditional system of Pakistan.     

Recombinant avian adeno-associated virus: transgene expression in vivo and enhancement of expression in vitro

Recombinant avian adeno-associated viruses coding for the LacZ gene were used to inoculate embryonating chicken eggs, to assess the usefulness of the system for the expression of a transgene in vivo. The results obtained indicate significantly higher levels of expression of the reporter gene at various time intervals in the embryos inoculated with the recombinant virus in comparison with the mock-inoculated controls. At the embryo level, significant differences were evident at 120 hr postinoculation; hatched chicks showed transgene expression up to 14 days of age. In a second experiment, different cell-line cultures were transfected with plasmids encoding for a reporter gene flanked by the avian adeno-associated virus inverted terminal repeats (ITR), either alone or in the presence of the major nonstructural proteins of the virus (Rep 78/68) to assess the ability of these proteins and DNA elements to enhance gene expression. Results indicate that the inclusion of the viral ITR alone or during coexpression of the Rep proteins significantly enhances the expression of the transgene in all cell lines tested, as evidenced by the detection of the beta-galacrosidase protein through chemiluminescence reactions and staining of transfected monolayers.     

Antimicrobial activity of gallium against virulent Rhodococcus equiin vitro and in vivo

Rhodococcus equi, a facultative intracellular bacterium, causes severe pneumonia in foals. Evidence suggests that most foals become infected very early in life, when they have immature or ineffective innate immune responses. This study evaluated the antimicrobial activity of gallium against R. equi, as a potential chemoprophylactic and therapeutic agent. Rhodococcus equi was grown in media with various concentrations of gallium nitrate (GN), with and without excess iron. GN significantly inhibited growth and killed R. equi, and these effects were abolished with excess iron. Antimicrobial effects of Ga appear to be related to its interference with iron metabolism. Mice were treated orally with gallium maltolate (GaM), 10 or 50 mg/kg BW, or distilled H2O prior to and after experimental infection with R. equi. Six days post-infection, organs were harvested and R. equi concentrations assessed, and serum gallium concentrations determined. GaM was absorbed in a dose-dependent manner, and R. equi tissue burdens were greater in control mice than in all GaM-treated mice. GaM may aid in the control of disease by preventing development of overwhelming R. equi tissue burdens prior to the establishment of requisite innate and adaptive immune responses.     

Modulation, in vivo and in vitro, of surface expression of CD18 by bovine neutrophils.

A series of experiments was designed to elucidate some of the factors that may influence surface expression of CD18 by bovine neutrophils. Expression of CD18 was determined by immunofluorescence flow cytometry. Neutrophils recovered from the uterus of cows (n = 9) after intrauterine administration of sterile oyster glycogen solution expressed (mean +/- SD) 123 +/- 21% more CD18 than did circulating neutrophils recovered simultaneously from the same cows (P = 0.003). In 8 cows given 20 mg of dexamethasone IM daily for 3 days, expression of CD18 on blood neutrophils was 29.6 +/- 8% less after treatment than before treatment (P = 0.0078). Neutrophils from 12 cows or bulls exposed to phorbol myristate acetate in vitro increased expression of CD18 by 137 +/- 37% (P = 0.0035). Likewise, exposure of neutrophils from 8 cattle to zymosan-activated bovine plasma increased CD18 exposure by 10.6 +/- 3.8% (P = 0.029). These findings indicate that expression of CD18 by bovine neutrophils is a dynamic system, capable of responding to inflammatory stimuli. Inadvertent activation of neutrophils may be responsible for some of the variance in expression observed when examining large groups of cattle for CD18 expression by neutrophils. The ability of bovine neutrophils to respond rapidly to various stimuli by increasing surface expression of CD18 indicates that a pool of intracellular CD18 may be available for inclusion in the plasmalemma, as has been reported for human neutrophils.     

Enhancement of antibacterial activity of tilmicosin against Staphylococcus aureus by solid lipid nanoparticles in vitro and in vivo

This study aimed to enhance the antibacterial activity of tilmicosin by solid lipid nanoparticles (SLN). Tilmicosin-loaded hydrogenated castor oil (HCO)-SLN was prepared using a hot homogenisation and ultrasonication method. The physicochemical characteristics of SLN were investigated by scanning electron microscopy (SEM) and photon correlation spectroscopy (PCS). The antibacterial activity of tilmicosin-SLN against Staphylococcus aureus was evaluated by growth inhibition and colony-counting method. A therapeutic study of tilmicosin-SLN was conducted by subcutaneous injection in a mouse mastitis model infected with S. aureus by teat canal infusion. Therapeutic efficacy was assessed by physical appearance of the mammary gland and measurement of colony-forming units (CFU) per gland. The results showed that the diameter, polydispersivity index, zeta potential, encapsulation efficiency and loading capacity of the nanoparticles were 343±26 nm, 0.33±0.08, -7.9±0.4 mV, 60.4±3.3% and 11.2±0.47%, respectively. Tilmicosin-SLN showed a sustained-release effect and sustained and enhanced antibacterial activity in vitro. SLN significantly enhanced the therapeutic efficacy of tilmicosin determined by lower CFU counts and a decreased degree of inflammation. These results demonstrated that the HCO-SLN is an effective carrier to enhance the antibacterial activity of tilmicosin.     

Transfer of maternal cytokines to suckling piglets: in vivo and in vitro models with implications for immunomodulation of neonatal immunity

Maternal cytokines may play instructive roles in development of the neonatal immune system. However, cytokines in colostrum and milk and their transfer from mothers to neonates have not been well documented, except for TGF-beta. Swine provide a unique model to study lactogenic cytokines because the sow's impermeable placenta prohibits transplacental passage. We investigated IL-6 and TNF-alpha (pro-inflammatory), IFN-gamma and IL-12 (Th1), IL-10 and IL-4 (Th2) and TGF-beta1 (Th3) concentrations in sow serum and colostrum/milk and serum of their suckling and weaned piglets and in age-matched colostrum-deprived gnotobiotic piglets. All cytokines were detected in colostrum/milk and correlated with concentrations in sow serum except for mammary-derived TNF-alpha and TGF-beta1. Detection of IL-12 and TGF-beta1 in pre-suckling and colostrum-deprived gnotobiotic piglet serum suggests constitutive production: other cytokines were undetectable confirming absence of transplacental transfer. Peak median cytokine concentrations in suckling piglet serum occurred at post-partum days 1-2 (IL-4>IL-6>IFN-gamma>IL-10). The effects in vitro of physiologically relevant concentrations of the two predominant lactogenic cytokines (TGF-beta1 and IL-4) on porcine naive B cell responses to lipopolysaccharide (LPS) and rotavirus (RV) were investigated. High (10 ng/ml) TGF-beta1 suppressed immunoglobulin secreting cell responses to LPS and rotavirus; low concentrations (0.1 ng/ml) promoted isotype switching to IgA antibody. Interleukin-4 induced inverse dose-dependent (0.1 ng>10 ng/ml) isotype switching to IgA and enhanced IgM secreting cell responses to LPS and rotavirus. In summary, we documented the transfer and persistence of maternal cytokines from colostrum/milk to neonates and their potential role in Th-2 biased IgA responses and reduced immunologic responsiveness of neonates.     

香豆素化合物体内外抗狂犬病病毒的效果

狂犬病是由狂犬病病毒(rabies virus,RABV)引起的一种以侵害中枢神经系统为特征的高度致死性人兽共患传染病,虽然人类尝试了多种方法用于治疗狂犬病,但到目前为止,狂犬病一旦出现临床症状,死亡率仍高达100%,因此,寻找有效的抗RABV药物仍是亟待解决的问题。已有研究显示香豆素化合物(coumarin,CM)具有一定的抗病毒作用,但其对RABV的作用目前尚没有研究。首先以BHK-21细胞为模型,通过SRB、直接免疫荧光(DFA)和病毒滴度等方法,在体外筛选具有良好抗RABV作用的CM,进而从直接灭活、阻滞吸附、不同时间点对病毒抑制效果等方面探讨CM体外抗RABV的作用机制。随后构建RABV感染动物模型,观察筛选获得的CM是否可以挽救病毒感染小鼠。结果显示,CM-3(9种CM分别标记为CM-1～9)可以有效抑制RABV在宿主细胞内的复制,但不能直接灭活病毒,也不能阻滞RABV对宿主细胞的吸附;其在RABV感染48h内均可以显著抑制病毒复制,最高抑制率可达60.0%。另外,结果显示在RABV感染后3～12h内用药,CM-3的抑制效果更为显著,抑制率均为50.0%以上;随后,通过体内试验证实CM-3(50mg/kg)可将感染小鼠的存活率由0%提高至12.5%。综上,结果提示,CM-3可能通过感染后抑制病毒复制的方式发挥抗RABV的作用,而此类结构的CM有可能作为潜在的药物用于狂犬病的治疗。     

Adenovirus-mediated expression of interleukin-1 receptor antagonist in swine cells in vitro and in vivo

Adenovirus-mediated gene delivery of anticytokines is a powerful tool for modulating the cytokine environment under conditions of respiratory disease. In order to determine the feasibility of cytokine modulation in the context of respiratory disease in swine, nonreplicating E1- and E3-deficient adenovirus constructs expressing a model protein, beta-galactosidase, and an anticytokine, the IL-1 receptor antagonist (IL-1Ra), were evaluated for in vitro expression in porcine PK15 cells, and in vivo following endotracheal instillation into the lungs. beta-Galactosidase and IL-1Ra were readily expressed in vitro in swine cells. Endotracheal administration of lacZ-containing adenovirus demonstrated that endothelial and epithelial cells in the alveolar spaces and bronchi of the middle and lower lobes were the principal sites of infection and expression, whereas beta-galactosidase staining was not observed in the upper lobe. Endotracheal administration of IL-1Ra recombinant adenovirus resulted in sustained expression of IL-1Ra into the alveolar spaces, where it was recovered in a concentration of 660 pg/ml in 500 ml of lavage fluid, equivalent to 330 ng IL-1Ra, in the lungs 7 days after treatment. Moreover, in vivo instillation of nonreplicating adenovirus did not induce an inflammatory response in the 1-week time frame of the study period. Lung weight as a percent of body weight, serum zinc, serum amyloid A, leukocyte differentials, neutrophil activity, and TNF levels all were the same between untreated pigs and pigs treated with either recombinant adenovirus. The results indicate that the delivery of IL-1Ra to swine lungs via nonreplicating, recombinant adenovirus may be an effective method for in vivo modulation of IL-1 activity and investigation of cytokine involvement in respiratory disease pathogenesis.     

Characterization of in vitro and in vivo antiviral activity of lactoferrin and ribavirin upon hantavirus.

Mechanisms of anti-hantaviral activities of bovine lactoferrin (LF) and ribavirin (Rbv) were investigated. Hantavirus focus formation at 48 hr was 15% of the control in cells treated with 400 microg/ml LF for 1 hr at 37 degrees C prior to viral infection. Post infection treatment with 100 microg/ml Rbv also inhibited the focus formation to 2.5% of the control. Combined LF pre- and Rbv post-infection treatment completely inhibited focus formation. Viral glycoprotein (G2) and nucleocapsid protein (NP) syntheses were delayed in LF pretreated cells up to 24 hr post infection (hpi) but became comparable to the control by 48 hpi. Further, LF inhibited viral shedding at 24 hpi but did not inhibit shedding after 48 hpi. However, Rbv was able to inhibit synthesis of viral proteins, (+) and (-) strand RNAs also inhibited viral shedding after 24 hr. These results suggest that LF inhibits viral adsorption to cells, while Rbv inhibits viral RNA synthesis. For in vivo trials of LF and Rbv, LF pre- and Rbv post-treatment were evaluated in suckling mice infected with hantavirus, of which 7% survived. LF concentrations of 40 and 160 mg/kg administered prior to viral challenge improved survival rates to 15% and 70%, respectively for single administration and 85% and 94%, respectively, for double administration. Rbv concentrations of 25 and 50 mg/kg gave survival rates of 68% and 81%, respectively. This suggests that both LF and Rbv are efficacious in hantavirus infection in vivo.