Determination of RBC Survival in C57BL/6 and C57BL/6-Tg(UBC–GFP) Mice

Although several methods for determining erythrocyte lifespan are used in research studies that involve mice, all involve the alteration of RBC to allow for its tracking over time, which may affect overall RBC survival. The aims of this study were to determine 1) whether sex affects RBC survival; 2) whether RBC survival differs between the biotin method and an alternative method that uses GFP; and 3) whether repeat exposure of mice to biotin results in an antibiotin antibody response or decreased RBC survival. The results suggest no difference in the RBC half-life between male and female C57BL/6 mice (22.9 ± 1.2 and 22.4 ± 0.9 d, respectively). In addition, RBC half-life did not differ between the biotin- and GFP-based methods (20.5 ± 2.1 d and 22.7 ± 2.1 d, respectively). Finally, retransfusion of mice 90 d after an initial transfusion with biotin-labeled RBC did not induce detectable antibiotin antibodies nor alter the half-life of transfused biotin-labeled RBC (initial transfusion, 22.0 ± 1.2 d; subsequent transfusion, 23.4 ± 1.4 d, respectively).Abbreviations: T_1/2, half-lifeRBC lifespan and senescence are important parameters used both clinically and in research studies of hereditary disorders of erythrocyte metabolism, transfusion medicine, and sepsis.^{8,21,27,32,35} Labeling RBC with a biotinylating reagent is a common method used to determine their circulating lifespan. Other methods involve using radioactive isotopes, such as ⁵¹Cr and ⁵⁹Fe.^7,20 Biotinylating reagents are preferred for various research applications with humans,^8,23,24 and are used in a variety of animal models.^{1,25,33,34,37} Once biotin attaches to RBC surface proteins, streptavidin (a protein derived from Streptomyces avidinii) that is labeled with a fluorescent dye is used to form a strong and rapid complex with biotin, thereby allowing for its detection through flow cytometry. Blood samples analyzed sequentially over a period of weeks will show a linear decline in biotin–streptavidin signal as labeled cells age and are cleared from the circulation through the reticuloendothelial system.The characteristics of an ideal label for performing RBC survival studies include: 1) stable presence on or within the cell throughout its normal lifespan; 2) specificity for RBC; 3) inertness, such that the cell does not become prone to accelerated destruction; 4) nonrecycling (that is, the label does not reenter the circulation and bind to new cells after destruction of the labeled RBC); and 5) easy and accurate measurement by using available assays. Radioactive isotopes and other labels fulfill several of these criteria, but their limitations include elution from RBC as well as safety concerns.^7,22 In contrast, biotin poses little to no risk of accumulation or toxicity. The sulfo- N-hydroxysuccinimide–biotin ester used for RBC tracking studies in humans and animals can be administered directly or through the transfusion of biotinylated RBC. Although it is generally accepted that biotinylation of RBC does not affect their function, antibodies to biotin have been demonstrated in some human studies, posing the question of whether repeated administration of biotin ester or biotinylated RBC could interfere with subsequent results within the same subject.^4,20 Repeat transfusions of biotinylated RBC to mice have not been described in the literature. One aim of this study was to determine whether exposure to biotinylated RBC induces an antibiotin antibody response in mice. Furthermore, we tested whether the survival of biotinylated RBC changed after repeat exposure.Recently GFP-expressing RBC have been used to track the posttransfusion survival and recovery of stored RBC administered to nonGFP-expressing recipient mice.^9,12,36 The C57BL/6-Tg(UBC–GFP)30Scha/J mouse strain is characterized by GFP expression under the control of a human ubiquitin C promoter. All tissues of these mice express GFP, including blood.²⁶ GFP expression appears to be consistent throughout life and does not otherwise alter the normal structure, physiology, or function of RBC. In addition, GFP is unaffected by ambient light contamination or degradation, drawbacks that are associated with fluorescent dyes.¹⁵ In addition, GFP allows for the separation of cell populations through flow cytometry.^9,11 Many qualities of GFP suggest that it may serve as a useful surrogate marker in place of other labeling techniques in mice. Therefore, we sought to evaluate the utility of UBC–GFP transgenic mice as an alternative to labeling RBC with biotin esters. Our aim for this work was to determine the survival of RBC in wild-type C57BL/6 mice and in the UBC–GFP strain and to compare methods for determining RBC half-life (T_1/2).     

Sex-restricted non-Mendelian inheritance of mouse Chromosome 11 in the offspring of crosses between C57BL/6J and (C57BL/6J × DBA/2J)F1 mice

We report on the observation of sex-restricted, non-Mendelian inheritance over a region of mouse Chromosome (Chr) 11, occurring in the offspring of crosses between two commonly used Mus musculus-derived inbred strains, C57BL/6J and DBA/2J. In the surviving backcross progeny of reciprocal matings between (C57BL/6J × DBA/2J)F₁ hybrids and the C57BL/6J parental strain, we observed the preferential appearance of C57BL/6J alleles along a region of Chr 11. The deviation from Mendelian predictions was observed only in female offspring from both reciprocal backcrosses, and not in males from either cross. The sex-specificity of the observed non-Mendelian inheritance points to an explanation based on embryonic or neonatal lethality. Our data add to previously obtained evidence for a Chr 11 locus or loci with sex-specific and allele-specific effects on viability. Received: 19 December 1997 / Accepted: 10 June 1998     

Quantitative trait loci for growth traits in C57BL/6J × DBA/2J mice

Quantitative trait loci (QTLs) for body weight and tail length are mapped in an F₂ population of 927 C57BL/6J × DBA/2J mice. We test the concordance between the locations of the mapped QTLs with those detected by changes of marker frequency under artificial selection in a previous experiment with the same base population. The directions of effects of the QTLs are generally in agreement, and in many cases significant QTLs are found in similar map positions, but there are also discrepancies between the two experiments. There are indications of age-specific QTL effects on growth. For body weight traits, the genetic variation in the F₂ appears to result from many loci with relatively small effects. For tail length at 10 weeks, however, a single QTL on Chromosome (Chr) 1 with a peak LOD score of ∼33 contributes most of the genetic variation detected, changes the trait value by about 6%, and explains about 20% of the phenotypic variance of the trait. Received: 4 August 1998 / Accepted: 17 November 1998     

A comparison between intratracheal and inhalation delivery of Aspergillus fumigatus conidia in the development of fungal allergic asthma in C57BL/6 mice

Allergic asthma is a debilitating disease of the airways characterized by airway hyperresponsiveness, eosinophilic inflammation, goblet cell metaplasia with associated mucus hypersecretion,?and airway wall remodelling events, particularly subepithelial fibrosis and smooth muscle cell hyperplasia. Animal models that accurately mimic these hallmarks of allergic airways disease are critical for studying mechanisms associated with the cellular and structural changes that lead to disease pathogenesis. Aspergillus fumigatus, is a common aeroallergen of human asthmatics. The intratracheal (IT) delivery of A. fumigatus conidia into the airways of sensitized mice has been described as a model of allergic disease. Here, we compared the IT model with a newly developed inhalation (IH) challenge model. The IH model allowed multiple fungal exposures, which resulted in an exacerbation to the allergic asthma phenotype. Increased recruitment of eosinophils and lymphocytes, the hallmark leukocytes of asthma, was noted with the IH model as compared to the IT model in which macrophages and neutrophils were more prominent. Immunoglobulin E (IgE) production was significantly greater after IH challenge, while that of IgG(2a) was higher after IT challenge. Airway wall remodelling was pronounced in IH-treated mice, particularly after multiple allergen challenges. Although the IT model may be appropriate for the examination of the played by innate cells in the acute response to fungus, it fails to consistently reproduce the chronic remodelling hallmarks of allergic asthma. The ability of the IH challenge to mimic these characteristics recommends it as a model suited to study these important events.     

Doxorubicin plus tumor necrosis factor α combination treatments in EL4-lymphoma-bearing C57BL/6 mice

 The therapeutic efficacy of a total of 42 single-agent or combination protocols involving doxorubicin (Adriamycin, ADM) and tumor necrosis factor α (TNFα) were evaluated in the syngeneic murine lymphoma model, C57BL/6-EL4. Combination treatments were the most effective and the therapeutic effects were schedule-dependent; e.g. it was generally advantageous for ADM to precede TNFα administration. Two protocols selected for further study were 4 mg/kg ADM i.v. on days 1 and 8 plus TNFα, i.v., at either 16 000 U (7 μg)/injection, on days 1 and 8 or 4000 U (1.7 μg)/injection, on days 11–15. Survival of mice bearing one of four EL4 sublines having different in vitro drug sensitivities was assessed. These sublines were E10 (ADM-sensitive/TNFα-resistant), E16 (sensitive/sensitive), ER2 (ADM-resistant/TNFα-sensitive) and ER13 (resistant/resistant). Between 80% and 100% long-term survivors (i.e. tumor free on day 60) were obtained with the two treatments in mice bearing ADM-sensitive sublines, even though one of these sublines, E10, was resistant to TNFα in vitro. Induction of long-term survival appeared, therefore, to correlate with in vitro defined sensitivity/resistance to ADM, but not to TNFα. Treatment-induced modulations of tumoricidal immune effector functions were also examined. Taken together, the results indicated that induction of long-term survival involved complex interactions of: (1) ADM-induced tumor modifications, including, but not limited to, tumor debulking, (2) combination-treatment-induced modifications of splenic cytolytic T cell and macrophage activities, and (3) the restoration of thymus cellularity. Finally, when long-term survivors resulting from treatment of E10- or E16-bearing mice were implanted with ER2 on day 120, the majority survived, indicating that long-term immune memory, capable of recognizing drug resistant variants, had been established. Received: 14 August 1997 / Accepted: 2 October 1997     

Biological activities of the LXRα and β agonist, 4β-hydroxycholesterol,and of its isomer, 4α-hydroxycholesterol,on oligodendrocytes: Effects on cell growth and viability,oxidative and inflammatory status

The biochemical and biological properties of 4β-hydroxycholesterol and of its isomer, 4α-hydroxycholesterol, are not well known. So, we determined the ability of 4α- and 4β-hydroxycholesterol to react with LXRα and LXRβ, and we characterized the activities of these oxysterols on oligodendrocytes which are myelin synthesizing cells. The effects of 4α- and 4β-hydroxycholesterol were studied on 158N murine oligodendrocytes to assess their activities on cell growth and viability, oxidative and inflammatory status. To this end different parameters were used: cell counting with trypan blue; identification of dead cells and cell cycle analysis with propidium iodide; evaluation of mitochondrial depolarization, lysosomal membrane integrity, actin depolimerization, nuclear morphology, and superoxide anion production after staining with JC-1, acridine orange, rhodamine-phalloidin, Hoechst 33342, and dihydroethidium, respectively; evaluation of ultrastructural changes by transmission electron microscopy, and cytokine quantification with a cytometric bead array. Only 4β-hydroxycholesterol is a LXRα and β agonist. No cytotoxic effects were found with 4α-hydroxycholesterol except a slight inhibition of cell growth at elevated concentrations. At high concentrations, 4β-hydroxycholesterol was not only able to inhibit cell growth, but also to induce cell death associated with a loss of mitochondrial transmembrane potential, dysfunctions of lysosomal membrane integrity, and superoxide anion overproduction. These side effects were lower than those observed with 7-ketocholesterol and 25-hydroxycholesterol used as positive controls. On oligodendrocyte murine primary cultures, only lysosomal membrane integrity was slightly affected under treatment with 4α- and 4β-hydroxycholesterol. So, 4α- and 4β-hydroxycholesterol have different biological activities. Their ability to induce cytotoxic effects on oligodendrocytes can be considered as weak comparatively to 7-ketocholesterol and 25-hydroxycholesterol.     

Do sires and juvenile male mice (C57BL/6) contribute to the rearing of the offspring?

Copulation and/or cohabitation with a pregnant female facilitate paternal behavior in male mice. However, their contribution to the rearing of the offspring is still not well understood. Our aims were to investigate the behavior of sires toward own or alien pups; the immediate consequences of the presence of fathers on the offspring and the behavior of the mother; and whether the exposure of juvenile males to newborn siblings, in an overlapping litters context, facilitates paternal behavior in C57BL/6 mice. We found that sires behaved paternally toward alien pups at both postpartum days 3 and 7; did not affect the behavior of the mother (e.g., licking and grooming, retrieval behavior, time in the nest, and crouching postures); and reduced the time offspring stayed alone in the nest. The exposure to newborn siblings did not promote paternal behavior in juvenile males. Therefore, sires are more paternal than usually described in the literature for laboratory mice, suggesting a facultative role in the rearing of the offspring. However, juvenile male mice, in contrast to juvenile females, could be adapted to leave the nest earlier without major contribution to the offspring.     

WFIKKN1 and WFIKKN2 bind growth factors TGFβ1, BMP2 and BMP4 but do not inhibit their signalling activity

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP domain, a follistatin domain, an immunoglobulin domain, two Kunitz-type protease inhibitor domains and an NTR domain. Recent experiments have shown that both proteins have high affinity for growth and differentiation factor (GDF)8 and GDF11. Here we study the interaction of WFIKKN proteins with several additional representatives of the transforming growth factor (TGF)β family using SPR measurements. Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN proteins bind TGFβ1, bone morphogenetic protein (BMP)2 and BMP4 with relatively high affinity (K(d) ～ 10(-6) m). To assess the biological significance of these interactions we studied the effect of WFIKKN proteins on the activity of GDF8, GDF11, TGFβ1, BMP2 and BMP4 using reporter assays. These studies revealed that WFIKKN1 and WFIKKN2 inhibited the biological activity of GDF8 and GDF11 in the nanomolar range, whereas they did not inhibit the activities of TGFβ1, BMP2 and BMP4 even in the micromolar range. Our data indicate that WFIKKN proteins are antagonists of GDF8 and GDF11, but in the case of TGFβ1, BMP2 and BMP4 they function as growth factor binding proteins. It is suggested that the physical association of WFIKKN proteins with these growth factors may localize their action and thus help to establish growth factor gradients in the extracellular space.     

Protective effect of sesamol against 60Co γ-ray-induced hematopoietic and gastrointestinal injury in C57BL/6 male mice

Abstract

Protection of γ-ray-induced injury in hematopoietic and gastrointestinal (GI) systems is the rationale behind developing radioprotectors. The objective of this study, therefore, was to investigate the radioprotective efficacy and mechanisms underlying sesamol in amelioration of γ-ray-induced hematopoietic and GI injury in mice. C57BL/6 male mice were pre-treated with a single dose (100 or 50 mg/kg, 30 min prior) of sesamol through the intraperitoneal route and exposed to LD_50/30 (7.5 Gy) and sublethal (5 Gy) dose of γ-radiation. Thirty-day survival against 7.5 Gy was monitored. Sesamol (100 mg/kg) pre-treatment reduced radiation-induced mortality and resulted survival of about 100% against 7.5 Gy of γ-irradiation. Whole-body irradiation drastically depleted hematopoietic progenitor stem cells in bone marrow, B cells, T cell subpopulations, and splenocyte proliferation in the spleen on day 4, which were significantly protected in sesamol pre-treated mice. This was associated with a decrease of radiation-induced micronuclei (MN) and apoptosis in bone marrow and spleen, respectively. Sesamol pre-treatment inhibited lipid peroxidation, translocation of gut bacteria to spleen, liver, and kidney, and enhanced regeneration of crypt cells in the GI system. In addition, sesamol pre-treatment reduced the radiation-induced pattern of expression of p53 and Bax apoptotic proteins in the bone marrow, spleen, and GI. This reduction in apoptotic proteins was associated with the increased anti-apoptotic-Bcl-x and PCNA proteins. Further, assessment of antioxidant capacity using ABTS and DPPH assays revealed that sesamol treatment alleviated total antioxidant capacity in spleen and GI tissue. In conclusion, the results of the present study suggested that sesamol as a single prophylactic dose protects hematopoietic and GI systems against γ-radiation-induced injury in mice.     

Effects of hydroxypropyl-β-cyclodextrin on the growth and morphology of Absidia coerulea

Cyclodextrin has been found to be an attractive novel solubilizer due to its unique material properties. Absidia coerulea is widely used in steroid bioconversion. The effects of hydroxypropyl-β-cyclodextrin (HP-β-CD) on the growth, morphology, and steroid-converting activity of A. coerulea CICC 40302 were systematically studied. HP-β-CD affected A. coerulea growth, resulting in changes in its spore morphology and mycelial morphology. It induced an increase in the spore germination rate and a decrease in cell biomass at the stationary phase. Optical microscopy revealed that HP-β-CD altered the mycelial morphology and reduced the pellet compactness of A. coerulea. A convenient and feasible computing method was used to measure pellet compactness, and it demonstrated that the compactness degree of the pellet decreased as HP-β-CD increased, which could be attributed to the modification of the physical properties of the fermentation medium. Moreover, the changing of mycelial morphology influenced steroid-converting activity. The results showed that HP-β-CD had multiple concentration-dependent effects on A. coerulea cells. HP-β-CD in the proper concentration range holds great potential as a biocompatible solubilizer.     

Inhibition of NF-κB activation by diethylcarbamazine prevents alcohol-induced liver injury in C57BL/6 mice

Induction of NF-κB-mediated gene expression has been identified in the pathogenesis of alcoholic liver disease (ALD). Diethylcarbamazine (DEC) is a piperazine derivative drug with anti-inflammatory properties. The present study was designed to evaluate the effect of DEC on NF-κB pathways in mice undergoing alcoholism induced hepatic inflammation. Forty male C57BL/6 mice were divided equally into four groups: control group (C); DEC-treated group, which received 50 mg/kg (DEC50); alcoholic group (EtOH), submitted to chronic alcohol consumption and the alcohol-DEC treated group (EtOH50), submitted to chronic alcoholism consumption plus DEC treatment. Histological analysis of the alcoholic group showed evident hepatocellular damage which was reduced in EtOH50 group. Immunohistochemistry and western blot results showed elevated expression of inflammatory markers such as MDA, TNF-α, IL-1β, COX-2 and iNOS in hepatocytes of EtOH group. However, low immunopositivity for these markers was detected following DEC treatment. In the EtOH group the activation of NF-κB was observed by an increase in the expression of both NF-κB and pNF-κB in hepatocytes. This expression was significantly reduced in livers of EtOH50 group. Protein expression of Iκβα was measured to determine whether activation of NF-κB might be the result of Iκβα degradation. It was observed that expression of this protein was low in EtOH group, while animals treated with DEC had a high expression of Iκβα. The results of the present study indicate that DEC alleviates alcoholic liver injury, in part by the inhibiting activation of NF-κB and by suppressing the induction of NF-κB-dependent genes.     

Effects of lead exposure on the expression of amyloid β and phosphorylated tau proteins in the C57BL/6 mouse hippocampus at different life stages

The objective of this study was to investigate the effects of lead exposure on spatial learning and memory capacity and the expression of amyloid β and phosphorylated tau proteins in the mouse hippocampus. A total of 24 adult C57BL/6 mice (12 of each sex) were mated at a 1:1 ratio. After delivery, the litters were normalised to 6 pups per litter. During the lactation period, the pups were randomly separated into four groups: control, early exposure, late exposure, or long-term exposure. These groups were not exposed to lead, exposed to lead from birth to week 24, exposed to lead from week 24 to week 48, or exposed to lead from birth to 48 weeks of age, respectively. Lead exposure was induced by providing Pb-contaminated drinking water at a concentration of 0.1%. All of the pups were fed until 72 weeks of age, at which time their spatial learning and memory capacity was evaluated via the Morris water maze test. Then, the lead levels in their blood and hippocampus were measured via graphite furnace atomic absorption spectrometry. The protein expression of amyloid β and phosphorylated tau in the hippocampus was detected via Western blot. The results revealed that the hippocampal and blood lead levels were significantly higher in all of the groups exposed to lead than the control group (P < 0.05). The spatial learning and memory performances of the lead-exposed groups were much poorer than those of the control group (P < 0.05). The expression levels of amyloid β and phosphorylated tau proteins were increased in the lead-exposed groups compared to the control group (P < 0.05). The enhanced expressions of amyloid β and phosphorylated tau proteins might contribute to the impairment in spatial learning and memory in the lead-exposed mice.     

Why does a sucrose choice reduce the consumption of alcohol in C57BL/6J mice?

To determine why animals reject alcohol when offered palatable solutions of sucrose, male C57BL/6J mice were challenged first with 5% sucrose then with 10% sucrose, while given continuous free-access to alcohol and water. The 5% sucrose dramatically reduced the intake of alcohol and increased the intake of total fluid by an average of 7.3 ml/day. The suppression of alcohol intake could not be attributed to a volumetric ceiling since access to 10% sucrose produced a further large increase in total intake (8.8 ml/day). The results support the interpretation that animals consume alcohol for characteristics it shares with sucrose.     

The development of pathological forms of behavior in submissive male C57BL/6J mice during agonistic zoosocial interactions. A possible model of depression?

It is shown that a long experience of defeats in daily zoosocial collisions (ZC) elicits changes in the structure of submissive behaviour. Male mice of C57BL/6J line after 20 defeats demonstrated poses of passive subordination instead of active defence and run away which they manifested in the first ZC. Moreover, new immobile poses appeared which were rare in the first ZC. Submissive animals (CA) demonstrated a decrease of travels in the open field test and an increase of immobility time in the Porsolt test. Chronic administration of imipramine (10 mg/kg, i.p., twice a day during two weeks against the background of repeated ZC) prevented an increase of depressivity, estimated in Porsolt test. Changes were noted in the content of serotonin and 5-HIAA in some brain structures of subordinated mice in comparison to control animals (five days of isolation). The data are discussed from a position of the development of depression in SA of C57BL/6J line as a result of a long nonavoided zoosocial stress.     

Protective specific immunity induced by cyclophosphamide plus tumor necrosis factor α combination treatment of EL4-lymphoma-bearing C57BL/6 mice

A combination treatment protocol initiated 12 days after tumor injection, when the tumor was large, by administering cyclophosphamide (CY, 150 or 250 mg/kg) intraperitoneally followed by intravenous tumor necrosis factor (TNF, 1000 units/injection) on days 13, 16, 18, 21, and 23, resulted in about 60% longterm survival (i.e., survival for at least 60 days) in the syngeneic C57BL/6 mouse/EL4 lymphoma model system. The establishment of a specific antitumor immune memory and its possible therapeutic relevance was verified by reinjecting 60-day survivors with EL4 cells; all 60-day survivors that had received the combination treatments rejected the implants and survived for a further 60 days. Thymic cellularity was reduced during treatment and its recovery appeared to correlate with long-term survival and immunity. Thymocytes from mice treated with the combination were found to express significant levels of specific anti-EL4 cytolytic activity following a 4-day stimulation culture with X-irradiated EL4 cells and low concentrations of interleukin-2. This response could not be generated with thymocytes from naive animals. In each case the effect seen with the combination of a moderate CY dose (150 mg/kg) with TNF was better than that seen with either dose of CY alone and equal to or better than that seen with the higher dose of CY combined with TNF. These results indicate that treatment with a single moderate dose of CY in combination with TNF is effective against a large, established tumor in this murine model. Furthermore, all the long-term survivors induced by this treatment developed protective immunity against reimplanted tumor and demonstrated a long-term specific immune memory in the thymus.This work was done in partial fulfillment of the M.S. degree requirements (C. M. K.) in the Program of Natural Sciences, Roswell Park Graduate Division, SUNY at Buffalo     

Adequacy of calcium and vitamin D reduces inflammation, β-catenin signaling,and dysbiotic Parasutterela bacteria in the colon of C57BL/6 mice fed a western-style diet

Adoption of an obesogenic diet low in calcium and vitamin D (CaD) leads to increased obesity, colonic inflammation, and cancer. However, the underlying mechanisms remain to be elucidated. We tested the hypothesis that CaD supplementation (from inadequacy to adequacy) may reduce colonic inflammation, oncogenic signaling, and dysbiosis in the colon of C57BL/6 mice fed a Western diet. Male C57/BL6 mice (4-weeks old) were assigned to 3 dietary groups for 36 weeks: (1) AIN76A as a control diet (AIN); (2) a defined rodent “new Western diet” (NWD); or (3) NWD with CaD supplementation (NWD/CaD). Compared to the AIN, mice receiving the NWD or NWD/CaD exhibited more than 0.2-fold increase in the levels of plasma leptin, tumor necrosis factor α (TNF-α) and body weight. The levels of plasma interleukin 6 (IL-6), inflammatory cell infiltration, and β-catenin/Ki67 protein (oncogenic signaling) were increased more than 0.8-fold in the NWD (but not NWD/CaD) group compared to the AIN group. Consistent with the inflammatory phenotype, colonic secondary bile acid (inflammatory bacterial metabolite) levels increased more than 0.4-fold in the NWD group compared to the NWD/CaD and AIN groups. Furthermore, the abundance of colonic Proteobacteria (e.g., Parasutterela), considered signatures of dysbiosis, was increased more than four-fold; and the α diversity of colonic bacterial species, indicative of health, was decreased by 30% in the NWD group compared to the AIN and NWD/CaD groups. Collectively, CaD adequacy reduces colonic inflammation, β-catenin oncogenic signaling, secondary bile acids, and bacterial dysbiosis in mice fed with a Western diet.     

Cytochemical localization of β-galactosidase in resident and inflammatory peritoneal macrophages from C57BL mice

Summary A cytochemical method for the detection of -galactosidase (-Gase) in mouse peritoneal macrophages was used to study the ultrastructural localization of this enzyme in these cells. It was found that the reaction product for -Gase was localized in the perinuclear cisternae, the endoplasmic reticulum, the Golgi complex, lysosomes, vesicles and on the cell surface of peritoneal macrophages from untreated C57BL mice. When examined by X-ray microanalysis the crystalline reaction product was found to contain bromine, an element present in the indolyl substrate which was used to identify -Gase. Injection of Proprionibacterium acnes (P. acnes) intraperitoneally or BCG intravenously caused a visible loss in -Gase from all the organelles and from the cell surface of the macrophages.Abbreviations used -Gase -galactosidase - RP reaction product - PNC perinuclear cisternae - RER rough endoplasmic reticulum     

Effect of the association of IGF-I, IGF-II, bFGF, TGF-β1, GM-CSF, and LIF on the development of bovine embryos produced in vitro

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-β1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-β1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P < 0.05) on Day 8 after in vitro fertilization and similar results to use of SOF + 10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-β1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer.