POSSIBLE MECHANISMS FOR REDUCTION OF CIRCULATING CONCENTRATIONS OF PROGESTERONE BY INTERFERON-ALPHA IN COWS - EFFECTS ON HYPERTHERMIA, LUTEAL CELLS, METABOLISM OF PROGESTERONE AND SECRETION OF LH

Experiments were performed to determine the mechanism by which recombinant bovine interferon-alpha(I)1 (rbIFN-alpha) causes an acute reduction in plasma concentrations of progesterone. In experiment 1, administration of a prostaglandin synthesis inhibitor blocked rbIFN-alpha-induced hyperthermia but did not prevent the decline in plasma concentrations of progesterone. The decline in progesterone concentrations caused by rbIFN-alpha was, therefore, not a direct consequence of the associated hyperthermia or of pathways mediated through prostaglandin synthesis. It is also unlikely that rbIFN-alpha acts to increase the clearance of progesterone since injection of rbIFN-alpha did not decrease plasma concentrations of progesterone in ovariectomized cows given an intravaginal implant of progesterone (experiment 2). In experiment 3, rbIFN-alpha did not affect basal and LH-induced release of progesterone from cultured luteal slices, indicating that rbIFN-alpha is unlikely to affect luteal function directly. Injection of rbIFN-alpha did, however, cause a decrease in plasma concentrations of LH in ovariectomized cows (experiment 4) that coincided temporally with the decrease in progesterone concentrations seen in cows having a functional corpus luteum. The present results strongly suggest that rbIFN-alpha acts to reduce secretion of progesterone by interfering with pituitary support for luteal synthesis of progesterone. The finding that rbIFN-alpha can inhibit LH secretion implies that interferon-alpha molecules should be considered among the cytokines that can regulate hypothalamic or pituitary function.

Partial urethral obstruction of rabbit urinary bladder: stereological evidence that the increase in muscle content is mostly driven by changes in number, rather than size, of smooth muscle cells

The effects of partial urethral obstruction on the detrusor muscle of rabbit urinary bladder were investigated using stereological sampling and estimation tools. Twelve female Norfolk rabbits (2.5-3.0 kg body weight) were divided into four groups: 3, 7 and 12 weeks after surgical intervention to produce a standard partial obstruction and unobstructed controls. Following removal, bladder axes (craniocaudal, dorsoventral and laterolateral) and organ weights were recorded. Bladders were prepared for light microscopy by multistage random sampling procedures. Stereological methods were used to estimate the volume of muscle and the packing density and total number of myocyte nuclei in each bladder. We also estimated mean myocyte volume and the mean cross-sectional area and length of myocytes. Group comparisons were made by one-way analysis of variance. Changes in bladder axes were mainly laterolateral and craniocaudal. Mean bladder weight increased roughly six-fold by 3 weeks and 17-fold by 12 weeks and was accompanied, on average, by 12- and 33-fold increases in total muscle volume. These variables did not differ at 3 and 7 weeks post-obstruction. Increases in muscle content were not accompanied by changes in packing densities but were associated with increases in the total numbers of myocyte nuclei (13-fold by 3 weeks, 28-fold by 12 weeks). Mean myocyte volume did not vary significantly between groups but cells in obstructed groups were shorter and wider. These findings support the notion that partial outflow obstruction leads to an increase in the number, but not mean volume, of myocytes. If due solely to myocyte mitosis, the total of 43 x 10(8) cells found at 12 weeks could be generated by the original complement of 15 x 10(7) cells if an average of only 2.1 x 10(6) new cells was produced every hour. In reality, even this modest proliferation rate is unlikely to be achieved because myocyte proliferation rates are very low and it is possible that new myocytes can arise by differentiation of mesenchymal or other precursor cells.

Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats

The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/osteoblasts/bone lining cells. To find such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and electron microscopy. Fragments of alveolar bone from young rats were fixed in Bouin and formaldehyde for morphology and for the TUNEL method. Glutaraldehyde-formaldehyde fixed specimens were processed for transmission electron microscopy. Results showed TUNEL positive round/ovoid structures on the bone surface and inside osteocytic lacunae. These structures - also stained by hematoxylin - were therefore interpreted, respectively, as osteoblasts/lining cells and osteocytes undergoing apoptosis. Osteoclasts also exhibited TUNEL positive apoptotic bodies inside large vacuoles; the nuclei of osteoclasts, however, were always TUNEL negative. Ultrathin sections revealed typical apoptotic images - round/ovoid bodies with dense crescent-like chromatin - on the bone surface, corresponding therefore to apoptotic osteoblasts/lining cells. Osteocytes also showed images compatible with apoptosis. Large osteoclast vacuoles often contained fragmented cellular material. Our results provide further support for the idea that osteoclasts internalize dying bone cells; we were however, unable to find images of osteoclasts in apoptosis. (C) 2001 Harcourt Publishers Ltd.

The dead core model applied to beads with immobilized cells in a fed-batch cephalosporin C production bioprocess

Viable cells immobilized in inert supports are currently studied for a wide range of bioprocesses. The intrinsic advantages of such systems over suspended cultures incite new research, including studies on fundamental aspects as well as on the industrial viability of these non-conventional processes. In aerobic culture of filamentous fungi, scale-up is hindered by oxygen mass transfer limitation through the support material and bioprocess kinetics must be studied together with mass transfer limitation. In this work, experimental and simulated data of cephalosporin C production were compared. Concentrations in the bulk fermentation medium and cellular mass profiles inside the bioparticles are focused. Immobilized cells were used in a tower bioreactor, operated in fed-batch mode. To describe the radial variation of oxygen concentration within the pellet, a dead core model was used. Despite the extremely low sugar concentrations, bioreaction rates in the pellets were limited by the dissolved oxygen concentration. Cell growth occurs only in the outer layers, a result also confirmed by scanning electron microscopy. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

SUPPORT OF PARACOCCIDIOIDES-BRASILIENSIS MULTIPLICATION BY HUMAN MONOCYTES OR MACROPHAGES - INHIBITION BY ACTIVATED PHAGOCYTES

The interaction of human monocytes or monocyte-derived macrophages and yeast-form Paracoccidioides brasiliensis was studied in vitro. Yeast cells were readily ingested by adherent monocytes or macrophages. Multiplication of P. brasiliensis, measured by growth as colony forming units (cfu) on a supplemented medium with good plating efficiency, was greater in monocyte co-cultures compared to the number of cfu obtained from complete tissue-culture medium (CTCM). Multiplication increased with time in macrophage cocultures, e.g., from two-six-fold in 24 h to nine-fold in 72 h. Microscopic observations indicated that ingested yeast cells multiplied inside macrophages. When monocytes were treated with supernate cytokines (CK) from concanavalin-A-stimulated mononuclear cells, then co-cultured with P. brasiliensis, multiplication was significantly inhibited compared with control monocyte co-cultures. Treatment of macrophages-derived from monocytes by culture in vitro for 3 days-for a further 3 days with CK resulted in maximal inhibition of multiplication over the subsequent 72 h. Similarly, when monocyte-derived macrophages (after culture for 7 days) were treated for 3 days with recombinant human gamma-interferon (IFN; 300 U/ml) or CK they restricted multiplication of P. brasiliensis by 65% and 95%, respectively, compared with control macrophages, Antibody to IFN abrogated the effect of IFN or CK treatment. These findings show that ingested P. brasiliensis can multiply in human monocytes or macrophages and that this multiplication can be restricted by activated monocytes or macrophages.

Immobilized cells of Acidithiobacillus ferrooxidans in PVC strands and sultite removal in a pilot-scale bioreactor

The biooxidation of ferrous ion into ferric ion by Acidithiobacillus ferrooxidans can be potentially used for the removal of H2S from industrial gases. In this work, Fe3+ ions were obtained through the oxidation of Fe2+ using the LR strain of At. ferrooxidans immobilized in PVC stands in a pilot-scale bioreactor, while H2S was removed in an absorption tower equipped with Rasching rings. At. ferrooxidans LR strain cells were immobilized by inoculating the bacterium in a Fe2+-mineral medium and percolating it through the support. After complete Fe2+ oxidation, which took around 90 h, the reactor was washed several times with sulfuric acid (pH 1.7) before a new cycle was started. Four additional cycles using fresh Fe2+ mineral medium were then run. During these colonization cycles, the time required for complete iron oxidation decreased, dropping to about 60 h in the last cycle. The batch experiments in the H2S gas removal trials resulted in a gas removal rate of about 98-99% under the operational conditions employed. In the continuous experiments with the bioreactor coupled to the gas absorption column, a gas removal efficiency of almost 100% was reached after 500 min. Precipitate containing mainly sulfur formed during the experimental trial was identified by EDX. (c) 2005 Elsevier B.V. All rights reserved.

Clavulanic acid production by immobilized cells if Streptomyces clavuligerus

A clavulanic acid production process with immobilized Streptomyces clavuligerus cells was investigated. Cells were immobilized in diatomaceous earth, calcium alginate gel as well as in the form of natural pellets and cultivated in shake flasks in a medium containing glycerol and soytone as the carbon and nitrogen sources, respectively. In all experiments growth occurred in the first 48 h and glycerol consumption after 72 h, while clavulanic acid production was observed between 48 and 60h, with gradual degradation after this period. The natural pellets presented higher product concentration as compared with the cells immobilized in supports. However, calcium alginate was found to be the best support in relation to cell retention capacity.

Combined TUNEL and TRAP methods suggest that apoptotic bone cells are inside vacuoles of alveolar bone osteoclasts in young rats

Although it is generally accepted that osteoclasts breakdown and resorb bone matrix, the possibility that they may also be able to engulf apoptotic osteoblasts/ lining cells and/or osteocytes remains controversial. Apoptosis of osteoblasts/ lining cells and/or osteocytes and interactions between these cells and osteoclasts are extremely rapid events that are difficult to observe in viva. A suitable in viva model for studying these events is the alveolar bone of young rats because it is continuously. Thus, sections of aldehyde fixed alveolar undergoing intense resorption/remodeling bone of young rats were stained by the combined terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and the tartrate-resistant acid phosphatase (TRAP) method for the simultaneous visualization of apoptotic cells and osteoclasts in the same section. The combined TUNEL and TRAP reactions, in the same section, greatly facilitated visualization of relationship between osteoclasts and apoptotic bone cells during alveolar bone remodeling. Our results showed that several TRAP-positive osteoclasts exhibited large vacuoles containing TUNEL positive apoptotic structures, probably derived from osteoblasts/lining cells and/or osteocytes. These results support the idea that alveolar bone osteoclasts are able to internalize dying apoptotic bone cells.

Follicular somatic cell factors and follicle development

Considerable attention is currently paid to oocyte-derived secreted factors that act upon cumulus and granulosa cells. Also important for follicle development are somatic cell-derived secreted factors. This is illustrated by the ability of granulosa cell-derived Kit ligand (KITL) to promote primordial follicle activation, and the loss of follicle development that accompanies KITL gene disruption. This review summarises our current understanding of somatic cell factors during both preantral and antral follicle growth, involving not only signalling from granulosa cells to the oocyte, but also signalling between granulosa and theca cells. Principal granulosa cell-derived factors include activin, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Theca cells also secrete BMPs and FGFs. The interplay between these factors is equally important for follicle growth as the activity of oocyte-derived factors.

Isolation and characterization of Wharton's jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Susceptibility of neuron-like cells derived from bovine Wharton's jelly to bovine herpesvirus type 5 infections

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A pro-inflammatory role of lymphoid cells in acute pleurisy in rats

The effect of viable splenic lymphoid cells and their constituents (filtrate) on carrageenan-induced acute pleurisy was investigated in rats. Suspensions of lymphoid cells administered intravenously to recipients just prior to initiation of pleurisy enhance both the volume of exudate and cell accumulation in the pleural cavity 3 h after the irritation. Similar results were observed when filtrate of disrupted lymphoid cells was injected either 30 or 5 min before the carrageenan, but not when administered 30 min afterwards. Suspensions of bone marrow cells, on the contrary, were ineffective in producing an enhancement of the parameters studied. When administered into the pleural cavity together with carrageenan, the lymphoid cell filtrate augmented the inflammatory response to the irritant. Nevertheless, it was ineffective, per se, to elicit any local change. It is suggested that lymphoid cells may play a pro-inflammatory role in the initiation of the process by enhancing both the fluid and the cellular components of inflammation.

Infection and apparent invasion of Vero cells by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis probably uses many different mechanisms to establish itself in the host and cause disease. In this work, we assess an in vitro model system which uses cultured mammalian cells to investigate the virulence factors of P. brasiliensis. We were able to demonstrate an invasion process of the yeast form of this fungus in Vero cell cultures. We deduced that the overall invasive process involved three steps: adhesion, followed by invasion of individual epithelial cells and spread to adjacent cells.

The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulosa cells.

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

The electro-oxidation of carbon monoxide and ethanol on supported Pt nanoparticles: The influence of the support and catalyst microstructure

The sluggish kinetics of ethanol oxidation on Pt-based electrodes is one of the major drawbacks to its use as a liquid fuel in direct ethanol fuel cells, and considerable efforts have been made to improve the reaction kinetics. Herein, we report an investigation on the effect of the Pt microstructure (well-dispersed versus agglomerated nanoparticles) and the catalyst support (carbon Vulcan, SnO2, and RuO2) on the rate of the electrochemical oxidation of ethanol and its major adsorbed intermediate, namely, carbon monoxide. By using several structural characterization techniques such as X-ray diffraction, X-ray absorption spectroscopy, and transmission electron microscopy, along with potentiodynamic and potentiostatic electrochemical experiments, we show that by altering both the Pt microstructure and the support, the rate of the electrochemical oxidation of ethanol can be improved up to a factor of 12 times compared to well-dispersed carbon-supported Pt nanoparticles. As a result of a combined effect, the interaction of Pt agglomerates with SnO2 yielded the highest current densities among all materials studied. The differences in the activity are discussed in terms of structural and electronic properties as well as by mass transport effects, providing valuable insights to the development of more active materials. © 2013 Springer-Verlag Berlin Heidelberg.