GM-CSF augments the immunosuppressive capacity of neonatal spleen cells in vitro

Addition of exogenous granulocyte-macrophage colony stimulating factor (GM-CSF) to cultures of adult murine spleen cells with sheep red blood cells (SRBC) results in an augmented plaque forming cell (PFC) response. The influence of GM-CSF on the ability of neonatal spleen cells to suppress the anti-SRBC plaque forming response of adult spleen cells was tested by adding GM-CSF to cultures of neonatal and adult spleen cells. The suppressive capacity of the neonatal spleen cells was augmented by exogenous GM-CSF. The augmented suppression of the neonatal spleen cells was dependent on a G-10 adherent population since the addition of GM-CSF to cultures containing G-10 passed neonatal spleen cells resulted in an augmented PFC response and not suppression. Neonatal splenic glass adherent cells were also capable of suppressing the response. Neonatal spleen cells or purified neonatal glass adherent spleen cells cultured in the presence of GM-CSF had markedly increased levels of PGE2 in the culture supernatant. Neonatal spleen cells cultured with GM-CSF had increased numbers of morphologically identifiable macrophages after 48 hr of culture. Both irradiation and G-10 passage of the neonatal spleen diminished the numbers of macrophages formed in response to GM-CSF, and both of these manipulations resulted in reversal of suppression in response to GM-CSF. Thus, the augmented suppressive capacity of neonatal spleen cells in response to GM-CSF is probably mediated by its ability to drive monocyte to macrophage differentiation as well as increase the suppressive capacity of the existing neonatal splenic macrophages by increasing their production of PGE2.     

Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.     

Identification of genes involved in apoptosis and dominant follicle development during follicular waves in cattle

We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 +/- 1.0 versus 7.6 +/- 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 +/-3 8.5 versus 24.1 +/- 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor beta, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for beta glycan, cyclo-oxygenase-1, tumor necrosis factor alpha, caspase-activated DNase, and DRAK-2, and in theca cells for beta glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.     

Improved single- and multi-contact life-time testing of dental restorative materials using key characteristics of the human masticatory system and a force/position-controlled robotic dental wear simulator

This paper presents a new in vitro wear simulator based on spatial parallel kinematics and a biologically inspired implicit force/position hybrid controller to replicate chewing movements and dental wear formations on dental components, such as crowns, bridges or a full set of teeth. The human mandible, guided by passive structures such as posterior teeth and the two temporomandibular joints, moves with up to 6 degrees of freedom (DOF) in Cartesian space. The currently available wear simulators lack the ability to perform these chewing movements. In many cases, their lack of sufficient DOF enables them only to replicate the sliding motion of a single occlusal contact point by neglecting rotational movements and the motion along one Cartesian axis. The motion and forces of more than one occlusal contact points cannot accurately be replicated by these instruments. Furthermore, the majority of wear simulators are unable to control simultaneously the main wear-affecting parameters, considering abrasive mechanical wear, which are the occlusal sliding motion and bite forces in the constraint contact phase of the human chewing cycle. It has been shown that such discrepancies between the true in vivo and the simulated in vitro condition influence the outcome and the quality of wear studies. This can be improved by implementing biological features of the human masticatory system such as tooth compliance realized through the passive action of the periodontal ligament and active bite force control realized though the central nervous system using feedback from periodontal preceptors. The simulator described in this paper can be used for single- and multi-occlusal contact testing due to its kinematics and ability to exactly replicate human translational and rotational mandibular movements with up to 6 DOF without neglecting movements along or around the three Cartesian axes. Recorded human mandibular motion and occlusal force data are the reference inputs of the simulator. Experimental studies of wear using this simulator demonstrate that integrating the biological feature of combined force/position hybrid control in dental material testing improves the linearity and reduces the variability of results. In addition, it has been shown that present biaxially operated dental wear simulators are likely to provide misleading results in comparative in vitro/in vivo one-contact studies due to neglecting the occlusal sliding motion in one plane which could introduce an error of up to 49% since occlusal sliding motion D and volumetric wear loss V(loss) are proportional.     

Subcellular Localization of Asparaginase and Asparagine Aminotransferase in Pisum sativum Leaves

Protoplasts isolated from young and mature pea leaves (Pisum sativum L.) were broken and their contents fractionated by differential centrifugation or on sucrose-density gradients. Asparaginase was found only in the cytosol of young leaves. Asparagine aminotransferase was found in both young and mature leaves and was localized exclusively in the peroxisome. This corroborates the observation that asparagine transamination is catalyzed by the serine:glyoxylate aminotransferase.     

Aspects of reproduction and their implications for the conservation of the endangered limpet,Patella ferruginea

Summary

The protandric limpet, Patella ferruginea G., is the most endangered marine species in the Western Mediterranean and is at serious risk of extinction. Nevertheless, its biology and ecology are little known. In the present work, several reproductive aspects are studied. Recruitment take place in June and the largest individuals are the most scarce. The sex ratio is slanted towards the largest sizes, and the species seems to show sex change at sizes from 60 mm upwards, although males can be observed up to 80 mm. There is no correlation between size of oocytes and shell length; however, the larger females contribute greatly to the reproductive event, with high fecundity and GSI values. The mean diameter of oocytes was observed to be 149.78 μm, whereas the heads of spermatozoa were 3.78 μm long. The results of the present study increase the maximum length for males by double that cited in previous literature and highlight the importance of strictly protecting females (i.e., most of the largest individuals, >60 mm), since their population percentage is very low, in order to develop adequate strategies to preserve the species.     

The survey on cellular and tissue-engineered therapies in Europe and neighboring Eurasian countries in 2014 and 2015

Background aims

With the support of five established scientific organizations, this report, the seventh of its kind, describes activity in Europe for the years 2014 and 2015 in the area of cellular and tissue-engineered therapies, excluding hematopoietic stem cell (HSC) treatments for the reconstitution of hematopoiesis.