Stereology and computer assisted three-dimensional reconstruction as tools to study probiotic effects of Aeromonas hydrophila on the digestive tract of germ-free Artemia franciscana nauplii.

AIMS: Validation of stereology and three-dimensional reconstruction for monitoring the probiotic effect of Aeromonas hydrophila on the gut development of germ-free Artemia franciscana nauplii. METHODS AND RESULTS: Germ-free Artemia nauplii were cultured using Baker's yeast and dead Aer. hydrophila. Live Aer. hydrophila were added on the first day to the treatment group. The gut length and volume were monitored on days two and four using stereology and three-dimensional reconstruction. Both methods showed comparable results. Stereology was least labour intensive to estimate volumes, while three-dimensional reconstructions rendered architectural and topographical data of the gut. Moreover, a positive effect of probiotic bacterium, Aer. hydrophila is likely. CONCLUSION: Slight increment in the growth of the digestive tract of A. franciscana nauplii exerted by probiotic bacteria could be detected using stereology and three-dimensional reconstruction. SIGNIFICANCE AND IMPACT OF THE STUDY: The gnotobiotic Artemia rearing system is unique to investigate the effects of micro-organisms on the development of nauplii. However, in the base of this model system, only survival counts and length measurements exist as monitoring tools. Therefore, additional tools such as stereology and three-dimensional reconstruction are prerequisite to obtain more powerful analysis.

Evaluation of the agreement between Brix refractometry and serum immunoglobulin concentration in neonatal piglets.

Neonatal piglets lack immunoglobulins at birth. Sufficient colostrum intake (CI) and immunoglobulin absorption are essential for an appropriate passive transfer of immunity via the colostrum. Most methods to measure immunoglobulins in serum of piglets are labour-intensive, expensive or imprecise and not designed for on-farm use. The present diagnostic test study evaluated digital Brix refractometry to measure immunoglobulins in serum of neonatal piglets and to suggest thresholds for different serum immunoglobulin concentration. Additionally, agreements between Brix refractometry and optical refractometer (serum total protein, STP) and between Brix refractometry and ELISA (immunoglobulin G, IgG) were also investigated. Forty-five sows and 269 piglets from three different farms were enrolled in the study. Piglets were weighed at birth and 24 h later to calculate the CI. Serum was collected at 24 h after birth and analysed for STP, γ-globulins (electrophoresis), % Brix and IgG. In piglets, median (interquartile range, IQR) CI was 412 (196) g per piglet. Median (IQR) STP, γ-globulin and % Brix concentrations in piglet serum were 60 (11) g/L, 35 (10) g/L and 8 (2) %, respectively. Average (±SD) IgG concentration was 49 ±â€¯23 g/L. Passing-Bablok regression revealed a strong concordance between % Brix and STP (Kendall's tau (Τ): 0.620, P < 0.0001, n = 267) and % Brix and γ-globulin concentration (Kendall's Τ: 0.575, P < 0.0001, n = 267). The agreement between the Brix refractometer and IgG concentration was poor (Kendall's Τ: 0.267, P < 0.0001, n = 269). Receiver operating characteristic curves were performed to evaluate test characteristics of Brix refractometry for three γ-globulin cut-off values, i.e. 10, 20 and 30 g/L. The % Brix cut-off values resulting in the optimal combination of sensitivity and specificity were 5.4 (100 and 98.5%), 7.0 (100 and 89.3%) and 7.9 (90.1 and 80.6%), respectively. In conclusion, digital Brix refractometry is a sufficiently fast and practical method to assess serum γ-globulin concentrations in neonatal piglets on-farm and to evaluate them by considering the thresholds found in this study. Further studies are needed to validate those thresholds regarding piglet's survival in the pre-weaning period.

A single dose of intravenous combretastatin A4-phosphate is reasonably well tolerated and significantly reduces tumour vascularization in canine spontaneous cancers.

Combretastatin A4-phosphate (CA4P) is an anti-tumour vascular targeting agent which selectively blocks tumour blood flow. Research on CA4P in rodent tumour models is extensive; however, knowledge of its effect on spontaneous cancer is scarce. This study was conducted in canine patients with spontaneous solid tumours. The goal was to assess the toxicity and efficacy of CA4P in various spontaneous tumour types. Eight dogs with spontaneous tumours were enrolled and treated with a single dose of 75 mg m-2 intravenous CA4P. The dogs were screened and monitored before and after injection. Pre- and post-treatment tumour blood flow was analysed in vivo by power Doppler ultrasound (PDUS) and contrast-enhanced ultrasound (CEUS). Vessel destruction and tumour necrosis were evaluated by histopathology. Clinically relevant toxicity was limited to one case of temporary tetraparesis; other adverse events were mild. Significant cardiovascular changes were mostly confined to changes in heart rate and cTnI levels. Macroscopic tumour size reduction was evident in 2 dogs. Based on PDUS and CEUS, CA4P induced a significant decrease in vascular index and tumour blood flow. Post-treatment, histopathology revealed a significant increase of necrotic tumoural tissue and a significant reduction in microvessel density in tumoural tissue. Anti-vascular and necrotizing effects of CA4P were documented in a variety of canine spontaneous cancers with only minimal side effects. This is the first study reporting the administration of CA4P to canine cancer patients with in vivo and ex vivo assessment, and a first step toward implementing CA4P in combination therapies in veterinary oncology patients. The use of CA4P in canine patients was approved and registered by the Belgian Federal Agency for Medicines and Health Products (FAMHP) (approval number 0002588, registration number 6518 ID 2F12).

Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes.

The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3'/5' integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof.

Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir.

HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting HIV DNA may aid patient monitoring and cure research. Digital PCR is a promising tool that enables direct absolute quantification with high sensitivity. With recent technological advances, several platforms are available to implement digital PCR in a clinical setting. Here, we compared two digital PCR platforms, the Quantstudio 3D (Life Technologies) and the QX100 (Bio-Rad) with a semi-nested qPCR on serial HIV DNA dilutions and DNA isolated from PBMCs of ART-suppressed patients. All three methods were able to detect target to the lowest levels of 2.5 HIV DNA copies. The QX100 excelled in having the least bias and highest precision, efficiency and quantitative linearity. Patient sample quantifications by the QX100 and semi-nested qPCR were highly agreeable by Bland-Altman analysis (0.01±0.32 log10). Due to the observation of false-positive signals with current digital PCR platforms however, semi-nested qPCR may still be preferred in a setup of low quantity detection to discriminate between presence or absence of HIV DNA.

Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores.

Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca(2+) signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca(2+) signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca(2+) signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca(2+) stores, thereby leaving less Ca(2+) available for release upon IP3- and ryanodine-receptor activation. The Ca(2+)-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca(2+) release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression.

Angiogenesis and collagen type IV expression in different endothelial cell culture systems.

In vitro angiogenesis assays constitute an important tool for studying the mechanisms of angiogenesis and for identification of pro- and anti-angiogenic substances. Therefore, endothelial cell and media systems used for in vitro angiogenesis assays are required to mimic the angiogenic process in vivo including endothelial capability to express collagen type IV as a component of the basement membrane. In this study, the expression of collagen type IV and its α chains (α1-6) was investigated in different endothelial cell culture systems in vitro qualitatively and quantitatively. These systems included four different batches of microvascular endothelial cells derived from the human skin, heart and lung, from which only two batches were found to be angiogenic and two batches were classified as non-angiogenic. Distribution of the transcripts of the α chains of collagen type IV was similar in all cell and media systems investigated. However, secretion and deposition of a stable extracellular network of collagen type IV could only be observed in the angiogenic cultures. In conclusion, the consecutive steps of the angiogenic cascade in vivo as well as in vitro depend on an increasing secretion and subsequent extracellular deposition of collagen type IV.

Porcine reproductive and respiratory syndrome virus infection is associated with an increased number of Sn-positive and CD8-positive cells in the maternal-fetal interface.

It is already known that porcine reproductive and respiratory syndrome virus (PRRSV) infection in lungs changes a local cell pattern and cytokine profile. However, there is no information about cellular and immunological events upon PRRSV infection in the maternal-fetal interface yet. The altered number and/or function of macrophages and NK cells in the maternal-fetal interface during infection may have a functional importance for virus replication. In addition, local cellular and immunological disbalance may also disrupt fragile homeostasis and contribute to the PRRSV-related reproductive disorders. Sialoadhesin (Sn)-positive macrophages are target cells for PRRSV and Sn overexpression has been observed upon chronic inflammatory and infectious diseases. It is also known that mouse Sn-positive macrophages in lymph nodes are able to closely interact with and activate NK cells in response to viral particles. Therefore, the main purpose of the present study was to examine if PRRSV infection is associated with altered Sn expression on endometrial and placental macrophages. In addition, CD8-positive cells (porcine endometrial NK cells were previously described as CD8(+)CD3(-) cells) were localized and quantified in the PRRSV-positive and control tissues. Tissue samples were obtained from three PRRSV-inoculated and three non-inoculated control sows at 100 days of gestation. Real-time RT-PCR showed a clear upregulation of Sn mRNA expression in the PRRSV-positive endometrium/placenta (p<0.05). Sn-, CD163- and CD14-specific immunofluorescence stainings revealed that PRRSV-inoculated sows had a significantly higher number of Sn-positive macrophages in the endometrium and placenta due to de novo Sn expression on local CD163-positive macrophages. Along with the increased number of Sn-positive macrophages an increased number of CD8-positive cells, which were mostly CD3-negative, was observed in the PRRSV-positive endometrium. The effects of the observed cellular changes on virus replication and potential contribution to placental damage and reproductive disorders are discussed.

Distribution of the Shadoo protein in the ovine brain assessed by immunohistochemistry.

Shadow of prion protein is a gene potentially involved in the pathogenesis of prion diseases. However, the Shadoo protein encoded by this gene has not yet been studied in sheep, an important species in prion matters. Therefore, we developed a polyclonal antibody against ovine Shadoo and assessed the presence and distribution of this protein in the ovine brain by immunohistochemistry. The strongest staining level was found in the cerebellum (especially in the Purkinje cells) and in the pons, but cerebrum, hippocampus, pituitary gland, medulla oblongata, thalamus and hypothalamus were also immunopositive. Remarkably, a typical granular pattern was seen in most of the tested brain tissues, which might indicate that Shadoo is primarily expressed at synapses. The results of this study and the availability of an ovine anti-Shadoo antibody can contribute to future research on the function of Shadoo and on its potential involvement in prion diseases.

Detection of hypoxia inducible factors and angiogenic growth factors during foetal endochondral and intramembranous ossification.

During skeletogenesis, the development of a new vascular network, i.e. angiogenesis, is triggered by hypoxia through the activation of the hypoxia inducible factors (HIFs) HIF-1alpha and HIF-2alpha. HIFs regulate the expression of several genes, including those coding for angiogenic growth factors such as VEGFA, angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2). The expression of HIFs and angiogenic growth factors is well documented in endochondral ossification, but few data exist on their expression during intramembranous ossification. In this study, the localization of these factors was examined using immunohistochemistry and RT-PCR in bones of porcine foetuses. Immunostaining for HIF-1alpha and HIF-2alpha was observed during endochondral ossification, whereas only HIF-2alpha was present at sites of intramembranous ossification. Furthermore, immunostaining for ANGPT2 was present at sites of endochondral and intramembranous ossification. In addition, gene transcripts for ANGPT1, ANGPT2 and VEGFA were detected with RT-PCR in laser capture microdissected isolates from both types of ossification. These results indicate that angiogenesis plays an important role during endochondral and intramembranous ossification. However, the different expression pattern of the HIF-alpha subunits suggests that alternative regulatory pathways trigger angiogenesis in these distinct types of ossification.

Electron microscopic study of the porcine choroid plexus epithelium.

The choroid plexus (CP) is a highly vascularized organ in the brain ventricles which acts as the main producer of cerebrospinal fluid (CSF). A study of the surface ultrastructure of the porcine CP was performed using scanning and transmission electron microscopy. The vascular walls of the capillaries were fenestrated. Epiplexus cells of different morphology were abundant on top of the epithelial surface. Two types of epithelial cells were present, characterized by the presence or absence of microvilli. Some epithelial cells contained cilia while other cells had large secretory protrusions called blebs. In the choroid epithelium of the lateral ventricles, some cells with large depressions were present. Cells with peduncles, such as recently discovered in the buffalo, could not be recognized. The variability of the choroidal surface structures clearly indicates the active role of the CP in the formation and maintenance of the CSF and its components.