Perturbation of microbiota in one-day old broiler chickens with antibiotic for 24 hours negatively affects intestinal immune development.

BACKGROUND: Gut microbial colonization and development of immune competence are intertwined and are influenced by early-life nutritional, environmental, and management factors. Perturbation of the gut microbiome at young age affects the crosstalk between intestinal bacteria and host cells of the intestinal mucosa. RESULTS: We investigated the effect of a perturbation of the normal early life microbial colonization of the jejunum in 1-day old chickens. Perturbation was induced by administering 0.8 mg amoxicillin per bird per day) via the drinking water for a period of 24 h. Effects of the perturbation were measured by 16S rRNA profiling of the microbiome and whole genome gene expression analysis. In parallel to what has been observed for other animal species, we hypothesized that such an intervention may have negative impact on immune development. Trends were observed in changes of the composition and diversity of the microbiome when comparing antibiotic treated birds with their controls. in the jejunum, the expression of numerous genes changed, which potentially leads to changes in biological activities of the small intestinal mucosa. Validation of the predicted functional changes was performed by staining immune cells in the small intestinal mucosa and a reduction in the number of macrophage-like (KUL01+) cells was observed due to a direct or indirect effect of the antibiotic treatment. We provide evidence that a short, early life antibiotic treatment affects both the intestinal microbiota (temporarily) and mucosal gene expression over a period of 2 weeks. CONCLUSION: These results underscore the importance of early life microbial colonization of the gut in relation to immune development and the necessity to explore the capabilities of a variety of early life dietary and/or environmental factors to modulate the programming for immune competence in broilers.

Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid.

BACKGROUND: Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents. RESULTS: The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10-1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni. CONCLUSION: It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

Mycoplasma detection by triplex real-time PCR in bronchoalveolar lavage fluid from bovine respiratory disease complex cases.

BACKGROUND: In this study we evaluated the RespoCheck Mycoplasma triplex real-time PCR for the detection in bronchoalveolar lavage fluid (BALF) of Mycoplasma (M.) dispar, M. bovis and M. bovirhinis, all three associated with bovine respiratory disease (BRD). Primers and probes of the RespoCheck Mycoplasma triplex real-time PCR are based on the V3/V4 region of the 16S rRNA gene of the three Mycoplasma species. RESULTS: The analytical sensitivity of the RespoCheck triplex real-time PCR was, as determined by spiking experiments of the Mycoplasma strains in Phosphate Buffered Saline, 300 colony forming units (cfu)/mL for M. dispar, and 30 cfu/mL for M. bovis or M. bovirhinis. The analytical sensitivity of the RespoCheck Mycoplasma triplex real-time PCRwas, as determined on purified DNA, 10 fg DNA per assay for M. dispar and 100 fg fo rM. bovis and M. bovirhinis. The analytical specificity of the RespoCheck Mycoplasma triplex real-time PCR was, as determined by testing Mycoplasmas strains (n = 17) and other bacterial strains (n = 107), 100, 98.2 and 99.1% for M. bovis, M. dispar and M. bovirhinis respectively. The RespoCheck Mycoplasma triplex real-time PCR was compared with the PCR/DGGE analysis for M. bovis, M. dispar and M. bovirhinis respectively by testing 44 BALF samples from calves. CONCLUSION: In conclusion, the RespoCheck PCR assay can be a valuable tool for timely and accurate detection of three Mycoplasma species associated with in bovine respiratory disease.

Early life microbial colonization of the gut and intestinal development differ between genetically divergent broiler lines.

BACKGROUND: Host genetic makeup plays a role in early gut microbial colonization and immune programming. Interactions between gut microbiota and host cells of the mucosal layer are of paramount importance for a proper development of host defence mechanisms. For different livestock species, it has already been shown that particular genotypes have increased susceptibilities towards disease causing pathogens. The objective of this study was to investigate the impact of genotypic variation on both early microbial colonization of the gut and functional development of intestinal tissue. From two genetically diverse chicken lines intestinal content samples were taken for microbiota analyses and intestinal tissue samples were extracted for gene expression analyses, both at three subsequent time-points (days 0, 4, and 16). RESULTS: The microbiota composition was significantly different between lines on each time point. In contrast, no significant differences were observed regarding changes in the microbiota diversity between the two lines throughout this study. We also observed trends in the microbiota data at genus level when comparing lines X and Y. We observed that approximately 2000 genes showed different temporal gene expression patterns when comparing line X to line Y. Immunological related differences seem to be only present at day 0, because at day 4 and 16 similar gene expression is observed for these two lines. However, for genes involved in cell cycle related processes the data show higher expression over the whole course of time in line Y in comparison to line X. CONCLUSIONS: These data suggest the genetic background influences colonization of gut microbiota after hatch in combination with the functional development of intestinal mucosal tissue, including the programming of the immune system. The results indicate that genetically different chicken lines have different coping mechanisms in early life to cope with the outside world.

Development of a DNA-based microarray for the detection of zoonotic pathogens in rodent species.

The demand for diagnostic tools that allow simultaneous screening of samples for multiple pathogens is increasing because they overcome the limitations of other methods, which can only screen for a single or a few pathogens at a time. Microarrays offer the advantages of being capable to test a large number of samples simultaneously, screening for multiple pathogen types per sample and having comparable sensitivity to existing methods such as PCR. Array design is often considered the most important process in any microarray experiment and can be the deciding factor in the success of a study. There are currently no microarrays for simultaneous detection of rodent-borne pathogens. The aim of this report is to explicate the design, development and evaluation of a microarray platform for use as a screening tool that combines ease of use and rapid identification of a number of rodent-borne pathogens of zoonotic importance. Nucleic acid was amplified by multiplex biotinylation PCR prior to hybridisation onto microarrays. The array sensitivity was comparable to standard PCR, though less sensitive than real-time PCR. The array presented here is a prototype microarray identification system for zoonotic pathogens that can infect rodent species.

Genetic diversity of Streptococcus suis isolates as determined by comparative genome hybridization.

BACKGROUND: Streptococcus suis is a zoonotic pathogen that causes infections in young piglets. S. suis is a heterogeneous species. Thirty-three different capsular serotypes have been described, that differ in virulence between as well as within serotypes. RESULTS: In this study, the correlation between gene content, serotype, phenotype and virulence among 55 S. suis strains was studied using Comparative Genome Hybridization (CGH). Clustering of CGH data divided S. suis isolates into two clusters, A and B. Cluster A isolates could be discriminated from cluster B isolates based on the protein expression of extracellular factor (EF). Cluster A contained serotype 1 and 2 isolates that were correlated with virulence. Cluster B mainly contained serotype 7 and 9 isolates. Genetic similarity was observed between serotype 7 and serotype 2 isolates that do not express muramidase released protein (MRP) and EF (MRP⁻EF⁻), suggesting these isolates originated from a common founder. Profiles of 25 putative virulence-associated genes of S. suis were determined among the 55 isolates. Presence of all 25 genes was shown for cluster A isolates, whereas cluster B isolates lacked one or more putative virulence genes. Divergence of S. suis isolates was further studied based on the presence of 39 regions of difference. Conservation of genes was evaluated by the definition of a core genome that contained 78% of all ORFs in P1/7. CONCLUSIONS: In conclusion, we show that CGH is a valuable method to study distribution of genes or gene clusters among isolates in detail, yielding information on genetic similarity, and virulence traits of S. suis isolates.

Olfactory ensheathing glia and Schwann cells exhibit a distinct interaction behavior with meningeal cells.

Schwann cells (SCs) and olfactory ensheathing glia (OEG) have both been used as cellular transplants to promote spinal cord repair. Both cell types support axonal regeneration and have beneficial effects on functional recovery. A significant difference between SCs and OEG is the effect of these cell types on astrocytes (ACs) present in the neural scar. In contrast to OEG, which associate and intermingle with ACs, SCs and ACs form separate cellular territories. Here, we show that OEG and SCs also interact differently with meningeal cells (MCs), another major cellular component of the neural scar. Whereas OEG intermingle with MCs in cocultures, SCs aggregate into well-defined cell clusters. Our data suggest that (a) soluble factor(s) as well as direct cellular contact are involved in the MC-induced SC clustering. Furthermore, the cluster formation of SCs in coculture with MCs is different from the previously reported segregation of SCs and ACs in coculture. The present results help to understand the differential behavior of both cell types after transplantation in the injured spinal cord and will be important to either determine which cell has optimal capacities to render the scar more permissive for regeneration, or to exploit the transplantation of both cell types in combination.

Enapotamab vedotin, an AXL-specific antibody-drug conjugate, shows preclinical antitumor activity in non-small cell lung cancer.

Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin-mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor-resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.

Comparative gene expression profiling of olfactory ensheathing glia and Schwann cells indicates distinct tissue repair characteristics of olfactory ensheathing glia.

Olfactory ensheathing glia (OEG) are a specialized type of glia that support the growth of primary olfactory axons from the neuroepithelium in the nasal cavity to the brain. Transplantation of OEG in the injured spinal cord promotes sprouting of injured axons and results in reduced cavity formation, enhanced axonal and tissue sparing, remyelination, and angiogenesis. Gene expression analysis may help to identify the molecular mechanisms underlying the ability of OEG to recreate an environment that supports regeneration in the central nervous system. Here, we compared the transcriptome of cultured OEG (cOEG) with the transcriptomes of cultured Schwann cells (cSCs) and of OEG directly obtained from their natural environment (nOEG), the olfactory nerve layer of adult rats. Functional data mining by Gene Ontology (GO)-analysis revealed a number of overrepresented GO-classes associated with tissue repair. These classes include "response to wounding," "blood vessel development," "cell adhesion," and GO-classes related to the extracellular matrix and were overrepresented in the set of differentially expressed genes between both comparisons. The current screening approach combined with GO-analysis has identified distinct molecular properties of OEG that may underlie their efficacy and interaction with host tissue after implantation in the injured spinal cord. These observations can form the basis for studies on the function of novel target molecules for therapeutic intervention after neurotrauma.

Olfactory ensheathing glia: their contribution to primary olfactory nervous system regeneration and their regenerative potential following transplantation into the injured spinal cord.

Olfactory ensheathing glia (OEG) are a specialized type of glia that guide primary olfactory axons from the neuroepithelium in the nasal cavity to the brain. The primary olfactory system is able to regenerate after a lesion and OEG contribute to this process by providing a growth-supportive environment for newly formed axons. In the spinal cord, axons are not able to restore connections after an injury. The effects of OEG transplants on the regeneration of the injured spinal cord have been studied for over a decade. To date, of all the studies using only OEG as a transplant, 41 showed positive effects, while 13 studies showed limited or no effects. There are several contradictory reports on the migratory and axon growth-supporting properties of transplanted OEG. Hence, the regenerative potential of OEG has become the subject of intense discussion. In this review, we first provide an overview of the molecular and cellular characteristics of OEG in their natural environment, the primary olfactory nervous system. Second, their potential to stimulate regeneration in the injured spinal cord is discussed. OEG influence scar formation by their ability to interact with astrocytes, they are able to remyelinate axons and promote angiogenesis. The ability of OEG to interact with scar tissue cells is an important difference with Schwann cells and may be a unique characteristic of OEG. Because of these effects after transplantation and because of their role in primary olfactory system regeneration, the OEG can be considered as a source of neuroregeneration-promoting molecules. To identify these molecules, more insight into the molecular biology of OEG is required. We believe that genome-wide gene expression studies of OEG in their native environment, in culture and after transplantation will ultimately reveal unique combinations of molecules involved in the regeneration-promoting potential of OEG.

Supplementation of fructooligosaccharides to suckling piglets affects intestinal microbiota colonization and immune development.

Emerging knowledge shows the importance of early life events in programming the intestinal mucosal immune system and development of the intestinal barrier function. These processes depend heavily on close interactions between gut microbiota and host cells in the intestinal mucosa. In turn, development of the intestinal microbiota is largely dependent on available nutrients required for the specific microbial community structures to expand. It is currently not known what the specificities are of intestinal microbial community structures in relation to the programming of the intestinal mucosal immune system and development of the intestinal barrier function. The objective of the present study was to investigate the effects of a nutritional intervention on intestinal development of suckling piglets by daily oral administration of fructooligosaccharides (FOS) over a period of 12 d (days 2-14 of age). At the microbiota community level, a clear "bifidogenic" effect of the FOS administration was observed in the colon digesta at day 14. The former, however, did not translate into significant changes of local gene expression in the colonic mucosa. In the jejunum, significant changes were observed for microbiota composition at day 14, and microbiota diversity at day 25. In addition, significant differentially expressed gene sets in mucosal tissues of the jejunum were identified at both days 14 and 25 of age. At the age of 14 d, a lower activity of cell cycle-related processes and a higher activity of extracellular matrix processes were observed in the jejunal mucosa of piglets supplemented with FOS compared with control piglets. At day 25, the lower activity of immune-related processes in jejunal tissue was seen in piglets supplemented with FOS. Villi height and crypt depth in the jejunum were significantly different at day 25 between the experimental and control groups, where piglets supplemented with FOS had greater villi and deeper crypts. We conclude that oral FOS administration during the early suckling period of piglets had significant bifidogenic effects on the microbiota in the colon and on gene expression in the jejunal mucosa by thus far unknown mechanisms.

Differential regulation of the CXCR2 chemokine network in rat brain trauma: implications for neuroimmune interactions and neuronal survival.

Chemokine receptors represent promising targets to attenuate inflammatory responses and subsequent secondary damage after brain injury. We studied the response of the chemokines CXCL1/CINC-1 and CXCL2/MIP-2 and their receptors CXCR1 and CXCR2 after controlled cortical impact injury in adult rats. Rapid upregulation of CXCL1/CINC-1 and CXCL2/MIP-2, followed by CXCR2 (but not CXCR1), was observed after injury. Constitutive neuronal CXCR2 immunoreactivity was detected in several brain areas, which rapidly but transiently downregulated upon trauma. A second CXCR2-positive compartment, mainly colocalized with the activated microglia/macrophage marker ED1, was detected rapidly after injury in the ipsilateral cortex, progressively emerging into deeper areas of the brain later in time. It is proposed that CXCR2 has a dual role after brain injury: (i) homologous neuronal CXCR2 downregulation would render neurons more vulnerable to injury, whereas (ii) chemotaxis and subsequent differentiation of blood-borne cells into a microglial-like phenotype would be promoted by the same receptor.