Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation.

PURPOSE: To determine whether bovine corneal endothelial (BCE) cells and keratocytes express the inducible form of nitric oxide synthase (NOS) after exposure to cytokines and lipopolysaccharide (LPS), and to study the regulation of NOS by growth factors. METHODS: Cultures of bovine corneal endothelial cells and keratocytes were exposed to increasing concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). At selected intervals after exposure, nitrite levels in the supernatants were evaluated by the Griess reaction. Total RNA was extracted from the cell cultures, and messenger RNA levels for inducible NOS (NOS-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Exposure of BCE cells and keratocytes to LPS and IFN-gamma resulted in an increase of nitrite levels that was potentiate by the addition of TNF-alpha. Analysis by RT-PCR demonstrated that nitrite release was correlated to the expression of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced nitrite release in both cells, whereas transforming growth factor-beta (TGF-beta) slightly potentiated it. Fibroblast growth factor-2 (FGF-2) inhibited LPS-IFN-gamma-induced nitrite release and NOS-2 messenger RNA accumulation in keratocytes but not in BCE cells. CONCLUSIONS: The results demonstrate that in vitro activation of keratocytes and BCE cells by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammatory corneal diseases in vivo.

Role of cytokines in secondary lymphoid organ development

Summary Secondary lymphoid organs are sites of antigen presentation, clonal expansion of B and lymphocytes, and affinity maturation of B lymphocytes. In the intestine, these immune functions occur mainly in Peyer's patches (PP). PP develop through the interplay of two main cell types, haematopoietic cells and meserichyrnal cells. One particular haematopoietic cell type was identified as the inductive cell type in the formation of both PP and lymph nodes and was therefore designated as lymphoid tissue inducer cell. For a successful PP organogenesis, the crucial molecular components involved in the crosstalk of inducer cells and their mesenchymal target cells are adhesion molecules, lymphotoxin (LT) family members, and cytokines. In particular, the interleukin 7 receptor (IL-7R) expressed on inducer cells is absolutely required. To investigate the contribution of the ligand for the IL-7R. the cytokine IL-7, in the process of PP formation, we analyzed double transgenic (TG) mice. These mice resulted from an interbreeding of an IL-7TG mouse strain where the transgene is under the control of the MHC class II promoter with a second transgenic mouse strain, which overexpresses a transactivator for MHC class II genes. Double TG offsprings revealed higher levels of IL-7 mRNA occuring earlier in embryogenesis. Consequently, double TG mice showed a striking phenotype with a 3- to 5-fold increase in PP numbers compared to single IL-7TG or control littermates. Analysis of embryonic double TG intestines demonstrated that the process of PP development was already elevated during development as early as the embryonic day 16.5. Importantly, inducer cells were significantly increased in numbers in these embryonic intestines. Furthermore, the expression of LT? mRNA, which at this early time point is exclusively expressed by inducer cells, was also increased in double TG animals. These data clearly indicate a direct influence of IL-7 on the expansion of lymphoid tissue inducer cells and on the availability of LT? leading to a higher frequency of developing PP in fetal life. Interestingly, in addition to an enhanced frequency of PP development, in double TG mice, three additional phenotypic differences were observed. i) Lymphocyte infiltration in various non-lymphoid organs, such as stomach, salivary gland, and liver. Subsequent analysis demonstrated that B lymphocytes were predominant within these tertiary lymphoid structures. ii) Ectopic lymph node-like structures containing both B and T lymphocytes were found near the inguinal lymph node. iii) Double TG mice had a severe bone resorption syndrome most likely as a consequence of the pro-osteoclastic effect of IL-7. Taken together, these results show that IL-7 plays a key role in the homeostasis of inducer cells, in the generation of PP in the gut, in the formation of ectopic lymphoid tissue, and in bone resorption. Résumé Les organes lymphoïdes secondaires sont les lieux de présentation des antigènes aux lymphocytes, permettant l'expansion des lymphocytes B et T et la maturation d'affinité des lymphocytes B. Dans l'intestin, ces fonctions immunitaires se déroulent dans les plaques de Peyer (PP). Ces plaques se développent grâce à l'interaction des cellules hématopoïétiques avec des cellules mésenchymales. Un type particulier de cellules hématopoïétiques a été identifié comme cellule inductrice dans la formation des PP et des ganglions lymphatiques et de ce fait a été désigné cellule inductrice des tissus lymphoïdes. Durant l'organogénèse des PP, les composants moléculaires cruciaux impliqués dans l'interaction des cellules inductrices et des cellules mésenchymales sont les molécules d'adhésion, les membres de la famille des lymphotoxines (LT) et les cytokines. En particulier, le récepteur de l'interleukine 7 (IL-7R) exprimé par les cellules inductrices est absolument nécessaire. Pour étudier le rôle du ligand de l'IL-7R, l'interleukine IL-7, dans la formation des PP, nous avons croisé une lignée de souris transgénique (TG) surexprimant IL-7 sous contrôle du promoteur MHC class Il avec une lignée de souris transgénique surexprimant un transactivateur des genes MHC class II. Les souris doubles TG présentent une concentration élevée d'ARNm de l'IL-7 durant l'embryogénèse, ce qui résulte en une augmentation du nombre de PP de 3 à 5 fois en comparaison aux souris ayant seul le transgène IL-7 et aux souris contrôles. L'analyse des intestins des souris doubles TG démontre que le processus de développement des PP était élevé dès le jour 16.5 du développement embryonnaire. L'augmentation du nombre des cellules inductrices dans ces intestins embryonnaires est signilicative. De plus l'expression de l'ARNm LT?, qui à ce stade précoce est exclusivement exprimé dans les cellules inductrices, est également augmenté dans les doubles TG. Ces résultats indiquent clairement une influence directe d'IL-7 sur l'expansion des cellules inductrices des tissues lymphoïdes et sur la synthèse de LT? induisant une augmentation des PP se développant durant la vie foetale. En plus du développement accru des PP dans les souris doubles TG, trois différences phénotypiques ont été observées. i) L'infiltration lymphocytaire dans différents organes non-lymphoïdes, comme l'estomac, les glandes salivaires et le foie. Des analyses complémentaires ont demontré que les lymphocytes B étaient prédominants dans ces structures lymphoïdes tertiaires. ii) Des structures de ganglions lymphatiques ectopiques contenant des lymphocytes B et T ont été trouvées près des ganglions lymphatiques inguinaux. iii) Les souris doubles TG présentent un syndrome de résorption osseuse sévère probablement dû à l'effet pro-osteoclaste d'IL-7. Globalement, ces résultats montrent que IL-7 joue un rôle clé dans l'homéostasie des cellules inductrices dans la génèse de PP de l'intestin, dans la formation des tissus lymphoïdes ectopiques et dans la résorption osseuse.

Associations between alcohol consumption and selected cytokines in a Swiss population-based sample (CoLaus study).

To assess the associations between alcohol consumption and cytokine levels (interleukin-1beta - IL-1β; interleukin-6 - IL-6 and tumor necrosis factor-α - TNF-α) in a Caucasian population. Population sample of 2884 men and 3201 women aged 35-75. Alcohol consumption was categorized as nondrinkers, low (1-6 drinks/week), moderate (7-13/week) and high (14+/week). No difference in IL-1β levels was found between alcohol consumption categories. Low and moderate alcohol consumption led to lower IL-6 levels: median (interquartile range) 1.47 (0.70-3.51), 1.41 (0.70-3.32), 1.42 (0.66-3.19) and 1.70 (0.83-4.39) pg/ml for nondrinkers, low, moderate and high drinkers, respectively, p<0.01, but this association was no longer significant after multivariate adjustment. Compared to nondrinkers, moderate drinkers had the lowest odds (Odds ratio=0.86 (0.71-1.03)) of being in the highest quartile of IL-6, with a significant (p<0.05) quadratic trend. Low and moderate alcohol consumption led to lower TNF-α levels: 2.92 (1.79-4.63), 2.83 (1.84-4.48), 2.82 (1.76-4.34) and 3.15 (1.91-4.73) pg/ml for nondrinkers, low, moderate and high drinkers, respectively, p<0.02, and this difference remained borderline significant (p=0.06) after multivariate adjustment. Moderate drinkers had a lower odds (0.81 [0.68-0.98]) of being in the highest quartile of TNF-α. No specific alcoholic beverage (wine, beer or spirits) effect was found. Moderate alcohol consumption is associated with lower levels of IL-6 and (to a lesser degree) of TNF-α, irrespective of the type of alcohol consumed. No association was found between IL-1β levels and alcohol consumption.

Biofilm formation by staphylococci on fresh, fresh-frozen and processed human and bovine bone grafts.

Biofilm formation is a multi-step process influenced by surface properties. We investigated early and mature biofilm of Staphylococcus aureus on 4 different biological calcium phosphate (CaP) bone grafts used for filling bone defects. We investigated standardised cylinders of fresh and fresh-frozen human bone grafts were harvested from femoral heads; processed humanand bovine bone grafts were obtained preformed. Biofilm formation was done in tryptic soy broth (TSB) using S. aureus (ATCC 29213) with static conditions. Biofilm density after 3 h (early biofilm) and 24 h (mature biofilm) was investigated by sonication and microcalorimetry. After 3 h, bacterial density was highest on fresh-frozenandfresh bone grafts. After 24 h, biofilm density was lowest on freshbone grafts (p < 0.001) compared to the other 3 materials, which did not differ quantitatively (p > 0.05). The lowest increase in bacterial density was detected on fresh bone grafts (p < 0.001). Despite normal shaped colonies, we found additional small colonies on the surface of the fresh and fresh-frozen samples by sonication. This was also apparent in microcalorimetric heat-flow curves. The four investigated CaP bone grafts showed minor structural differences in architecture but marked differences concerning serum coverage and the content of bone marrow, fibrous tissue and bone cells. These variations resulted in a decreased biofilm density on freshand fresh-frozenbone grafts after 24 h, despite an increased early biofilm formation and might also be responsible for the variations in colony morphology (small colonies). Detection of small colony variants by microcalorimetry might be a new approach to improve the understanding of biofilm formation.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Une IgA à coefficient de sédimentation 11 S dans les sécrétions externes de l'espèce bovine

The sedimentation coefficient of a secretory IgA found in bovine colostrum and saliva is compared with that of IgG and IgM from the same colostrum. The IgA fraction gives a value of 10.8 S, whereas the major part of the IgG has a value of 7.1 S and the IgM 19.2 S. The sedimentation coefficient of the free secretory piece has also been determined: its value is 4.95 S.

Survey of Infectious Etiologies of Bovine Abortion during Mid- to Late Gestation in Dairy Herds.

Bovine abortion of unknown infectious etiology still remains a major economic problem. Thus, we investigated whether Brucella spp., Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Coxiella burnetii are associated with abortion and/or stillbirth in Tunisian dairy cattle. Using a pan-Chlamydiales PCR, we also investigated the role of Chlamydiaceae, Waddlia chondrophila, Parachlamydia acanthamoebae and other members of the Chlamydiales order in this setting. Veterinary samples taken from mid to late-term abortions from twenty dairy herds were tested. From a total of 150 abortion cases collected, infectious agents were detected by PCR in 73 (48.66%) cases, 13 (8.66%) of which represented co-infections with two infectious agents. Detected pathogens include Brucella spp (31.3%), Chlamydiaceae (4.66%), Waddlia chondrophila (8%), Parachlamydia acanthamoebae (5.33%), Listeria monocytogenes (4.66%) and Salmonella spp. (3.33%). In contrast, Campylobacter spp. and Coxiella burnetii DNA were not detected among the investigated veterinary samples. This demonstrates that different bacterial agents may cause bovine abortion in Tunisia. This is the first report suggesting the role of Parachlamydia acanthamoebae in bovine abortion in Africa. Further studies with a larger number of samples are necessary to confirm whether this emerging pathogen is directly linked to abortion in cattle.

Involvement of Parachlamydia in bovine abortions in Scotland.

Bovine abortion represents a major animal welfare issue and a cause of substantial economic loss yet the rate of successful diagnosis remains low. Chlamydia-related organisms including Parachlamydia have recently emerged as putative cattle abortifacients. Placental tissue samples and fetal lung from bovine abortion submissions across Scotland in Spring 2011 were investigated by histopathology for the presence of suspect Chlamydia-related organisms. Evidence of Chlamydia-related organisms was observed in 21/113 (18.6%) placenta samples. Thirteen of the suspect cases and 18 histopathology negative cases were analysed by molecular and immunohistochemical methods. All samples were PCR positive for Parachlamydia but sequencing revealed high homology between identified environmental 16S sequences in all but three cases. Parachlamydial antigen was detected in 10/31 placental samples (32.2%) with pathology consistent with chlamydial infection. This work supports the need for further surveillance investigations and experimental studies to determine the role of Parachlamydia in bovine abortion.

Histological behavior of glutaraldehyde cross-linked bovine collagen injected into the human bladder for the treatment of vesicoureteral reflux

Endoscopic subureteral collagen injection has become an accepted means for the treatment of vesicoureteral reflux in children. The aim of this study was to evaluate the histological behavior of glutaraldehyde cross-linked bovine collagen implants. The specimens were harvested from 29 patients who underwent reimplant surgery 2 to 30 months (mean 9.5) after unsuccessful subureteral injection therapy. In addition to routine hematoxylin and eosin staining, a new staining method (solophenyl red 3BL) able to demonstrate selectively neoformation of types I and III human collagen, was applied. Invasion of host fibroblasts into the bovine implant and the formation of endogenous types I and III collagen were demonstrated in all 29 cases. Adverse histological reactions were rare and, if present, they were predominantly of an inflammatory nature.

Involvement of connexin 43 in meiotic maturation of bovine oocytes.

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.

Development of a well-defined medium for the in vitro maturation of immature bovine cumulus-oocyte complexes.

PURPOSE: Our purpose was to develop a well-defined medium for the in vitro maturation (IVM) of immature bovine cumulus-oocyte complexes (COC). METHODS: The COC were cultured in the presence of three protein supplementations: fetal bovine serum (FBS), bovine serum albumin, and Synthetic Serum Substitute. The embryos obtained after in vitro fertilization of IVM oocytes were cocultured with Vero cells and their development to the morula and blastocyst stages was studied. RESULTS: When FBS was absent from the IVM medium, a significantly lower fertilization rate was observed, followed by a decrease in the percentage of embryos reaching the blastocyst stage. When FBS was replaced by a defined protein supplementation, the best results were obtained with Synthetic Serum Substitute. CONCLUSIONS: Adequate protein supplementation of the IVM medium optimizes the fertilization rate and the development of bovine IVM oocytes. The implication of these results in the human field is discussed.