Site of action for endotoxin-induced cortisol release in the suppression of preovulatory luteinizing hormone surges

The study was conducted to identify the mechanisms of endotoxin/cortisol action in the suppression of preovulatory LH surges in heifers infused with Escherichia coli (E. coli ) endotoxin. The hypotheses tested were that 1) endotoxin stimulates the release of progesterone, possibly from the adrenal leading to the LH blockade; 2) cortisol released in response to endotoxin infusion blocks the synthesis of estradiol at the ovarian level, culminating in a failure of the LH surge. Eight Holstein heifers were given two injections of prostaglandin F(2alpha) (PG), 11 d apart, to synchronize estrus. Starting from 25 h after the second injection of PG (PG-2), the uterus of each heifer was infused either with 5 ml of pyrogen-free water (control, n = 3) or with E. coli endotoxin (5 mug/kg of body weight) in 5 ml of pyrogen-free water (treated, n = 5), once every 6 h for 10 treatments. Blood samples were obtained every 15 min for 1 h before infusion and again 2 h after each infusion, then hourly until 1 h before the next infusion. After the tenth infusion, blood was collected daily until estrus. Serum progesterone concentrations remained at baseline values (< 1 ng/ml) in control and treated heifers. The total amount of progesterone measured starting 24 to 84 h after PG-2 injection was not different between control and treated heifers (P 0.05). In the control heifers, serum estradiol concentrations remained basal (< 10 pg/ml) until 4 h before the LH surge. Serum estradiol concentrations increased to 20 +/- 5.6 pg/ml, 4 h before the LH surge in control heifers (LH surge occurred 60 to 66 h after the PG-2 injection). There were no changes in serum estradiol concentrations in treated heifers during the sampling period, and the concentrations remained < 10 pg/ml. The total amount of estradiol measured in control heifers was higher (P < 0.05) than in treated heifers. The results if this study suggest that increases in cortisol concentrations after the infusion of endotoxin might block the synthesis of estradiol at the ovarian level, resulting in the failure of a preovulatory LH surge to occur.     

Plasma endotoxin and concentrations of stable metabolites of prostacyclin, thromboxane A2, and prostaglandin E2 in postpartum dairy cows

The presence of endotoxin in plasma and patterns of stable metabolites of prostacyclin (PC), thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) were determined during the first postpartum estrous cycles in sixteen dairy cows. These included 8 cows with uterine infections which exhibited shortened luteal phases (SC) and 8 cows which had normal luteal phases (NC) after the first post partum ovulations. Endotoxin was consistently detected in all SC cows during the abbreviated estrous cycles while plasma samples of NC cows were free of endotoxin. Plasma concentrations of TXA2 metabolite was higher in SC cows (p less than 0.05) (1785-3452 pg/ml) compared to NC cows (723-1240 pg/ml). Similarly, plasma concentrations of PC metabolite was higher in SC cows (p less than 0.07) (423-1847 pg/ml) compared to NC cows (159-325 pg/ml). In contrast, plasma concentrations of PGE2 metabolite was higher in NC cows (p less than 0.05) (850-2219 pg/ml) compared to SC cows (455-628 pg/ml). The results of this study suggest that postpartum uterine infections mediate the release of prostaglandins from the uteri by means of the endotoxin and endotoxin appears to stimulate selectively the production of PC and TXA2 favoring early demise of corpora lutea formed after first postpartum ovulations in dairy cows.     

Protective effect of Toll-like receptor 4 in pulmonary vaccinia infection

Innate immune responses are essential for controlling poxvirus infection. The threat of a bioterrorist attack using Variola major, the smallpox virus, or zoonotic transmission of other poxviruses has renewed interest in understanding interactions between these viruses and their hosts. We recently determined that TLR3 regulates a detrimental innate immune response that enhances replication, morbidity, and mortality in mice in response to vaccinia virus, a model pathogen for studies of poxviruses. To further investigate Toll-like receptor signaling in vaccinia infection, we first focused on TRIF, the only known adapter protein for TLR3. Unexpectedly, bioluminescence imaging showed that mice lacking TRIF are more susceptible to vaccinia infection than wild-type mice. We then focused on TLR4, the other Toll-like receptor that signals through TRIF. Following respiratory infection with vaccinia, mice lacking TLR4 signaling had greater viral replication, hypothermia, and mortality than control animals. The mechanism of TLR4-mediated protection was not due to increased release of proinflammatory cytokines or changes in total numbers of immune cells recruited to the lung. Challenge of primary bone marrow macrophages isolated from TLR4 mutant and control mice suggested that TLR4 recognizes a viral ligand rather than an endogenous ligand. These data establish that TLR4 mediates a protective innate immune response against vaccinia virus, which informs development of new vaccines and therapeutic agents targeted against poxviruses.     

Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-beta. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection.     

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.     

Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi

Microsporidia are obligate intracellular parasites that were thought to be an ancient eukaryotic lineage based on molecular phylogenies using ribosomal RNA and translation elongation factors. However, this ancient origin of microsporidia has been contested recently, as several other molecular phylogenies suggest that microsporidia are closely related to fungi. Most of the protein trees that place microsporidia with fungi are not well sampled, however, and it is impossible to resolve whether microsporidia evolved from a fungus or from a protistan relative of fungi. We have sequenced beta-tubulins from 3 microsporidia, 4 chytrid fungi, and 12 zygomycete fungi, expanding the representation of beta-tubulin to include all four fungal divisions and a wide diversity of microsporidia. In phylogenetic trees including these new sequences, the overall topology of the fungal beta-tubulins generally matched the expected relationships among the four fungal divisions, although the zygomycetes were polyphyletic in some analyses. The microsporidia consistently fell within this fungal diversification, and not as a sister group to fungi. Overall, beta-tubulin phylogeny suggests that microsporidia evolved from a fungus sometime after the divergence of chytrids. We also found that chytrid alpha- and beta-tubulins are much less divergent than are tubulins from other fungi or microsporidia. In trees in which the only fungal representatives were the chytrids, microsporidia still branched with fungi (i.e., with chytrids), suggesting that the affiliation between microsporidian and fungal tubulins is not an artifact of long-branch attraction.