Subclinical endometritis in beef cattle in early and late postpartum: Cytology, bacteriology, haptoglobin and test strip efficiency to evaluate the evolution of the disease.

In cows, detrimental effects on fertility are mainly caused by clinical and subclinical endometritis (SEM). As demonstrated in previous work, Piedmontese cattle are affected by a higher rate of infertility and presence of SEM. The objective of this study is to assess the pattern of SEM at 30 and 60 days postpartum by evaluating the correlation between uterine cytology and microbiology, analyzing SEM consequences on reproductive career and verifying the reliability of rising inflammatory proteins - haptoglobin and the test strip test. Seventy healthy cows were enrolled and sampled at 30 and 60 days postpartum; cytology and bacteriology as well as haptoglobin and test strip were evaluated. The ROC curve for cytology set the optimal cut-off at 6.5% at 30 days and 2.5% at 60 days for a Partum-to-Conception (PC) interval of 120 days. The cytological positivity was negatively correlated with fertility, at 30 days, but not at 60 days. A positive bacteriological test was not correlated with an increase in the PC at either 30 or 60 days postpartum. The presence of a calving parlor affect the fertility (P < 0.05) but not the presence of parity or suckling calf and parity. The ROC curve for strip test protein at 30 days postpartum set a cut-off of 2% for PC. No difference in serum haptoglobin was observed between negative or positive cytology/bacteriology in postpartum cattle. The test strip results for proteins have demonstrated a utility at 30 days postpartum for screening the cows that are at risk of developing an increased PC > 120 days.

Breast cancer vaccines: a clinical reality or fairy tale?

The characterization of tumor antigens recognized by immune effector cells has opened the perspective of developing therapeutic vaccines in the field of breast cancer. The potential advantages of the vaccines are: (i) the induction of a robust immune response against tumors that are spontaneously weekly immunogenic; (ii) the tumor specificity for some antigens; (iii) the good tolerance and safety profile and (iv) the long-term immune memory, critical to prevent efficiently tumor recurrence. Most trials evaluating breast cancer vaccines have been carried out in patients with extended metastatic breast cancer, characterized by aggressive tumors, resistant to standard cytotoxic treatments, so that clinical efficacy was difficult to achieve. However, some significant immune responses against tumor antigens induced upon vaccinations were recorded. The aim of this review is to analyze the activity of vaccination strategies in current clinical trials. Data of clinical activity have been observed by using vaccines targeting HER2/neu protein, human telomerase reverse transcriptase, carcinoembryonic antigen and carbohydrate antigen given after stem cell rescue. The review discusses possible future directions for vaccine development and applications in the adjuvant setting.

IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development.

To identify functions of the IKKalpha subunit of IkappaB kinase that require catalytic activity, we generated an Ikkalpha(AA) knockin allele containing alanines instead of serines in the activation loop. Ikkalpha(AA/AA) mice are healthy and fertile, but females display a severe lactation defect due to impaired proliferation of mammary epithelial cells. IKKalpha activity is required for NF-kappaB activation in mammary epithelial cells during pregnancy and in response to RANK ligand but not TNFalpha. IKKalpha and NF-kappaB activation are also required for optimal cyclin D1 induction. Defective RANK signaling or cyclin D1 expression results in the same phenotypic effect as the Ikkalpha(AA) mutation, which is completely suppressed by a mammary specific cyclin D1 transgene. Thus, IKKalpha is a critical intermediate in a pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1.

Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway.

In mammals, the canonical nuclear factor kappaB (NF-kappaB) signaling pathway activated in response to infections is based on degradation of IkappaB inhibitors. This pathway depends on the IkappaB kinase (IKK), which contains two catalytic subunits, IKKalpha and IKKbeta. IKKbeta is essential for inducible IkappaB phosphorylation and degradation, whereas IKKalpha is not. Here we show that IKKalpha is required for B cell maturation, formation of secondary lymphoid organs, increased expression of certain NF-kappaB target genes, and processing of the NF-kappaB2 (p100) precursor. IKKalpha preferentially phosphorylates NF-kappaB2, and this activity requires its phosphorylation by upstream kinases, one of which may be NF-kappaB-inducing kinase (NIK). IKKalpha is therefore a pivotal component of a second NF-kappaB activation pathway based on regulated NF-kappaB2 processing rather than IkappaB degradation.

NF-kappaB activation in response to toxical and therapeutical agents: role in inflammation and cancer treatment.

The NF-kappaB transcription factor is ubiquitously expressed and controls the expression of a large number of genes. Experimental data clearly indicate that NF-kappaB is a major regulator of the inflammatory reaction by controlling the expression of pro-inflammatory molecules in response to cytokines, oxidative stress and infectious agents. We demonstrated that NF-kappaB activation by IL-1beta follows three distinct cell-specific pathways. Moreover, our studies indicated that in one model of inflammatory diseases, horse recurrent airway obstruction (RAO), the extent of NF-kappaB basal activity correlates with pulmonary dysfunction. Another role of NF-kappaB activity protects cancer cells against apoptosis and could participate in the resistance to cancer treatment. However, we did not observe any increased cytotoxicity after treatment with anticancer drugs or TNF-alpha of cells expressing a NF-kappaB inhibitor. Therefore, we can conclude that the inhibition of apoptosis by NF-kappaB is likely to be cell type and stimulus-dependent. Further studies are required to determine whether NF-kappaB could be a target for anticancer treatments.

Non-enzymatic triggering of the ceramide signalling cascade by solar UVA radiation.

Ceramide is a key component of intracellular stress responses. Evidence is provided for a novel mechanism of ceramide formation that mediates solar ultraviolet (UV) A radiation-induced expression of the intercellular adhesion molecule (ICAM)-1. Similarly to UVA radiation, ceramide stimulation of human keratinocytes induced ICAM-1 mRNA expression and activated the ICAM-1 promoter through transcription factor AP-2. Ceramide-activated AP-2 and ceramide-induced ICAM-1 reporter gene activation were abrogated through deletion of the AP-2 binding site. UVA radiation increased the level of ceramide in keratinocytes and inhibition of sphingomyelin synthesis prevented UVA radiation-induced ICAM-1 expression. Hitherto, two pathways have been identified for ceramide accumulation: hydrolysis from sphingomyelin through neutral and acid sphingomyelinases, and de novo synthesis by ceramide synthase. UVA radiation did not activate any of these enzymes. Ceramide generation in UVA-irradiated cells, however, was inhibited by singlet oxygen quenchers and mimicked in unirradiated cells by a singlet oxygen-generating system. In addition, UVA radiation and singlet oxygen both generated ceramide in protein-free, sphingomyelin-containing liposomes. This study indicates that singlet oxygen triggers a third, non-enzymatic mechanism of ceramide formation.

Mechanisms of persistent NF-kappa B activity in the bronchi of an animal model of asthma.

In most cells trans-activating NF-kappaB induces many inflammatory proteins as well as its own inhibitor, IkappaB-alpha, thus assuring a transient response upon stimulation. However, NF-kappaB-dependent inflammatory gene expression is persistent in asthmatic bronchi, even after allergen eviction. In the present report we used bronchial brushing samples (BBSs) from heaves-affected horses (a spontaneous model of asthma) to elucidate the mechanisms by which NF-kappaB activity is maintained in asthmatic airways. NF-kappaB activity was high in granulocytic and nongranulocytic BBS cells. However, NF-kappaB activity highly correlated to granulocyte percentage and was only abrogated after granulocytic death in cultured BBSs. Before granulocytic death, NF-kappaB activity was suppressed by simultaneous addition of neutralizing anti-IL-1beta and anti-TNF-alpha Abs to the medium of cultured BBSs. Surprisingly, IkappaB-beta, whose expression is not regulated by NF-kappaB, unlike IkappaB-alpha, was the most prominent NF-kappaB inhibitor found in BBSs. The amounts of IkappaB-beta were low in BBSs obtained from diseased horses, but drastically increased after addition of the neutralizing anti-IL-1beta and anti-TNF-alpha Abs. These results indicate that sustained NF-kappaB activation in asthmatic bronchi is driven by granulocytes and is mediated by IL-1beta and TNF-alpha. Moreover, an imbalance between high levels of IL-1beta- and TNF-alpha-mediated IkappaB-beta degradation and low levels of IkappaB-beta synthesis is likely to be the mechanism preventing NF-kappaB deactivation in asthmatic airways before granulocytic death.

Correlation between nuclear factor-kappaB activity in bronchial brushing samples and lung dysfunction in an animal model of asthma.

Asthma is a chronic inflammatory disease of the airways, in which many inflammatory genes are overexpressed. Transcription factor, nuclear factor-kappaB (NF-kappaB), which is thought to control the transcriptional initiation of inflammatory genes, has been poorly investigated in asthma. In the present report, bronchial cells (BCs), recovered by bronchial brushing in healthy and heaves-affected horses (i.e., an animal model of asthma), were assessed for NF-kappaB activity. Small amounts of active NF-kappaB were present in BCs of healthy horses, whereas high levels of NF-kappaB activity was found during crisis (i.e., acute airway obstruction) in all heaves-affected horses. Three weeks after the crisis, the level of NF-kappaB activity found in BCs of heaves-affected horses was highly correlated (p < 0.01) to the degree of residual lung dysfunction. Unexpectedly, active NF- kappaB complexes found in BCs of heaves-affected horses were mainly p65 homodimers, rather than classic p65-p50 heterodimers. At last, intercellular adhesion molecule-1 (ICAM-1) expression paralleled p65 homodimers activity in these cells. These results demonstrate that the kinetics of NF-kappaB activity is strongly related to the course of the disease and confirm the relevance of NF-kappaB as a putative target in asthma therapy. Moreover, uncommon p65 homodimers could transactivate, in BCs, a subset of genes, such as ICAM-1, characteristic of chronic airway inflammation.

Cell type-specific role for reactive oxygen species in nuclear factor-kappaB activation by interleukin-1.

The role of reactive oxygen intermediates (ROIs) in nuclear factor-kappaB (NF-kappaB) activation remains a matter of controversy. We have studied whether ROIs played any role in NF-kappaB induction by interleukin-1beta (IL-1beta) in different cell types. Our studies indicated three different pathways. IL-1beta stimulation of lymphoid cells generates ROIs, which are required for IkappaB-alpha degradation and NF-kappaB activation. The source of these ROIs is the 5-lipoxygenase (5-LOX) enzyme. In monocytic cells, ROIs are also produced in response to IL-1beta and necessary for NF-kappaB induction, but their source appears to be the NADPH oxidase complex. Finally, epithelial cells do not generate ROIs after IL-1beta stimulation, but do rapidly activate NF-kappaB. Interestingly, transfection of epithelial cells with the 5-LOX and 5-LOX activating protein expression vectors restored ROI production and ROI-dependent NF-kappaB activation in response to IL-1beta. Our data thus indicate that ROIs are cell type-specific second messengers for NF-kappaB induction by IL-1beta.

Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.

Role of the protein kinase C lambda/iota isoform in nuclear factor-kappaB activation by interleukin-1beta or tumor necrosis factor-alpha: cell type specificities.

It has previously been reported that distinct signaling pathways can lead to nuclear factor (NF)-kappaB activation following stimulation of different cell types with inflammatory cytokines. As the role of atypical protein kinase C (PKC) isoforms in NF-kappaB activation remains a matter of controversy, we investigated whether this role might be cell type-dependent. Immunoblots detected atypical PKC expression in all the analyzed cell lines. The PKC inhibitor calphostin C inhibited NF-kappaB activation by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta in Jurkat or NIH3T3 cells but not in MCF7 A/Z cells. Cell transfections with a PKC lambda/iota dominant negative mutant abolished TNF-alpha-induced NF-kappaB-dependent transcription in NIH3T3 and Jurkat cells but not in MCF7 A/Z cells. Similarly, the same mutant blocked NF-kappaB-dependent transactivation after IL-1beta stimulation of NIH3T3 cells, but was ineffective after IL-1beta treatment of MCF7 A/Z cells. In MCF7 A/Z cells, however, the PKC lambda/iota dominant negative mutant could abolish transactivation of an AP-1-dependent reporter plasmid after stimulation with TNF-alpha but not with IL-1beta. These data thus confirm that transduction pathways for NF-kappaB activation after cell stimulation with TNF-alpha or IL-1beta are cell-type specific and that atypical PKC isoforms participate in this pathway in NIH3T3 and Jurkat cells.

Pyropheophorbide-a methyl ester-mediated photosensitization activates transcription factor NF-kappaB through the interleukin-1 receptor-dependent signaling pathway.

Pyropheophorbide-a methyl ester (PPME) is a second generation of photosensitizers used in photodynamic therapy. We demonstrated that PPME photosensitization activated NF-kappaB transcription factor in colon cancer cells. Unexpectedly, this activation occurred in two separate waves, i.e. a rapid and transient one and a second slower but sustained phase. The former was due to photosensitization by PPME localized in the cytoplasmic membrane which triggered interleukin-1 receptor internalization and the transduction pathways controlled by the interleukin-1 type I receptor. Indeed, TRAF6 dominant negative mutant abolished NF-kappaB activation by PPME photosensitization, and TRAF2 dominant negative mutant was without any effect, and overexpression of IkappaB kinases increased gene transcription controlled by NF-kappaB. Oxidative stress was not likely involved in the activation. On the other hand, the slower and sustained wave could be the product of the release of ceramide through activation of the acidic sphingomyelinase. PPME localization within the lysosomal membrane could explain why ceramide acted as second messenger in NF-kappaB activation by PPME photosensitization. These data will allow a better understanding of the molecular basis of tumor eradication by photodynamic therapy, in particular the importance of the host cell response in the treatment.

Multiple redox regulation in NF-kappaB transcription factor activation.

The well-known Rel/NF-kappaB family of vertebrate transcription factors comprises a number of structurally related, interacting proteins that bind DNA as dimers and whose activity is regulated by subcellular location. This family includes many members (p50, p52, RelA, RelB, c-Rel, ...), most of which can form DNA-binding homo- or hetero-dimers. All Rel proteins contain a highly conserved domain of approximately 300 amino-acids, called the Rel homology domain (RH), which contains sequences necessary for the formation of dimers, nuclear localization, DNA binding and IkappaB binding. Nuclear expression and consequent biological action of the eukaryotic NF-kappaB transcription factor complex are tightly regulated through its cytoplasmic retention by ankyrin-rich inhibitory proteins known as IkappaB. The IkappaB proteins include a group of related proteins that interact with Rel dimers and regulate their activities. The interaction of a given IkappaB protein with a Rel complex can affect the Rel complex in distinct ways. In the best characterized example, IkappaB-alpha interacts with a p50/RelA (NF-kappaB) heterodimer to retain the complex in the cytoplasm and inhibit its DNA-binding activity. The NF-kappaB/IkappaB-alpha complex is located in the cytoplasm of most resting cells, but can be rapidly induced to enter the cell nucleus. Upon receiving a variety of signals, many of which are probably mediated by the generation of reactive oxygen species (ROS), IkappaB-alpha undergoes phosphorylation at serine residues by a ubiquitin-dependent protein kinase, is then ubiquitinated at nearby lysine residues and finally degraded by the proteasome, probably while still complexed with NF-kappaB. Removal of IkappaB-alpha uncovers the nuclear localization signals on subunits of NF-kappaB, allowing the complex to enter the nucleus, bind to DNA and affect gene expression. Like proinflammatory cytokines (e.g. IL-1, TNF), various ROS (peroxides, singlet oxygen, ...) as well as UV (C to A) light are capable of mediating NF-kappaB nuclear translocation, while the sensor molecules which are sensitive to these agents and trigger IkappaB-alpha proteolysis are still unidentified. We also show that a ROS-independent mechanism is activated by IL-1beta in epithelial cells and seems to involve the acidic sphingomyelinase/ceramide transduction pathway.

Distinct signal transduction pathways mediate nuclear factor-kappaB induction by IL-1beta in epithelial and lymphoid cells.

We previously demonstrated that IL-1beta-mediated induction of the nuclear factor-kappaB (NF-kappaB) transcription factor proceeds through the production of reactive oxygen intermediates in lymphoid cells, while it occurs independently of any oxidative stress in epithelial transformed cells. Indeed, inhibition of receptor internalization as well as NH4Cl and chloroquine blocked IL-1beta-mediated induction of NF-kappaB in OVCAR-3 and in other epithelial cell lines but not in lymphoid cells, indicating that distinct pathways are involved. Conversely, while we observed phospholipase A2 activity in both cell types following IL-1beta stimulation, specific inhibitors of this enzyme inhibited NF-kappaB induction only in lymphoid cells. Moreover, expression of the 5-lipoxygenase (5-LOX) enzyme was not detected in epithelial cells, and inhibition of this enzyme blocked NF-kappaB induction by IL-1beta only in lymphoid cells. This study thus indicates that the activation of NF-kappaB following IL-1beta treatment involves the activation of phospholipase A2 and 5-LOX and the production of reactive oxygen intermediates (ROIs) in lymphoid cells, while in epithelial cells, another pathway predominates and could involve the acid sphingomyelinase. Moreover, arachidonic acid could induce NF-kappaB in epithelial and lymphoid cells, but this activation involved the 5-LOX enzyme and the production of ROIs only in lymphoid cells. The inefficiency of the ROI pathway in epithelial cells is probably the consequence of both low ROI production due to undetectable expression of 5-LOX and rapid degradation of hydrogen peroxide due to high catalase activity.

Interleukin-1 beta induces nuclear factor kappa B in epithelial cells independently of the production of reactive oxygen intermediates.

A large body of work has been devoted to tumor necrosis factor alpha or interleukin-1 beta (IL-1 beta) signaling leading to the activation of the transcription factor nuclear factor-kappa B (NF-kappa B) in various cell types. Several studies have indicated that NF-kappa B activation depends strictly on the production of reactive oxygen intermediates. In this report, we first demonstrated that IL-1 beta is a potent activator of NF-kappa B in various epithelial transformed cell lines (OVCAR-3, SKOV-3, MCF7 A/Z). In these cells, IL-1 beta rapidly induces NF-kappa B through a complete degradation of I kappa B-alpha, while H2O2 activates NF-kappa B with slower kinetics through a partial degradation of I kappa B-alpha, p100 and p105. We showed that IL-1 beta-mediated induction of NF-kappa B in OVCAR-3 and in other epithelial cell lines does not proceed through the production of reactive oxygen intermediates, while the same cytokine activates NF-kappa B in lymphoid cells through the intracellular generation of H2O2. Our study demonstrated that several signaling pathways lead to the activation of NF-kappa B, following IL-1 beta treatment in different cell types.

Highly-expressed p100/p52 (NFKB2) sequesters other NF-kappa B-related proteins in the cytoplasm of human breast cancer cells.

Several observations have suggested that NF-kappa B transcription factors could be involved in carcinogenesis. To investigate the possibility that members of the NF-kappa B family participate in the molecular control of the transformed phenotype, we examined the expression of these proteins in human breast cancer cell lines as well as in primary tumors. Western Immunoblots demonstrated high expression of the p52 precursor p100 (NFKB2) in several breast cancer cell lines while human mammary epithelial cells express this protein only faintly. Eighteen primary breast tumors out of 24 displayed significant expression of the p100/p52 protein. In MDA-MB-435 cells, overexpressed p100 and p52 are predominantly cytoplasmic and coimmunoprecipitation experiments demonstrated that p100 sequesters the heterodimer p50/p65 in the cytoplasm. We demonstrate that most p65 protein is complexed with p100 in these cells while it is complexed predominantly with I kappa B-alpha in cell lines expressing less p100. Our data strengthen the hypothesis that NF-kappa B could be involved in carcinogenesis and suggest that the p100/p52 NF-kappa B subunit could play a role in the development of human breast cancers, possibly by sequestering other NF-kappa B-related proteins in the cytoplasm.