New blocking antibodies impede adhesion, migration and survival of ovarian cancer cells, highlighting MFGE8 as a potential therapeutic target of human ovarian carcinoma.

Milk Fat Globule--EGF--factor VIII (MFGE8), also called lactadherin, is a secreted protein, which binds extracellularly to phosphatidylserine and to αvß3 and αvß5 integrins. On human and mouse cells expressing these integrins, such as endothelial cells, phagocytes and some tumors, MFGE8/lactadherin has been shown to promote survival, epithelial to mesenchymal transition and phagocytosis. A protumoral function of MFGE8 has consequently been documented for a few types of human cancers, including melanoma, a subtype of breast cancers, and bladder carcinoma. Inhibiting the functions of MFGE8 could thus represent a new type of therapy for human cancers. Here, we show by immunohistochemistry on a collection of human ovarian cancers that MFGE8 is overexpressed in 45% of these tumors, and we confirm that it is specifically overexpressed in the triple-negative subtype of human breast cancers. We have established new in vitro assays to measure the effect of MFGE8 on survival, adhesion and migration of human ovarian and triple-negative breast cancer cell lines. Using these assays, we could identify new MFGE8-specific monoclonal antibodies, which efficiently blocked these three tumor-promoting effects of MFGE8. Our results suggest future use of MFGE8-blocking antibodies as new anti-cancer therapeutics in subgroups of ovarian carcinoma, and triple-negative breast carcinoma patients.

Escherichia coli induces bovine neutrophil cell death independent from caspase-3/-7/-1, but with phosphatidylserine exposure prior to membrane rupture.

Neutrophils are essential for the innate immune response against bacterial pathogens and play a key role during the early phases of infection, including mastitis and endometritis in cows. When directly challenged with bacteria, neutrophils undergo phagocytosis induced cell death (PICD). The molecular mechanisms of this cell death modality are poorly understood, especially for bovine neutrophils. Therefore, this study aimed to determine the mechanisms and hallmarks of PICD in bovine neutrophils after in vitro challenge with Escherichia coli (E. coli). Our data show that various apoptotic hallmarks such as blebbing, chromatin condensation and executioner caspase (C)-3/-7 activity are only observed during constitutive bovine neutrophil apoptosis. In contrast, bovine neutrophil PICD is characterized by production of reactive oxygen species (ROS), pro-inflammatory C-1 activation, nuclear factor (NF)-κB activation, and interleukin (IL)-1ß and IL-6 secretion. Nevertheless, under both conditions these phagocytes undergo cell death with the exposure of phosphatidylserine (PS). Although PS exposure is generally attributed to the anti-inflammatory features of executioner caspase-dependent apoptosis, it surprisingly preceded plasma membrane rupture during bovine neutrophil PICD. Moreover, C-1 inhibition strongly affected IL-1ß production but not the PICD kinetics. This indicates that the secretion of the latter pro-inflammatory cytokine is a bystander effect rather than a regulator of PICD in bovine neutrophils, in marked contrast to the IL-1ß-dependent pyroptosis reported for macrophages.

Inflammatory mediators in Escherichia coli-induced mastitis in mice.

Escherichia coli (E. coli) infections in mouse mammary glands are rarely described and poorly characterized. In order to investigate the host immune response during coliform mastitis, several inflammatory parameters were evaluated at 24 and 48h following inoculation of mouse mammary glands with E. coli. Successfully challenged mice showed high values of the acute phase protein serum amyloid A (SAA) in blood. Systemic concentrations of the major inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were also increased as compared to control mice, while interleukin-1 (IL-1) levels remained negligible. Infected mammary glands showed a significant increase of all cytokine levels as compared to control glands. In accordance, mammary expression of the biologically inactive proform of IL-1beta was strongly up-regulated. Remarkably, data obtained in wild type as well as caspase-1 knockout mice showed that IL-1beta maturation seemed to occur independently from caspase-1. Finally, E. coli infection also triggered activation of the nuclear transcription factor-kappaB (NF-kappaB) in the mammary gland. In conclusion, the current study provides novel insights on the contribution of major regulatory proteins to the acute inflammatory host response at the local and systemic level during E. coli mastitis in mice.

In vivo imaging of NF-kappaB activity during Escherichia coli-induced mammary gland infection.

In mammary gland infections, the contribution of NF-kappaB is not well defined, and was therefore investigated following intramammary inoculation of Escherichia coli. Non-invasive real-time in vivo imaging of the transcription factor activation was performed in mammary glands of transgenic mice expressing luciferase under the control of NF-kappaB. Bacterial inoculation resulted in a major increase in luminescence as compared with control glands. This activation was confirmed by immunohistochemical nuclear staining of the NF-kappaB p65 subunit in mammary epithelium of infected glands, while nuclear p50 was not detected. The systemic response to the intramammary inoculation of Escherichia coli was also studied. NF-kappaB activation in the liver increased over time, and a relatively mild but longer-lasting response was observed as compared with the acute hepatic response of mice receiving lipopolysaccharide. This systemic reaction was confirmed by increased circulating levels of the acute phase protein serum amyloid A, tumour necrosis factor-alpha and interleukin-6. In addition, high concentrations of both cytokines in the mammary gland inoculated with bacteria showed that the infection was also well established at the local level. These results indicate that in vivo monitoring of NF-kappaB activation is an attractive novel approach to study mammary gland inflammation, and that this transcription factor is imperative in the early stages of the host immune response towards coliform intramammary infections, both at the local and systemic level.

Immunoassay of urinary retinol binding protein as a putative renal marker in cats.

The presence of low molecular weight retinol binding protein (RBP) in urine reflects tubular damage. Therefore, RBP has been used as a renal marker in humans and dogs. Using an anti-human RBP antibody (Ab), this study first demonstrates feline urinary RBP by Western blot analysis and then evaluates its potential as a renal marker in cats by enzyme-linked immunosorbent assay (ELISA). Urine was taken by cystocentesis, centrifuged and stored at -80 degrees C until analysis. Urinary RBP levels were compared in clinically healthy cats (H), chronic renal failure patients (CRF) and cats with hyperthyroidism (HT). The detection of a band at the same position as the human RBP standard with Western blot analysis, indicated that RBP was present in the urine of CRF and HT patients but minimally present in H cats. The data obtained with ELISA were in accordance with these observations. RBP levels were expressed as RBP:creatinine (RBP:c) ratios following normalisation with urinary creatinine. The functional assay sensitivity was 1.37 microg/l RBP. Parallelism between the trend lines of the human RBP standard curve and the curves obtained from sequentially diluted urine samples indicated that feline RBP was recovered. The mean intra-assay coefficient of variance was 7% and the standardised agreement index revealed satisfactory day-to-day repeatability. The RBP:c ratio in all H cats (n=10) was below the assay sensitivity. The groups of CRF and HT patients had increased mean RBP:c ratios of 1.6+/-0.5x10(-2) microg/mg (mean+/-SEM, n=10) and 1.4+/-0.4x10(-2) microg/mg (n=13), respectively. Both groups showed a large variation in the relative RBP concentrations of individual cats. In conclusion, RBP is demonstrated for the first time in urine from most CRF and HT patients and the validated ELISA allows its evaluation as a putative renal marker in cats.

Flow cytometric assessment of estrogen receptor beta expression in bovine blood neutrophils.

The optimisation of a flow cytometric protocol for the determination of the estrogen receptor beta (ERbeta) expression in bovine blood neutrophils is described. The following final incubation conditions were obtained: fixation with 0.25% formaldehyde and 70% methanol, both for 1 h; permeabilisation with 0.05% Triton X-100, overnight labelling at 4 degrees C with the primary antibody diluted at 10 microg/ml and subsequent labelling for 30 min on ice with the fluorescein isothiocyanate-conjugated secondary antibody at 8 microg/ml. Of the three anti-human or anti-rat ERbeta primary antibodies evaluated, only PA1-311 was found to cross-react with bovine cells. Immunoblot analysis supports the obtained results. The flow cytometric technique allows reproducible quantitative determination of the ERbeta protein in neutrophils and may be a valuable tool for future expression studies in these cells of the innate immune system.

NF-kappaB inhibition accelerates apoptosis of bovine neutrophils.

Apoptosis is one of the major events that contribute to the regulation of the immune system. For human neutrophils, evidence has been produced that the transcription factor NF-kappaB is critical in influencing the ultimate outcome of a cell's fate. However, such research has not yet been performed on bovine neutrophils. This urged us to examine the possible involvement of NF-kappaB in apoptosis of these cells. At first, we investigated whether p65 and p50, the most important members of the NF-kappaB family, are expressed in isolated blood neutrophils. The presence of both members was demonstrated on the RNA and protein level. Then the effect on bovine neutrophil apoptosis of gliotoxin, a potent and specific inhibitor of NF-kappaB, was examined. The rate of constitutive apoptosis was found to be greatly accelerated by inhibition of NF-kappaB. Furthermore, gliotoxin dramatically augmented the limited pro-apoptotic effect of TNF-alpha, an important inflammatory mediator. The results were obtained in six cows by annexin-V-FITC staining of externalized phosphatidylserine and subsequent flow cytometric analysis. Additional measurement of caspase-3/7 activity and evaluation of morphological criteria confirmed the outcome of this experiment. Finally, NF-kappaB activity was assessed under these conditions. The activity of p50 was found to be minimally affected by gliotoxin, while significantly lower active p65 values were observed. Still, the highest percentage of apoptosis, which was caused by incubation with both gliotoxin and TNF-alpha, did not correspond to the lowest activity of p65. We conclude that NF-kappaB p65 promotes the survival of bovine neutrophils by delaying the initiation of apoptosis.

Regulation of the transcriptional activity of the nuclear factor-kappaB p65 subunit.

Nuclear factor-kappaB (NF-kappaB) is well known for its role in inflammation, immune response, control of cell division and apoptosis. The function of NF-kappaB is primarily regulated by IkappaB family members, which ensure cytoplasmic localisation of the transcription factor in the resting state. Upon stimulus-induced IkappaB degradation, the NF-kappaB complexes move to the nucleus and activate NF-kappaB-dependent transcription. Over the years, a second regulatory mechanism, independent of IkappaB, has become generally accepted. Changes in NF-kappaB transcriptional activity have been assigned to phosphorylation of the p65 subunit by a large variety of kinases in response to different stimuli. Here, we give an overview of the kinases and signalling pathways mediating this process and comment on the players involved in tumour necrosis factor-induced regulation of NF-kappaB transcriptional activity. Additionally, we describe how other posttranslational modifications, such as acetylation and methylation of transcription factors or of the chromatin environment, may also affect NF-kappaB transcriptional activity.