Immunological Homeostasis at the Ovine Placenta May Reflect the Degree of Maternal Fetal Interaction.

Successful mammalian pregnancies are a result of complex physiological, endocrinological, and immunological processes that combine to create an environment where the mother is tolerant to the semi-allogeneic fetus. Our knowledge of the mechanisms that contribute to maternal tolerance is derived mainly from human and murine studies of haemochorial placentation. However, as this is the most invasive type of placentation it cannot be assumed that identical mechanisms apply to the less invasive epitheliochorial placentation found in other species such as ruminants. Here, we examine three features associated with reproductive immune regulation in a transformed ovine trophoblast cell line and ex-vivo ovine reproductive tissues collected at term, namely: major histocompatibility complex (MHC) expression, Indoleamine 2,3 dioxygenase-1 (IDO-1) expression, and Natural Killer (NK) cell infiltration. High levels of MHC class I protein expression were detected at the surface of the trophoblast cell line using a pan-MHC class I specific monoclonal antibody. The majority of MHC class I transcripts isolated from the cell line clustered with classical MHC alleles. Transcriptional analysis of placental tissues identified only classical MHC class I transcripts. We found no evidence of constitutive transcription of IDO-1 in either the trophoblast cell line or placental tissues. Ex-vivo tissues collected from the materno-fetal interface were negative for cells expressing NKp46/NCR1. Collectively, these observations suggest that the relatively non-invasive synepitheliochorial placentation found in sheep has a more limited requirement for local immunoregulation compared to the more invasive haemochorial placentation of primates and rodents.

The effects of macrophage migratory inhibitory factor on acute-phase protein production in primary human hepatocytes.

Macrophage inhibitory factor (MIF) is a pituitary peptide released during the physiological stress response, a T-cell product secreted during the antigen-specific response and a pro-inflammatory macrophage cytokine secreted after LPS stimulation. It has become apparent that MIF is central to the regulation of the inflammatory response and is implicated in the pathogenesis of a variety of acute and chronic inflammatory conditions. This is, at least in part, due to the apparent counter-regulation of the anti-inflammatory actions of glucocorticoids, including the reversal of glucocorticoid-mediated IL-6 release inhibition. This study examines the effect of recombinant MIF on regulation of the acute phase response in isolated human hepatocytes. MIF alone increased C-reactive protein (CRP) release in a dose-dependent manner < or = 0.1 ng/ml after which the effects of MIF were attenuated. In combination with IL-6 both CRP and and alpha-1-antichymotrypsin (ACT) release were increased above levels found with either IL-6 or MIF treatment alone. Dexamethasone attenuated the effects of MIF upon CRP production but increased the MIF stimulated release of ACT. The study demonstrates that the effects of MIF upon the acute phase response are complex and can differentially modulate the production of acute phase proteins depending on the presence of other factors.

TNF-alpha induced DNA damage in primary murine hepatocytes.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, usually arising from a background of chronic inflammatory disease. Tumor necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine produced in response to tissue injury, endotoxin exposure or infection and TNF-alpha signalling in hepatocytes is associated with an increase in oxidative stress. DNA is vulnerable to reactive oxygen species (ROS)-induced damage, which is highly mutagenic. Cells respond to DNA damage through the stabilisation of the tumor suppressor p53, which maintains genomic fidelity through induction of a cell cycle arrest in order to allow repair or elimination of the damaged cell through apoptosis. This study was carried out to determine if TNF-alpha caused oxidative DNA damage in primary cultures of murine hepatocytes and whether any damage would result in the induction of the tumor suppressor p53 and cell-cycle arrest. Using a modified Comet assay, to measure DNA damage we have demonstrated that TNF-alpha causes the formation of 8-oxo-deoxyguanosine (8-oxodG), an established marker of oxidative DNA damage, and a lesion associated with chronic hepatitis in human livers. In addition, the increase in DNA damage did not result in p53 stabilisation and TNF-alpha caused an increase in cell-cycle progression. We believe that this study indicates a possible putative role for TNF-alpha in the early stages of malignant transformation of hepatocytes.

Absence of correlation between serum CRP levels and mitochondrial D-loop DNA mutations in gastro-oesophageal adenocarcinoma.

INTRODUCTION: Both inflammation and mitochondrial DNA (mtDNA) mutation are thought to play a role in the many human cancers. The aim of this study was to evaluate the relationship between inflammation and accumulation of mitochondrial DNA (mtDNA) mutations in the D-loop region in carcinogenesis of gastro-oesophageal adenocarcinomas. MATERIALS AND METHODS: Blood samples of 20 patients with gastro-oesophageal adenocarcinoma were taken for measurement of serum C-reactive protein (CRP) concentration. Direct sequencing of mtDNA in the D-loop region was done in the 20 adenocarcinoma samples and their corresponding surrounding non-cancerous tissue. Sequences were compared with existing mtDNA databases to identify mutations. RESULTS: mtDNA mutations in the D-loop region occur commonly with almost identical frequency in both non-cancerous tissue (3.0 ± 1.6) and adenocarcinoma (3.1 ± 1.9) (P = 0.916, paired t-test). CRP levels are not predictive of the number of D-loop mutations in both adenocarcinoma (ß: -0.131; 95% CI: -2.354-1.364; P = 0.583) and non-cancerous tissue samples (ß: 0.130; 95% CI: -1.125-1.933; P = 0.586). Five new mutations were identified that were not recorded previously in mtDNA databases. CONCLUSION: D-loop mtDNA mutations are common in both gastro-oesophageal adenocarcinoma and surrounding non-cancerous tissue. However, the accumulation of such mutations appears to occur independent of systemic inflammation. The frequency of D-loop mutations is likely not useful as a marker for carcinogenesis in gastro-oesophageal adenocarcinoma.

Frequency of the mitochondrial DNA 4977bp deletion in oesophageal mucosa during the progression of Barrett's oesophagus.

PURPOSE: The mechanisms of the progression of Barrett's oesophagus (BO) to oesophageal adenocarcinoma (OA) are poorly understood. The frequency of the 4977bp deletion in mitochondrial DNA (mtDNA) was investigated in specimens ranging from normal oesophageal tissue to OA in order to investigate whether this deletion represents a useful biomarker of disease progression. METHODS: The presence of the 4977bp deletion was screened by PCR amplification from 70 specimens in total. RESULTS: The frequency of specimens with the 4977bp deletion increased in relation to the degree of dysplasia (8.3% in normal squamous epithelium; 15.4% in BO; 40% in low grade dysplasia (LGD); 69.2% in high-grade dysplasia and 90% in para-tumoural tissue). However, the frequency of the deletion reduced sharply in OA specimens (16.7%; p<0.001). CONCLUSION: The mtDNA 4977bp deletion may be useful as a biomarker to detect the severity of dysplasia but not the presence of OA.

Effect of high-fat enteral nutrition on hepatocyte injury in response to hemorrhagic shock in the rat.

BACKGROUND: High-fat enteral nutrition reduces the inflammatory response following hemorrhagic shock in the rat. AIMS: We hypothesized that this intervention might also ameliorate the remote organ injury to the liver associated with this model. METHODS: Male Sprague-Dawley rats were either starved or fed low-fat or high-fat isocaloric isonitrogenous feed prior to nonlethal hemorrhagic shock induced by a 40% reduction in the blood volume. Animals were sacrificed at 90 minutes or 24 hours after injury. Liver cell damage was assessed by histology and long polymerase chain reaction (PCR) to detect mitochondrial DNA damage. Stress protein expression was measured by Western blot and mRNA expression by real-time PCR and immunohistochemistry. RESULTS: Animals fed a low-fat diet had the same severity of liver injury as starved animals and increased expression of stress proteins. Animals fed a high-fat diet had minimal liver injury, no evidence of mitochondrial DNA damage, and significantly lower expression of stress proteins. This effect is associated with preservation of hepatocellular morphology, attenuation of mitochondrial DNA damage, and a reduced stress protein response to injury. CONCLUSIONS: High-fat enteral nutrition protects the liver from the remote effects of hemorrhagic shock, but the mechanism of this effect is not yet known.

Immunity in the female sheep reproductive tract.

Immune surveillance in the female reproductive tract is dependent on the interplay of many factors that include the expression of pattern recognition receptors on epithelial cells, resident leukocyte populations and hormones, none of which are uniform. The lower reproductive tract must accommodate the presence of commensal organisms whereas the upper reproductive tract is sterile. However, the upper female reproductive tract has its own immunological challenge in that it must tolerate the presence of a semi-allogeneic fetus if pregnancy is to succeed. So, immune activation and effector mechanisms to control pathogens may be qualitatively and quantitatively different along the reproductive tract. Our knowledge of innate and adaptive immunity in the sheep is less comprehensive than that of human or mouse. Nevertheless, comparative studies suggest that there are likely to be conserved innate immune sensory mechanisms (e.g. Toll-like receptors) and defence mechanisms (anti-proteases, defensins) that combine to limit infection in its early stages while shaping the adaptive response that leads to immunological memory and long-term protection. There are many pathogens that target the reproductive tract, and in particular the placenta, where specialised immunoregulatory mechanisms are operational. Among such pathogens are bacteria belonging to the genera Chlamydia/Chlamydophila that chronically infect the reproductive tracts of sheep and humans and ultimately cause disease through inflammation and tissue damage. An understanding of the immunological microenvironment of the reproductive tract is important for the design of novel control strategies to control chlamydial disease.