The source of circulating erythropoietin (EPO), the mediators and the mechanisms involved in the upregulation of EPO gene expression during acute-phase reaction are still poorly understood. Acute-phase reaction was induced by either intramuscular turpentine oil (TO) or intraperitoneal lipopolysaccharide (LPS) administration into wild-type and interleukin (IL)-6 knockout (KO) mice. Animals were killed at different time points and blood, liver and muscle tissue were collected. Serum levels of EPO were measured by enzyme-linked immunoadsorbent assay; liver and injured muscle samples were processed for RNA isolation and for protein analysis. EPO, hypoxia-inducible factors 1a and 2a (HIF-1a and HIF-2a) mRNA were analyzed by RT-PCR and the protein levels were analyzed by western blot and electrophoretic mobility shift assay. HIF-1a and HIF-2a localization was performed through immunofluorescence staining. EPO, HIF-1 and HIF-2 gene and protein expression levels were also analyzed in isolated mouse hepatocytes after stimulation with IL-6. In the wild-type animals, EPO serum levels increased dramatically at 12 h after the insults together with the hepatic gene expression. In TO-treated animals, the EPO gene expression reached an 8.2-fold increase at 12 h, and in LPS-treated mice a similar induction was recorded at 6 h (about 4.5-fold increase). In the IL-6KO strain, the upregulation after the inflammatory stimuli was much lower (only 2.0-fold increase). A progressive upregulation of HIF-1a and HIF-2a was detectable until 6 h after the insults, but only HIF-1a upregulation was reduced in IL-6KO mice. In isolated hepatocytes, stimulation with a single dose of IL-6 induced a nuclear accumulation of HIF-1a, in parallel with an increase of EPO mRNA. No effect on HIF-2a expression was found. IL-6 appears to be the main regulator of EPO gene expression and a major contributor for HIF-1a induction in hepatocytes and Kupffer cells during acute-phase response. The increase of HIF-2a, predominantly expressed in endothelial cells and fibroblast-like cells, seems not to be affected by the lack of IL-6.

The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of inflammation in the irradiated rat liver.

The liver and the spleen are the organs in which cellular material and aged erythrocytes are eliminated from the blood. Within the liver, Kupffer cells (KCs) are mainly responsible for this task, as such KCs have a pivotal role in iron metabolism. The aim of this study is to investigate the changes of hepatic gene expression in two models of KC phagocytosis. Gadolinium chloride (GD) or zymosan was injected intraperitoneally into rats and to endotoxin-resistant mice (C3H/HeJ). The animals were killed at different time points and their livers were immediately frozen in liquid nitrogen for RNA isolation and immunohistological studies. RNA was analyzed by real-time PCR and northern blot. Sera were used to measure transaminases, hepcidin and iron levels. The expression of iron metabolism genes, hepcidin, hemojuvelin (Hjv), ferroportin-1 (Fpn-1) and of the inflammatory cytokines IL-6, IL-1b, TNF-a and IFN-g was determined. Although phagocytosed material was detected in ED-1-and C1q-positive cells, no inflammatory cells were identified within the liver parenchyma. Serum levels of hepcidin, iron and transaminases did not differ from those of control animals. Both GD and zymosan induced an upregulation of hepcidin-gene expression in rat liver as early as 3 h, reaching a maximum 6 h after treatment. Hjv-and Fpn-1-gene expression was downregulated at the same time. IL-6 was by far the most induced acute-phase-cytokine in GD-and zymosan-treated livers, although IL-1b and TNF-a were also strongly upregulated by zymosan and to a lesser extent by GD. Similar results were obtained in the C3H/HeJ mouse strain excluding the possible role of contaminating endotoxin. This study shows that phagocytosis upregulates hepcidin-gene expression and downregulates Hjv-and Fpn-1-gene expression within the liver. These changes in iron-regulating-gene expression may be mediated by the locally produced acute-phase-cytokines.

An acute-phase response is the systemic reaction of an organism to insult (e.g. infection, trauma and burning). It represents the 'first line' of defence of the body to tissue-damaging attacks. In the present work, we used a rat model of an intra-muscular turpentine oil (TO) injection to analyse erythropoietin (EPO) gene expression changes in the liver, one of the main target organs of acute-phase cytokines. EPO began to increase in the serum of TO-treated animals 6 h after injection and reached a maximum at 24 h (125+/-20 pg/ml). The detection of total RNA by polymerase chain reaction analysis showed that the levels of EPO gene expression in the liver were considerably increased between 2 and 12 h by up to 20-fold at the peak after TO administration, followed by a gradual decrease over the next 48 h, although the values remained significantly higher compared with the control group. In the kidney, after a sudden slight increase, the values declined progressively to 3.5-fold decrease at 12 h after the injection. In the liver, a parallel upregulation of the hypoxia-inducible factor-1 (HIF-1) alpha gene was observed (up to 4.7-fold increase), while HIF-2 alpha gene expression remained unaltered. On the other hand, the protein of both genes became detectable after the injection and increased progressively over 24 h, with a subsequent decline. These results suggest that EPO may be added to the increasing group of positive acute-phase proteins and the liver might represent the major source of the hormone under these conditions in the rat.

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Background: Since the identification of HPV as the necessary cause of cervical cancer, knowledge of its natural history for cervical infection; genotypes, load and form, is essential to prevent and predict cervical carcinoma. A cross sectional study was conducted to see the natural history and genotypes of HPV in cervical neoplastic lesions. Materials and Methods: Two diagnostic modalities, IHC and PCR, were performed on 102 diagnosed cases of cervical premalignant and malignant lesions (Cervical Neoplastic Lesions: CNL) and lesions by histopathology. Results: Out of the102 cases there were 12, 44,40 and 6 cases of Squamous cell Papilloma(SCP), Squamous Cell Dysplasia(CD), Squamous Cell Carcinoma(SCC) and Adenocarcinoma(ADC) respectively. There were 82 CNL, which showed positive HPV reaction on IHC while 88 CNL showed presence of HPV (56/88:64% HPV-18 and 32/88:36% HPV-18) on PCR. There was significant association between the presence of HPV and p53 mutation among different CNL. There was no statistical difference in lesions with respect to positivity of HPV and P53 suggesting that the expression of this suppressor gene was affected by the presence of the HPV genome, p-value>0.05.IHC scoring of CNL was not significantly affected with HPV infection (p-value>0.05). The overall sensitivity, Specificity and accuracy of IHC against PCR was 93%,70,89% merits and demerits of each modality has also been given in detailed. The major mode of infection of HPV-16 was host nuclear integration (p:0.000) with different variant with predominant E-350G genotype(p:0.000). High Viral load was seen in infected cervical tissues. Conclusion: HPV-18&16 were detected;E-350 G genotype was most common form while predominant way of infection was host nuclear integration of HPV. The etiological involvement of HPV in the development of CNL was found because of high viral load and significant association with p53 protein expression with HPV infection. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A115.