Entomologic and Virologic Investigation of Chikungunya, Singapore

Data from longitudinal analyses can be useful in the design and implementation of control strategies

The Early Clinical Features of Dengue in Adults: Challenges for Early Clinical Diagnosis

Dengue infection in adults has become increasingly common throughout the world. As most of the clinical features of dengue have been described in children, we undertook a prospective study to determine the early symptoms and signs of dengue in adults. We show here that, overall, dengue cases presented with high rates of symptoms listed in the WHO 1997 or 2009 classification schemes for probable dengue fever thus resulting in high sensitivities of these schemes when applied for early diagnosis. However, symptoms such as myalgia, arthralgia, retro-orbital pain and mucosal bleeding were less frequently reported in older adults. This trend resulted in reduced sensitivity of the WHO classification schemes in older adults even though they showed increased risks of hospitalization and severe dengue. Instead, we suggest that older adults who present with fever and leukopenia should be tested for dengue, even in the absence of other symptoms. This could be useful for early clinical diagnosis in older adults so that they can be monitored and treated for severe dengue, which is especially important when an antiviral drug becomes available

Role of cyclin E in the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC), or primary liver cancer, is the fifth most common cancer worldwide and the third most common cause of cancer mortality (El-Serag 2012). The development of HCC is thought to be a multi-staged process that involves several risk factors including the chronic hepatitis B and C infection, carcinogen exposure, metabolic disease, excessive alcohol consumption and male gender. Accumulation of genetic and epigenetic alterations with DNA-damaged hepatocytes can also contribute to the molecular pathogenesis of HCC. A better understanding of molecular mechanisms associated with HCC could ultimately improve our current strategies for screening and targeted therapy of this disease. Array comparative genomic hybridisation studies in murine diethylnitrosamine (DEN)-induced HCC have identified ccne1 (cyclin E) as a candidate gene associated in accelerated liver carcinogenesis (Teoh et al. 2008). In order to investigate the role of cyclin E and its impact on hepatocyte cell cycle regulation in early HCC development, we employed a well-known and highly reproducible rodent model of DEN-induced hepatocarcinogenesis. C57BL/6J male mice were injected with DEN (10 mg/kg i.p.), age day 12-15. In this model, male animals develop hepatocyte dysplasia at 6 mths and HCC in >90%. Transcript and protein expression of cyclin E was evident as early as 6 mths in dysplastic nodules (DNs), and significantly increased in HCCs. In contrast, there was little or undetectable cyclin E in normal liver and liver surrounding HCCs at all timepoints. Glutathione S transferase-pi form and cyclin E expression co-localised in DNs from liver in mice at 6 and 9 mths. Cyclin E/cdk2 kinase activity was also significantly upregulated in DNs, while increased proliferative activity by cyclin D1 and proliferating cell nuclear antigen (PCNA) in HCCs was observed at 9 mths, a timepoint where there was maximal p53 and p21 tumour suppressor expression. Aberrant cyclin E protein expression, including low molecular weight (LMW) isoforms were detected in HCCs and liver surrounding HCCs. Interestingly, sequencing analyses of p53 revealed a 1093-1361 nucleotide deletion in up to 90% of DNs, causing dysfunctional p53 nuclear localisation and export signalling. Because p53 directly signals to p21, co-immunoprecipitation studies were performed and revealed preferential binding of p21 to cyclin D1, rather than cyclin E, thereby allowing "escape" from the G1/S checkpoint. To directly test whether cyclin E regulates p53 expression in HCCs derived from DEN-treated male mice at 9 mths, we conducted cyclin E knockdown in primary HCC cells. This strategy resulted in increased p53 and p21 expression, as well as significant diminution of Bcl-xL, the p53-induced anti-apoptotic marker. Cell viability tetrazolium/formazan assay was significantly impaired in cyclin E RNAi targeted primary HCC cells. Conversely, chemical inhibition of p53 by pfithrin-α, augmented cyclin E, PCNA and Bcl-xL protein expression whilst cell viability was restored following co-treatment with MG-262, a 26S proteasome inhibitor. In contrast, overexpressing cyclin E in naïve primary hepatocytes enhanced PCNA expression, increased hepatocyte viability, downregulated p53 and its downstream signalling intermediate, p21. We next determined whether miRNA-34, a co-regulator of cyclin E and p53, was instrumental in the reciprocity between cyclin E and p53 as key "drivers"of hepatocarcinogenesis. Dysplastic liver and HCCs obtained from DEN-injected male mice were assayed for miR-34a,b,c. miR-34a and c were significantly upregulated in HCCs and dysplastic liver compared with normal liver. Similar trends were noted for miR-34a,b,c in human hepatitis C-related HCCs when compared with normal human liver. Importantly, this was associated with significantly enhanced cyclin E and p53 mRNA expression in human HCCs compared to normal and cirrhotic liver. In this murine model, there was disproportional and upregulation of functionally active cyclin E, miR-34a,c in DNs and early HCCs with congruent loss of p53 function associated with cell cycle checkpoint failure, diminished apoptosis and increased proliferative drive. In human HCV-related HCCs, miR-34a, p53 and cyclin E transcript levels were universally upregulated. When we performed in vitro experiments in murine primary hepatocytes and primary HCC cells, knocking down or overexpressing cyclin E did not affect miR-34 expression. However, stabilising p53 with MG-262 enhanced miR-34a,c expression (though not significant), whilst inhibiting p53 using pfithrin-α significantly reduced miR-34a,c. miR-34 may provide a plausible link to increased cyclin E expression, activity and increased proliferative drive in dysplastic and neoplastic liver in mice and humans. Gender disparity in human HCC is well described, with a strong male predominance. However, the role of sex hormones in hepatocarcinogenesis remains poorly defined. In order to determine if there are gender differences in the expression of cyclin E, effects on cell cycle regulators and tumour suppressors, dysplastic liver and HCCs were studied in DEN-treated C57BL/6J female mice. These mice displayed a significant reduction in dysplastic hepatocytes compared with intact DEN-treated males at 3, 6 mths, while HCC incidence, number and size of tumours were significantly diminished in females at up to 15 mths. In carcinogen-treated female mice, cyclin E (native and LMW isoforms) protein expression and kinase activity were reduced compared to males at 6-12 mths, with concomitant reduction in hepatocyte proliferation by PCNA and cyclin D1 expression. Unlike male mice, G1/S checkpoint is evident by robust p53-mediated apoptosis. To ascertain if these differences are attributable to the effects of oestradiol/progesterone (E/P) and/or testosterone, we conducted hormonal manipulation studies using the same carcinogen-model by performing ovariectomy in female animals and orchidectomy in male mice, in which some animals received E/P or testosterone supplementation. Castration of DEN-injected male mice resulted in a loss of cyclin E LMW isoforms compared to intact males, diminution of cyclin E kinase activity and phospho-retinoblastoma expression. There was also induction of p53-mediated apoptosis in dysplastic hepatocytes, leading to a reduction in number of DNs by 6 mths. These anti-proliferative and pro-apoptotic effects were magnified by E/P replacement in castrated DEN-treated males. In contrast, testosterone-replacement in ovariectomised-female mice exhibited accelerated hepatocarcinogenesis compared to intact female DEN-treated animals and displayed LMW cyclin E isoforms similar to those detected in DEN-injected intact males. In further analyses, there was increased oestrogen receptor-α (ERα) transcript and protein expression in HCCs derived from DEN-injected intact male mice, and in dysplastic liver from castrated male mice replaced with E/P. E/P replacement and testosterone withdrawal were associated with ERα expression, the loss of cyclin E LMW isoforms, intact cdk2 expression, functional G1/S checkpoint control and induction of p53-mediated apoptotic cell death in preneoplastic hepatocytes. Further, oestrogen (E2) stimulation had varying effects on cell cycle regulation and viability in primary hepatocytes and HCC cells. As there was little to no significant correlation between ERα and its downstream target, c-myc transcript levels, we propose that E2/ERα signalling may be operative via other pathways to subsequently activate p53. These findings open up tantalising avenues to further explore the inter-regulatory signalling pathways between E2/ERα, cell cycle regulators and the tumour suppressor, p53

Testosterone regulation of cyclin E kinase is a key factor determining gender differences in hepatocarcinogenesis

Background & Aim: While gender differences in hepatocellular carcinoma (HCC) are profound, the mechanism is unclear. Using castration and hormone replacement strategies, we tested whether these gender differences are attributable to testosterone or estradiol/progesterone (E/P) effects on cell cycle regulators and p53. Methods: We studied dysplastic liver and HCCs in intact and castrated diethylnitrosamine (DEN)-injected C57BL/6J male and female mice, with or without hormonal replacement. Effects of sex steroids on proliferation and survival of primary hepatocytes and primary HCC cells were also characterized. Results: DEN-injected females displayed fewer dysplastic foci and slower onset of HCC than males, with smaller/more differentiated tumors and fewer metastases. Castration of DEN-injected males reduced cyclin E kinase and augmented hepatocyte apoptosis compared with intact males; E/P enhanced these effects. In intact females, cyclin E kinase activity was less than in males; testosterone administered to ovariectomized females up-regulated cyclin E, increased cyclin E kinase and accelerated hepatocarcinogenesis. In vitro, testosterone increased expression of cell cycle regulators (cyclin D1, cyclin E and cyclin-dependent kinase 2) and reduced p53 and p21, which enhanced hepatocyte viability. In contrast, estradiol both suppressed hepatocyte cell cycle markers, up-regulated p53 and reduced viability of hepatocytes and HCC cells. Conclusions: Testosterone is the positive regulator of hepatocyte cell cycle via cyclin E, while estradiol plays a negative role by effects of p53 and p21. Together, both sex hormones determine the male predominance of gender differences in hepatocarcinogenesis

Deriving murine transformed hepatocytes in primary culture: novel technique and potential utility

Introduction: There has been a heavy reliance on the use of hepatoma cell lines to study the pathways of hepatocarcinogenesis. Cell lines are transformed, immortalized, and do not mimic the natural pathobiological characteristics of hepatocellular carcinoma (HCC). Aim: This study aims to develop novel techniques of deriving and culturing primary dysplastic hepatocytes (DHs) and HCC cells for interventional and functional studies. Methods: Primary hepatocytes (PHs) are derived from naïve male mice using the hepatic portal vein perfusion method; primary DHs and HCCs from diethylnitrosamine-injected mice are isolated via fluorescence activated cell sorting and mechanical disruption. Cell viability is determined using MTT assay and protein expression by immunoblotting. Cell cycle phase analysis is performed using propidium iodide staining followed by fluorescence activated cell sorting, while p53 and mdm2 chemical inhibition with pifithrin-α and Nutlin-3 are used, respectively. Results: While the viability of PHs is limited to 5 days on collagen-coated plates, primary DHs can be maintained in culture for up to 4 weeks with minimal proliferative activity. In contrast, primary HCC cells undergo active hepatocyte proliferation, evident by several passages and positive proliferating cell nuclear antigen expression. Cell cycle phase analyses confirm that majority of PHs are maintained in cell cycle arrest phase (G0/G1) with only 2 ± 0.02% cells in G2/M phase. However, sorted DHs display an augmented population of G2/M cells (30 ± 0.03%) and such populations are enhanced in primary HCCs (56 ± 0.1%). Pifithrin-α administration results in decreased p53 mRNA in HCC cells but elicits no changes in DHs. puma mRNA is decreased in both cell types. Inhibition of p53 exhibits increased mdm2 and downregulation of usp18 mRNA expression in both cell types. Nutlin-3 treatment suppresses mdm2 mRNA only in DHs but not HCCs. p53 and usp18 mRNA are downregulated in both cell types. Conclusion: Isolated murine dysplastic hepatocytes exhibit distinct molecular mechanisms/properties compared to HCC cells and may prove to be an appropriate in vitro resource to elucidate the molecular changes early in hepatocarcinogenesis

Deriving and testing of dysplastic murine hepatocytes: a new platform in liver cancer research

Dysplastic hepatocytes (DH) represent altered hepatocytes with potential for malignant transformation. To date, most research on pathways to hepatocarcinogenesis has focused on use of “hepatoma” cell-lines derived from hepatocellular carcinoma (HCC). We describe a novel technique for deriving/culturing DH and demonstrate their utility for functional studies in vitro, compared to primary hepatocytes (PH) and HCC. PH and DH were prepared by portal vein collagenase perfusion from C57BL/6 J mice. DH were subsequently subjected to FACS. HCC from diethylnitrosamine (DEN)-injected mice were mechanically isolated. Cell cycle analyses were performed by flow cytometry and PCNA immunohistochemisty. To establish utility of DH, we studied pathways of p53 turnover, apoptosis and cell proliferation using pfithrin-α (PFT) and nutlin-3. Like PH, DH were minimally proliferative compared to HCC. Only 30±0.03% of DH were in G2/M phase versus 51±0.01% of HCC; this difference corroborated with PCNA-immunostaining of dysplastic nodules from DEN-injected mice. In DH and HCC, nutlin-3 suppressed p53 mRNA, induced p53 and mdm2 activation but paradoxically resulted in increased anti-apoptotic and proliferative activity. Primary murine DH display distinctive biological characteristics compared with PH and HCC. As an intermediate cell type to HCC, they offer a new pathobiologically relevant primary cell culture system with which to interrogate the molecular changes in hepatocarcinogenesi

Cyclin E facilitates dysplastic hepatocytes to bypass G(1)/S checkpoint in hepatocarcinogenesis

Background and Aim: By array-comparative genomic hybridization, we demonstrated cyclin E as one of seven genes associated with hepatocellular carcinoma (HCC) development in Ku70 DNA repair-deficient mice. We therefore explored the hypothesis that during hepatocarcinogenesis, cyclin E kinase can overcome the inhibitory effects of p53 and establish whether abnormal miRNA(mi-R)-34, a co-regulator of cyclin E and p53, can account for their interactions as " drivers" of HCC. Methods: Dysplastic hepatocytes (DNs) and HCCs were generated from diethylnitrosamine (DEN)-injected C57BL/6 male mice at 3-12 months. Results: Cyclin E/cdk2 was barely expressed in normal liver, but was readily detected in dysplastic hepatocytes, localizing to glutathione-S transferase pi-form positive cells dissected by laser-dissection. Cyclin E kinase activity preceded cyclin D1, proliferating cell nuclear antigen expression in DNs and HCCs despite maximal p53 and p21 expression. We confirmed that cyclin E, rather than cyclin D1, is the proliferative driver in hepatocarcinogenesis by immunoprecipitation experiments demonstrating preferential binding of p21 to cyclin D1, allowing cyclin E-mediated " escape" from G1/S checkpoint. We then showed cyclin E was responsible for regulating wild-type p53 by knockdown experiments in primary HCC cells; cyclin E-knockdown increased p53 and p21, diminished anti-apoptotic Bcl-XL and reduced cell viability. Conversely, blocking p53 augmented cyclin E, Bcl-XL expression and increased proliferation. Physiological interactions between cyclin E/p53/ p21 were confirmed in primary hepatocytes. miR-34a, c were upregulated in dysplastic murine, human liver and HCCs compared with normal liver, and appeared to be linked to cyclin E/p53. Conclusion: Upregulation of functionally active cyclin E via miR34 with loss of p53 function is associated with cell-cycle checkpoint failure increasing proliferative drive that favors hepatocarcinogenesis