Hepatic expression of proteasome subunit alpha type-6 is upregulated during viral hepatitis and putatively regulates the expression of ISG15 ubiquitin-like modifier, a proviral host gene in hepatitis C virus infection.

The interferon-stimulated gene 15 (ISG15) plays an important role in the pathogenesis of hepatitis C virus (HCV) infection. ISG15-regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV-infected (n = 54) and uninfected (n = 10) or HBV-infected controls (n = 23). Primary human hepatocytes (PHH) were treated with Toll-like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type-6 (PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific siRNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV-infected patients. In contrast to interferon-stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock-down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV.

Apparatus for measuring pressure-driven transport through channels at high Knudsen numbers.

The pressure-driven gas flow through micro- and nano-porous structures is particularly interesting for innovative technologies such as microelectromechanical and nano-mechanical-electrical systems. The classical continuum assumption breaks down for rarefied flow through channels with a characteristic dimension comparable to the mean free path of the gas. Theories based on molecular interactions have been formulated to predict the flow at high Knudsen numbers. Measuring rarefied gas flow experimentally is a challenge since only a few studies have been able to determine flowrates in the molecular flow regime. Here we present the design of an experimental apparatus, which can be used to measure the flow of gases through nano- and microscale channels in the flow regimes where molecular effects are critical. The equations used to design the apparatus are given, focusing on the slip and transition flow regimes (together sometimes called "Intermediate flow regime"). A channel with a diameter of 325 µm ± 5µm and a length of 2 mm was tested experimentally with the apparatus for a wide range of Knudsen numbers (10-2 < Kn < 1 × 105) demonstrating its suitability through the slip and transition regime (2.23 × 10-2 < Kn < 2.26).

Formation and repair kinetics of Pt-(GpG) DNA adducts in extracted circulating tumour cells and response to platinum treatment.

BACKGROUND: Pt-(GpG) intrastrand crosslinks are the major DNA adducts induced by platinum-based anticancer drugs. In the cell lines and mouse models, the persistence of these lesions correlates significantly with cell damage. Here we studied Pt-(GpG) DNA adducts in circulating tumour cells (CTC) treated with cisplatin in medium upfront to systemic therapy from patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Blood was drawn before systemic treatment and the CD45/CD15-depleted fraction of mononuclear cells was exposed to cisplatin, verified for the presence of CTC by pan-cytokeratin (pCK) staining and immunoanalysed for the level of Pt-(GpG) in DNA. RESULTS: Immunostaining for pCK, CD45 and subsequently for Pt-(GpG) adducts in the cisplatin-exposed cells (ex vivo) at different time points depicted distinct differences for adduct persistence in CTC between responders vs non-responders. CONCLUSION: Pt-(GpG) adducts can be detected in CTC from NSCLC patients and assessing their kinetics may constitute a clinically feasible biomarker for response prediction and dose individualisation of platinum-based chemotherapy. This functional pre-therapeutic test might represent a more biological approach than measuring protein factors or other molecular markers.

Influence of age on bronchoscopic findings in healthy beagle dogs.

The aims of this study were to evaluate the effect of age on bronchoscopic features and bronchoalveolar lavage fluid (BALF) cellularity in dogs. Thirty healthy beagle dogs from three age groups were included: young dogs (10 months to 4.5 years of age; n=8), middle-aged dogs (5-8 years old; n=13) and older dogs (>8 years; n=9). Haematology, thoracic radiography, bronchoscopy and bronchoalveolar lavage were performed; bronchoscopic findings were scored and BALF total and differential cell counts were determined. The total bronchoscopic score was higher in older dogs; these dogs had more irregular bronchial mucosa, more prominent mucosal vessels and bronchiectasis. Younger dogs had a higher percentage of neutrophils in BALF compared with middle-aged and old dogs and a higher percentage of lymphocytes in BALF compared with middle-aged dogs. The results show that age has an effect on bronchoscopic features of airways and the composition of BALF in the dog.

Lack of prognostic significance of serum DNA methylation of DAPK, MGMT, and GSTPI in patients with non-small cell lung cancer.

BACKGROUND AND OBJECTIVES: To further improve the screening, diagnosis and therapy of patients with non-small cell lung cancer (NSCLC) additional diagnostic tools are desperately warranted. Aim of this study was to investigate the potential of the DNA methylation of DAPK, MGMT, and GSTPI in serum of patients with NSCLC as a prognostic molecular marker in this disease. METHODS: Seventy-six patients with NSCLC were included in this study. The analysis of DNA methylation in serum of patients was performed on pre-operative samples. Following DNA isolation and bisulfite-treatment, DNA methylation was analyzed by quantitative-methylation-specific real-time PCR with beta-actin as the internal reference gene. RESULTS: DNA methylation was detectable with following frequencies: DAPK 68.4%, MGMT 7.9%, GSTPI 0%. There were no associations between DNA methylation status and histology, tumor stage, grading or gender detectable. With a mean follow-up of 19.7 months the median survival was 26.3 months. There were no associations between the status of DNA methylation in patient's serum and prognosis detectable. CONCLUSION: The analysis of DNA methylation in serum of patients with NSCLC by quantitative-methylation-specific real-time PCR is technically feasible. Although our results suggest quantification of DNA methylation in serum not of prognostic significance in this disease, further studies are warranted to determine the future potential of this molecular approach.

Survivin mRNA in peripheral blood is frequently detected and significantly decreased following resection of gastrointestinal cancers.

BACKGROUND AND OBJECTIVES: We evaluated if mRNA expression of survivin, an inhibitor of apoptosis, can be used to detect circulating tumor cells in peripheral blood of patients with various gastrointestinal cancers and if they decrease following complete surgical resection. METHODS: Blood samples from 40 gastrointestinal cancer patients were analyzed prior and following surgical resection by direct quantitative real-time reverse transcriptase-PCR (RT-PCR) assays. RESULTS: Survivin mRNA expression was pre-operatively detected in 35 of 40 cancer patients (88%). Post-operative survivin levels were significantly lower than pre-operative levels in 59% of resected patients and were non-detectable in 38% (Wilcoxon rank test: P < 0.04). CONCLUSIONS: This is the first report showing that direct quantitative real-time RT-PCR analysis of survivin mRNA expression in peripheral blood of patients with gastrointestinal cancers is technically feasible. Survivin mRNA levels fall significantly following complete resection and might become a molecular marker for the completeness of surgical resection.

Quantitative evaluation of tourniquet leak during i.v. regional anaesthesia of the upper and lower limbs in human volunteers.

Although it is accepted that during i.v. regional anaesthesia (IVRA) local anaesthetic can leak under the tourniquet into the systemic circulation, no published study has evaluated this leak quantitatively. In volunteers, during two random sessions, we have simulated IVRA using standard techniques with a radiolabelled compound which is chemically similar to lignocaine and has comparable tissue distribution (0.1 mg of HIDA labelled with 100 muCi of 99mTc in 40 ml of saline). The decrease in radioactivity was measured with a gamma camera for the 20 min of tourniquet inflation and for the 20 min of washout after cuff deflation. While the tourniquet was inflated, the leak for the lower limb (mean 29 (SD 8) %) was significantly greater (P < 0.004) than the leak for the upper limb (15 (5) %). Moreover, in each of 10 volunteers, the leak was always greater for the lower than the upper limb. During the first 3 min after tourniquet deflation the loss of radioactivity was 58 (8) % of the maximal amount for the upper limb and 39 (8) % for the lower limb (P < 0.001). As the leak under the tourniquet was significantly greater for the lower than the upper limb, we conclude that IVRA for the lower limb can be associated more frequently with a shorter duration of successful anaesthesia and/or failure.

Peripartum cardiomyopathy: a case report.

Peripartum cardiomyopathy is characterized by congestive left ventricular failure appearing during the last trimester of pregnancy or during the 6 months following delivery, without any specific etiology or pre-existing cardiac disease. The present report describes the case history of a woman presenting with severe cardiac failure immediately after the delivery of twins.

[Pharmacology of cardiopulmonary resuscitation]. / Pharmacologie de la réanimation cardiorespiratoire.

Among the adrenergic receptor agonists, epinephrine, at alpha and beta stimulating doses, remains the drug of choice for cardiopulmonary resuscitation. However, alpha adrenergic agonists such as phenylephrine, methoxamine and dopamine (at alpha stimulating doses) result in similar success rates of resuscitation as epinephrine. In the opposite, beta adrenergic agonists, without or with only low alpha stimulating effect, such as isoproterenol or dobutamine are significantly less efficient. There are few data indicating that sodium bicarbonate improves outcome. Moreover it carries the risk of adverse effects. It may be of benefit in case of preexisting metabolic acidosis or during prolonged resuscitation with documented acidosis. Calcium remains indicated in case of hypocalcaemia, hyperkalaemia or calcium channel blocker intoxication. Severe ionized hypocalcaemia can occur after out-of-hospital cardiac arrest.