Václav Zvoníček

Differential effects of insulin and dexamethasone on pulmonary surfactant-associated genes and proteins in A549 and H441 cells and lung tissue

In this study, the effects of insulin and dexamethasone on the expression and mRNA transcription of 4 pulmonary surfactant-associated proteins [surfactant protein (SFTP)A, SFTPB, SFTPC and SFTPD] were examined. The commercially available cell lines, A549 and H441, were used as acceptable models of lung surfactant-producing cells. Subsequently, the effects of insulin on the expression of surfactant-associated proteins were examined in patients with lung adenocarcinoma during lung resection. Our results demonstrated the inhibitory effects of insulin on the transcription of the SFTPB, SFTPC and SFTPD genes in H441 cells and the SFTPB gene in A549 cells. Treatment with insulin significantly decreased the protein expression of SFTPA1 and SFTPA2 in the H441 cells and that of proSFTPB in the A549 cells. Dexamethasone promoted the transcription of the SFTPB, SFTPC and SFTPD genes in the A549 and H441 cells and reduced the transcription of the SFTPA1 and SFTPA2 genes in the H441 cells (SFTPA mRNA expression was not detected in A549 cells). Furthermore, we demonstrated that the mRNA levels of the selected genes were significantly lower in the cell lines compared to the lung tissue. A549 and H441 cells represent similar cell types. Yet, in our experiments, these cells reacted differently to insulin and/or dexamethasone treatment, and the mRNA levels of their main protein products, surfactant-associated proteins, were significantly lower than those in real tissue. Therefore, the results obtained in this study challenge the suitability of A549 and H441 cells as models of type II pneumocytes and Clara cells, respectively. However, we successfully demonstrate the possibility of studying the effects of insulin on pulmonary surfactant-associated genes and proteins in patients with lung adenocarcinoma.

Temperature corrected thromboelastography in hypothermia: is it necessary?

Context Hypothermia is known to influence thromboelastography (TEG). TEG reproducibility is generally low. Objective The aim of this study was to evaluate the rationale of TEG temperature adjustment in patients during hypothermia. We hypothesised that temperature adjustment would not be important because of low TEG reproducibility. Design Prospective observational study. Setting Single-centre, secondary care study performed 01/2009 to 07/2010. Patients Survivors of cardiopulmonary resuscitation in whom therapeutic hypothermia (32 to 34°C) was indicated for 24 h were recruited to the study which lasted 36 h. Four hundred samples from 30 patients (22 men and eight women) were obtained. No specific exclusion criteria were defined. Main outcome measures Temperature adjusted and non-adjusted Kaolin–Heparinase and Rapid-TEG were done at 12-h intervals during the first 36 h. Results Bland–Altman plots were used for analysis. During hypothermia, the bias of adjusted measurements was greater in clot formation variables for both Kaolin–Heparinase-TEG (from −15 to −19%) and Rapid-TEG (−9 to −25%) compared to normothermia (from −3 to 3% for Kaolin–Heparinase-TEG and −10 to 2% for Rapid-TEG). Bias of clot strength variables was not influenced by temperature adjustment (median −1%). The 95% limits of agreement were wide for clot formation variables and independent of temperature. In Kaolin–Heparinase-TEG (R −42 to 40% normothermia, −47 to 18% hypothermia) and in Rapid-TEG (R −117 to 97% normothermia, −114 to 95% hypothermia). Limits of agreement of clot strength variables were narrower and independent of temperature in Kaolin–Heparinase-TEG (MA −16 to 13% normothermia, −9 to 10% hypothermia) and also in Rapid-TEG (MA −27 to 24% normothermia, −18 to 20% hypothermia). Conclusion Although TEG analysis with temperature adjusted to the in-vivo value during hypothermia yields results with small systematic bias, the importance of temperature adjustment in clinical routine is low because of the precision limits of TEG measurement itself. Therefore, we see no need to perform TEG analysis at the in-vivo temperature.

Simvastatin impairs the induction of pulmonary fibrosis caused by a western style diet: a preliminary study.

The role of an atherogenic diet in causing pulmonary fibrosis has received little attention and simvastatin has been shown to reduce pulmonary fibrosis in animal models. To determine if an atherogenic diet can induce pulmonary fibrosis and whether simvastatin treatment is beneficial by up‐regulating heat shock protein 70 and 90. New Zealand white rabbits (n = 15) were divided: Group 1 (control); Group 2 (MC) received a normal rabbit diet with 1% methionine plus 0.5% cholesterol (atherogenic diet). Group 3 received the same diet as the MC group plus 5 mg/kg/day simvastatin orally (MCS). After 4 weeks, the lungs were collected and analysed. Picrosirus red staining of lung interstitial collagen content showed that the atherogenic diet increased fibrosis 2.9‐fold (P < 0.05), bronchiole adventitial collagen was increased 2.3‐fold (P < 0.05) and bronchiole epithelium was increased 34‐fold (P < 0.05), and simvastatin treatment severely reduced this effect (P < 0.05). Western blot analysis showed that the atherogenic diet significantly reduced lung Hsp70 protein by 22% (P < 0.05) and Hsp90 protein by 18% (P < 0.05) and simvastatin treatment did not affect this result. However, aortic hyper‐responsiveness to vasoconstrictors (angiotensin II and phenylephrine) were markedly reduced by simvastatin treatment. We report that an atherogenic diet stimulates pulmonary fibrosis and reduces lung Hsp70/Hsp90 protein concentration. Simvastatin impairs this by mechanisms unrelated to Hsp70/Hsp90, but possibly a reduction in angiotensin II receptor or alpha adrenergic receptor pathways. These results could have implications in idiopathic pulmonary fibrosis.

Intravenous insulin therapy during lung resection does not affect lung function or surfactant proteins

BackgroundThe surgical resection of lung disrupts glucose homeostasis and causes hyperglycemia, as in any other major surgery or critical illness. We performed a prospective study where we carefully lowered hyperglycemia by insulin administration during the surgery, and for the first time we monitored immediate insulin effects on lung physiology and gene transcription.MethodsThe levels of blood gases (pH, pCO2, pO2, HCO3-, HCO3- std, base excess, FiO2, and pO2/FiO2) were measured at the beginning of surgery, at the end of surgery, and two hours after. Samples of healthy lung tissue surrounding the tumour were obtained during the surgery, anonymized and sent for subsequent blinded qPCR analysis (mRNA levels of surfactant proteins A1, A2, B, C and D were measured). This study was done on a cohort of 64 patients who underwent lung resection. Patients were randomly divided, and half of them received insulin treatment during the surgery.ResultsWe demonstrated for the first time that insulin administered intravenously during lung resection does not affect levels of blood gases. Furthermore, it does not induce immediate changes in the expression of surfactant proteins.ConclusionAccording to our observations, short insulin treatment applied intravenously during resection does not affect the quality of breathing.

Respiratory induced heart rate and blood pressure variability during mechanical ventilation in critically ill and brain death patients

We analysed respiratory induced heart rate and blood pressure variability in mechanically ventilated patients with different levels of sedation and central nervous system activity. Our aim was to determine whether it is possible to distinguish different levels of sedation or human brain activity from heart rate and blood pressure. We measured 19 critically ill and 15 brain death patients ventilated at various respiratory frequencies - 15, 12, 8 and 6 breaths per minute. Basal and deeper sedation was performed in the critically ill patients. We detected and analysed heart rate and blood pressure parameters induced by ventilation. Results: Respiratory induced heart rate variability is the unique parameter that can differentiate between brain death patients and sedated critically ill patients. Significant differences exist, especially during slow deep breathing with a mean period of 10 seconds. The limit values reflecting brain death are: baroreflex lower than 0.5 ms/mmHg and tidal volume normalised heart rate variability lower than 0.5 ms/ml. Reduced heart rate variability parameters of brain death patients remain unchanged even after normalisation to respiration volume. However, differences between basal and deep sedation do not appear significant on any parameter.

The impact of sedation on pulse pressure variation.

OBJECTIVE Pulse pressure variations (PPV) are mainly influenced by ventilation. The impact of sedation on PPV is not known. The aim of the study was to test the influence of sedation on pulse pressure variation in mechanically ventilated critically ill patients and to compare PPV in critically ill and brain dead patients. Beside the absolute value of PPV, the adjusted values of pulse pressure were used to eliminate influence of ventilation. DESIGN AND INTERVENTION Mechanically ventilated patients received four different breath frequencies. At each frequency airway pressure was adjusted to keep the end-tidal CO2 stable. In critically ill patients the frequencies were applied at basal (bispectral index - BIS median 38) and deeper sedation (BIS 29). MAIN OUTCOME MEASURES Simultaneous haemodynamic and respiratory data including oesophageal pressure were recorded, adjusted PPV were calculated as PPV/VT, PPV/dPair, PPV/dPes where VT is tidal volume, dPair and dPes are airway and oesophageal driving pressures. SETTING University Hospital, ICU. PARTICIPANTS 30 critically ill and 23 patients with a diagnosis of brain death. RESULTS The pulse pressure variation did not change significantly during deep sedation compared to basal sedation (median 10.3 vs 10.9%) whereas PPV/dPair increased from 0.7 to 0.8%/cmH2O and PPV/dPes from 1.9%/cmH2O to 2.4%/cmH2O (p=0.04). Patients with a diagnosis of brain death had higher PPV and adjusted PPV than critically ill patients. CONCLUSION Deeper sedation increases values of adjusted pulse pressure variation.