Post-Traumatic Stress Response and Appendicitis in Children-Clinical Usefulness of Selected Biomarkers.

Acute appendicitis is an inflammatory process which is one of the most frequent global causes of surgical interventions in children. The goal of the study was to determine whether acute phase proteins, that is, C-reactive protein (CRP), procalcitonin (PCT) and neutrophil gelatinase-associated lipocalin (NGAL), interleukin 6 (IL-6), transforming growth factor-beta1 (TGF-ß1) and cortisol (HC) play a role in the pathomechanism of post-trauma stress response of the organism and to establish the impact of the applied surgical procedure and/or of inflammation on their concentrations. An additional purpose was to establish the clinical usefulness of the studied biomarkers in the diagnostics of appendicitis. CRP concentrations were quantified via the immunoturbidimetric method, while the levels of IL-6 and PCT were assessed using a bead-based multiplexed immunoassay system in a microplate format (Luminex xMAP technology); NGAL, TGF-ß1 and cortisol concentrations were determined via the enzyme-linked immunosorbent assay (ELISA) technique. All the investigated biomarkers were assayed twice, i.e., immediately before the surgery and 12-24 h after its completion. Significant increases in CRP, IL-6 and PCT concentrations were found in all children subjected to laparoscopic surgeries (p = 0.001, p = 0.006, and p = 0.009, respectively) and open (classic) surgeries (p = 0.001, p = 0.016, and p = 0.044, respectively) compared to the initial concentrations. The patients undergoing classical surgery moreover presented with significant (p = 0.002, and p = 0.022, respectively) increases in NGAL and TGF-ß1 levels after the procedures. In a group of children undergoing laparoscopic surgery, the appendicitis induced an increase in cortisol concentration, whereas in patients undergoing classical surgery the increase in the levels of this biomarker was caused by the type of performed surgical procedure. Simultaneously assaying the levels of CRP, NGAL and IL-6 (p = 0.008, p = 0.022, and p = 0.000, respectively) may prove useful in clinical practice, enabling the diagnosis of appendicitis in paediatric patients reporting to a hospital with abdominal pains, in addition to data from anamnesis and from clinical or ultrasound examination. The performed study confirms the participation of examined biomarkers in the pathomechanism of post-injury stress reaction of the organism to surgical trauma.

High-light-inducible proteins HliA and HliB: pigment binding and protein-protein interactions.

High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and ß-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a 'generalist' that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of 'specialists' (HliA, B or D) dictate interactions with proteins other than Hlips.

Respiratory Burst and TNF-α Receptor Expression of Neutrophils after Sepsis and Severe Injury-Induced Inflammation in Children.

Systemic inflammatory response syndrome (SIRS) is defined as the systemic host response to infection or a non-infectious factor. The purpose of this study was to evaluate the involvement of reactive oxygen species (ROS) in severe inflammation and to assess the discrimination strength of the neutrophil BURSTTEST assay regarding its etiology in three groups of patients (sepsis, burns, and bone fractures) who met the SIRS criteria. The neutrophil activation (respiratory burst of granulocytes as well as p55 and p75 tumor necrosis factor (TNF-α) receptor expression) was evaluated twice using flow cytometry, and the results were compared with healthy controls and among SIRS subjects. A decreased oxygen metabolism in neutrophils after E.coli stimulation and increased TNF-α receptor expression were found in septic and burned patients on admission, while ROS production augmented and TNF-α receptor expression diminished with the applied therapy. The significant differences in neutrophil respiratory burst intensity among septic and burned patients and those with sepsis and bone fractures were found (however, there were not any such differences between patients with thermal and mechanical injuries). This study indicates that the neutrophil BURSTTEST evaluation might be a clinically reliable marker for differentiating the SIRS etiology.

Paraoxonase 1 Activity, Polymorphism and Atherosclerosis Risk Factors in Patients Undergoing Coronary Artery Surgery.

BACKGROUND: Paraoxonase1 (PON1), an enzyme connected to high density lipoproteins (HDL) particles, plays an important role in protecting arteries against atherosclerosis. The serum activity and concentration of PON1 depends on several genetic polymorphisms as well as environmental factors. MATERIALS AND METHODS: Investigated population consisted of 71 patients aged 43⁻76 years with confirmed coronary heart disease (CHD). Established risk factors of CHD such as hypertension, elevated total cholesterol and LDL cholesterol (LDL-C), low HDL cholesterol (HDL-C), diabetes mellitus, obesity, smoking and premature CHD in family history were assessed. PON1 genotype for ⁻108C/T promotor region was determined by polymerase chain reaction-restriction fragments length polymorphism (PCR⁻RFLP) method. Paraoxonase activity towards paraoxon and arylesterase activity towards phenyl acetate were measured spectrophotometrically. RESULTS: Significant correlations between diabetes mellitus and paraoxonase activity (R = ⁻0.264, p = 0.026) and between the premature coronary heart disease in family history and PON1 activity (R = ⁻0.293, p = 0.013) were found. In multivariate analysis, PON1 paraoxonase activity was independently of confounding factors associated with diabetes (OR = 0.985; p = 0.024) and premature CHD in family history (OR = 0.983; p = 0.027). PON1 activity towards aryl acetate positively correlated with HDL-C level (R = 0.255, p = 0.032). In patients treated with statins, PON1 paraoxonase activity was significantly (p = 0.033) higher than in patients without treatment. CONCLUSIONS: In diabetic patients with CHD, paraoxonase activity is lower than in normoglycemic patients despite similar lipid profiles. Diabetes and positive family history in patients with overt CHD are associated with the serum PON1 activity, which might be an additional factor helpful in evaluating cardiovascular risk in this group of patients.

Dynamic changes of paraoxonase 1 activity towards paroxon and phenyl acetate during coronary artery surgery.

BACKGROUND: Serum paraoxonase 1 (PON1), an enzyme associated with high - density lipoproteins (HDL) particles, inhibits the oxidation of serum lipoproteins and cell membranes. PON1 activity is lower in patients with atherosclerosis and in inflammatory diseases. The systemic inflammatory response provoked during cardiopulmonary bypass grafting may contribute to the development of postoperative complications. The aim of the present study was to estimate the dynamic changes in paraoxonase 1 (PON1) activity towards paraoxon and phenyl acetate during and after coronary artery surgery. METHODS: Twenty six patients with coronary heart disease undergoing coronary artery bypass grafting (CABG) were enrolled into the study. Venous blood samples were obtained preoperatively, after aortic clumping, after the end of operation, at 6, 18, 30 and 48 h after operation. Paraoxonase activity was measured spectrophotometrically in 50 mM glycine/NaOH buffer (pH 10.5) containing 1.0 mM paraoxon, and 1.0 mM CaCl2. Arylesterase activity was measured in 20 mM TrisCl buffer (pH 8.0) containing 1 mM phenyl acetate and 1 mM CaCl2. RESULTS: PON1 activity toward paraoxon and phenyl acetate significantly decreased after aorta cross clumping and increased directly after operation. PON1 activity towards paraoxon in preoperative period and PON1 activity towards phenyl acetate in seventh stage of experiment tended to inversely correlate with the occurrence of postoperative complications. CONCLUSION: The paraoxonase 1 plasma activity is markedly reduced during CABG surgery.

Evaluation of potential prognostic value of Bmi-1 gene product and selected markers of proliferation (Ki-67) and apoptosis (p53) in the neuroblastoma group of tumors.

INTRODUCTION: Cancer in children is a very important issue in pediatrics. The least satisfactory treatment outcome occurs among patients with clinically advanced neuroblastomas. Despite much research, the biology of this tumor still remains unclear, and new prognostic factors are sought. The Bmi-1 gene product is a currently highly investigated protein which belongs to the Polycomb group (PcG) and has been identified as a regulator of primary neural crest cells. It is believed that Bmi­1 and N-myc act together and are both involved in the pathogenesis of neuroblastoma. The aim of the study was to assess the potential prognostic value of Bmi-1 protein and its relations with mechanisms of proliferation and apoptosis in the neuroblastoma group of tumors. MATERIAL/METHODS: 29 formalin-fixed and paraffin-embedded neuroblastoma tissue sections were examined using mouse monoclonal antibodies anti-Bmi-1, anti-p53 and anti-Ki-67 according to the manufacturer's instructions. RESULTS: There were found statistically significant correlations between Bmi-1 expression and tumor histology and age of patients. CONCLUSIONS: Bmi-1 seems to be a promising marker in the neuroblastoma group of tumors whose expression correlates with widely accepted prognostic parameters. The pattern of BMI-1 expression may indicate that the examined protein is also involved in maturation processes in tumor tissue.

Prognostic value of paraoxonase 1 in patients undergoing coronary artery bypass grafting surgery.

BACKGROUND: The aim of this study was to evaluate whether -108C/T polymorphism of the paraoxonase 1 (PON1) gene and the plasma enzyme activity are risk factors for adverse cardiac events after coronary artery bypass grafting (CABG). MATERIAL AND METHODS: Seventy-one patients with coronary heart disease (CHD) undergoing CABG were enrolled in the study. Genomic DNA was extracted from the venous blood using the Gen Elute™ Blood Genomic DNA kit (Sigma) according to the manufacturer's instructions. PON1 activity was measured in 50 mM glycine/NaOH buffer (pH 10.5) containing 1.0 mM paraoxon, and 1.0mM CaCl2. RESULTS: The mean PON1 activity toward paraoxon and toward phenyl acetate was equal (166.5 ± 86.9 U/ml and 96.0 ± 47.2 U/ml, respectively) in patients with CHD. The -108C/T polymorphism of PON1 gene was tested. In CABG patients, PON1 activities in dependence on genotypes were significantly different and equalled 266.2 ± 117.9 U/ml for CC, 178.8 ± 64.7 U/ml for CT, and 98.9 ± 59.2 U/ml for TT genotype. Patients with PON1 activity lower than 193.5 U/ml exhibited significantly increased risk of a serious cardiac event in comparison with patients with PON1 activity higher or equal to this value (p=0.03). Additionally, TT genotype was significantly associated with shorter time of event-free survival in comparison with CT and CC genotypes (p=0.009). CONCLUSIONS: The PON1 polymorphism and enzyme plasma activity are associated with CHD occurrence. High PON1 activity connected with the presence of CC and CT genotypes decreases the recurrence of symptoms of coronary heart disease and improve prognosis after CABG.

Hippocampal volumes in patients exposed to low-dose radiation to the basal brain. A case-control study in long-term survivors from cancer in the head and neck region.

BACKGROUND: An earlier study from our group of long time survivors of head and neck cancer who had received a low radiation dose to the hypothalamic-pituitary region, with no signs of recurrence or pituitary dysfunction, had their quality of life (QoL) compromised as compared with matched healthy controls. Hippocampal changes have been shown to accompany several psychiatric conditions and the aim of the present study was to test whether the patients' lowered QoL was coupled to a reduction in hippocampal volume. METHODS: Patients (11 men and 4 women, age 31-65) treated for head and neck cancer 4-10 years earlier and with no sign of recurrence or pituitary dysfunction, and 15 matched controls were included. The estimated radiation doses to the basal brain including the hippocampus (1.5 - 9.3 Gy) had been calculated in the earlier study. The hippocampal volumetry was done on coronal sections from a 1.5 T MRI scanner. Measurements were done by two independent raters, blinded to patients and controls, using a custom method for computer assisted manual segmentation. The volumes were normalized for intracranial volume which was also measured manually. The paired t test and Wilcoxon's signed rank test were used for the main statistical analysis. RESULTS: There was no significant difference with respect to left, right or total hippocampal volume between patients and controls. All mean differences were close to zero, and the two-tailed 95% confidence interval for the difference in total, normalized volume does not include a larger than 8% deficit in the patients. CONCLUSION: The study gives solid evidence against the hypothesis that the patients' lowered quality of life was due to a major reduction of hippocampal volume.

[Immunohistochemical assessment of molecule Ki67 in nasal polyps tissue]. / Immunohistochemiczna ocena molekuly Ki67 w tkance polipów nosa.

Immunohistochemical assessment of proliferation marker--Ki67 in nasal polyps tissue and in healthy nasal mucosa was performed. There was no statistical significance difference between percentage of Ki67 positive cells in nasal polyps and nasal mucosa. These cells were observed mainly in epithelial layer and few in subepithelial layer. In examined group Ki67 expression was higher in planoepithelial metaplasia. Assessment of cells proliferation processes could be sensitive and early indicator of neoplasmatic transformation. Examined subgroup in witch Ki67 expression was also higher, was subgroup with eosinophilic infiltrations. Cells proliferation processes in nasal polyps tissue are very important occurrence and require further examinations.

Apoptosis of lymphocytes and sialic acid plasma level in patients undergoing cardiac surgery.

Increasing evidence shows that extensive tissue trauma and surgical stress are related to physical alterations of cells and cell death. It was previously reported that total sialic acid (SA) plasma concentration is elevated in patients undergoing coronary artery surgery. Shedding or secreting of SA from the cell membrane surface or releasing intracellular SA may induce apoptosis. It is possible that the terminal SA residues of carbohydrate moieties facilitate recognition and removal of apoptotic cells by phagocytes. The aim of the present study was to estimate the dynamic changes in rate ofapoptosis oflymphocytes and total sialic acid plasma level during coronary artery surgery. In 17 patients undergoing coronary artery bypass grafting surgery plasma total SA concentration was measured and the percentage of apoptotic lymphocytes was determined before operation, after aorta clumping, after the end of operation and at 6, 18, 30 and 48 h after operation. Plasma total SA concentration decreases after aortic clumping and then increases gradually during a 48 hr observation period. The percentage of apoptotic cells increases during and after surgery with the exception of a sample taken at 18 hours after operation. The findings indicate the bimodal character of apoptosis and dynamic increase in total SA plasma level, which may be considered a result of mechanical damage taken place during operation or inflammatory response to surgical trauma.