Control of hyperglycaemia in paediatric intensive care (CHiP): study protocol

Background

There is increasing evidence that tight blood glucose (BG) control improves outcomes in critically ill adults. Children show similar hyperglycaemic responses to surgery or critical illness. However it is not known whether tight control will benefit children given maturational differences and different disease spectrum.

Methods/Design

The study is an randomised open trial with two parallel groups to assess whether, for children undergoing intensive care in the UK aged ≤ 16 years who are ventilated, have an arterial line in-situ and are receiving vasoactive support following injury, major surgery or in association with critical illness in whom it is anticipated such treatment will be required to continue for at least 12 hours, tight control will increase the numbers of days alive and free of mechanical ventilation at 30 days, and lead to improvement in a range of complications associated with intensive care treatment and be cost effective. Children in the tight control group will receive insulin by intravenous infusion titrated to maintain BG between 4 and 7.0 mmol/l. Children in the control group will be treated according to a standard current approach to BG management. Children will be followed up to determine vital status and healthcare resources usage between discharge and 12 months post-randomisation. Information regarding overall health status, global neurological outcome, attention and behavioural status will be sought from a subgroup with traumatic brain injury (TBI). A difference of 2 days in the number of ventilator-free days within the first 30 days post-randomisation is considered clinically important. Conservatively assuming a standard deviation of a week across both trial arms, a type I error of 1% (2-sided test), and allowing for non-compliance, a total sample size of 1000 patients would have 90% power to detect this difference. To detect effect differences between cardiac and non-cardiac patients, a target sample size of 1500 is required. An economic evaluation will assess whether the costs of achieving tight BG control are justified by subsequent reductions in hospitalisation costs.

Discussion

The relevance of tight glycaemic control in this population needs to be assessed formally before being accepted into standard practice.

Trial Registration

Current Controlled Trials ISRCTN61735247     

2,3,7,8-TCDD exposure, endothelial dysfunction and impaired microvascular reactivity

Vascular function was examined in subjects with long-term high level of serum 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) during their follow-up visits. Their earlier mean peak TCDD level at the time of exposure in 1965-1968 was estimated in the range of 3300-74 000 pg/g lipids. Ten former pesticide production workers heavily exposed to TCDD (age 57 +/- 2 years, TCDD about 170 pg/g lipids) were examined in 2001. Extended group of 15 TCDD-exposed men (age 59 +/- 3 years, TCDD about 130 pg/g lipids) underwent the same examination in 2004. Findings were compared with a control group of 14 healthy men (age 54 +/- 2 years). Skin microvascular reactivity (MVR) was measured by laser Doppler perfusion monitoring in the forearm during post-occlusive reactive hyperemia (PORH) and thermal hyperemia (TH). Several parameters of MVR in men exposed to TCDD were significantly impaired, compared with the control group and further progression of the impairment of MVR has been observed between years 2001 and 2004. Serum concentration of E-selectin and inhibitor of tissue plasminogen activator 1 (PAI-1) was significantly higher in exposed subjects (56.0 +/- 18.4 ng/mL versus 40.0 +/- 12.0 ng/mL, P = 0.022 and 90.9 +/- 33.3 ng/mL versus 45.0 +/- 18.0, P = 0.002, respectively). In addition, PORH in the forearm was significantly negatively associated with SOD activity (r = -0.77, P = 0.009) as well as the velocity of perfusion increase during TH (r = -0.68, P = 0.03) and TH% (r = -0.78, P = 0.008). Our data document the presence of endothelial dysfunction in TCDD-exposed men.     

Helicobacter pylori infection and other bacteria in pancreatic cancer and autoimmune pancreatitis

Helicobacter pylori (H. pylori) is an infectious agent influencing as much as 50% of the world’s population. It is the causative agent for several diseases, most especially gastric and duodenal peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma of the stomach. A number of other, extragastric manifestations also are associated with H. pylori infection. These include neurological disorders, such as Alzheimer’s disease, demyelinating multiple sclerosis and Parkinson’s disease. There is also evidence for a relationship between H. pylori infection and such dermatological diseases as psoriasis and rosacea as well as a connection with infection and open-angle glaucoma. Generally little is known about the relationship between H. pylori infection and diseases of the pancreas. Most evidence about H. pylori and its potential role in the development of pancreatic diseases concerns pancreatic adenocarcinoma and autoimmune forms of chronic pancreatitis. There is data (albeit not fully consistent) indicating modestly increased pancreatic cancer risk in H. pylori-positive patients. The pathogenetic mechanism of this increase is not yet fully elucidated, but several theories have been proposed. Reduction of antral D-cells in H. pylori-positive patients causes a suppression of somatostatin secretion that, in turn, stimulates increased secretin secretion. That stimulates pancreatic growth and thus increases the risk of carcinogenesis. Alternatively, H. pylori, as a part of microbiome dysbiosis and the so-called oncobiome, is proven to be associated with pancreatic adenocarcinoma development via the promotion of cellular proliferation. The role of H. pylori in the inflammation characteristic of autoimmune pancreatitis seems to be explained by a mechanism of molecular mimicry among several proteins (mostly enzymes) of H. pylori and pancreatic tissue. Patients with autoimmune pancreatitis often show positivity for antibodies against H. pylori proteins. H. pylori, as a part of microbiome dysbiosis, also is viewed as a potential trigger of autoimmune inflammation of the pancreas. It is precisely these relationships (and associated equivocal conclusions) that constitute a center of attention among pancreatologists, immunologists and pathologists. In order to obtain clear and valid results, more studies on sufficiently large cohorts of patients are needed. The topic is itself sufficiently significant to draw the interest of clinicians and inspire further systematic research. Next-generation sequencing could play an important role in investigating the microbiome as a potential diagnostic and prognostic biomarker for pancreatic cancer.     

Autologous hematopoietic stem cell transplantation in adult acute lymphoblastic leukemia: still not out of fashion

The role of autologous hematopoietic stem cell transplantation (autoHSCT) in adult acute lymphoblastic leukemia (ALL) is still unclear. We retrospectively analyzed the results of the autoHSCT and maintenance therapy, with oral 6-mercaptopurine and methotrexate, in comparison to conventional-dose chemotherapy in the consolidation treatment of adult ALL and lymphoblastic lymphoma (LBL). The patients, with HLA identical sibling donor, underwent allogeneic transplantation, while the others were treated with autoHSCT and maintenance therapy with oral 6-mercaptopurine and methotrexate, or by conventional-dose chemotherapy (patient’s decision, no autologous hematopoietic stem cells harvest). Sixty consecutive adult patients (median age 35.2 years; range 17.3 to 70.7) with ALL (n = 52), LBL (n = 7), and acute biphenotypic leukemia (n = 1) were treated in our center from 1997 to 2007. Patients treated with chemotherapy alone (n = 35) had a shorter median progression-free survival (PFS) compared to patients who underwent autoHSCT plus maintenance therapy (n = 18), 8.4 and 46.8 months, respectively (p = 0.017). Patients treated with chemotherapy alone had also a shorter median overall survival (OS) compared to patients treated with autoHSCT: 13.0 vs. 46.8 months (p = 0.046). The differences remained statistically significant even after excluding patients with Ph positivity. We can conclude that, in our case, autoHSCT followed by maintenance chemotherapy is a good option for adult patients with ALL and, in standard-risk and high-risk patients, provides more favorable OS and PFS rates compared to patients treated by chemotherapy alone. However, we are aware of the fact that our analysis may have been distorted by the fact that the analysis is retrospective, that treatment with autoHSCT was based on patient’s decision, and that chemotherapy may have been administered to negatively selected patients.     

Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.Invasive infections caused by mucormycetes started to occur more frequently in the last decade and are connected with rapid progression and high mortality rates. Early diagnostics and targeted treatment are crucial. Most mucormycosis cases (over 90%) are caused by Rhizopus spp., followed by Mucor spp., Lichtheimia spp., Rhizomucor pusillus, and, rarely, some other species (2, 9, 11, 16).Definitive diagnosis of mucormycosis is usually made after histopathological proof of mucormycete-like hyphae in involved tissue; the causative agent can be determined only by culture (13). So far, no serological test is available and radiological methods are nonspecific.Molecular detection of mucormycetes is complicated by several factors, and we still do not have any standard protocol. Few methods for the detection of mucormycetes have been published, and only some have been evaluated using clinical samples (1, 5, 10, 14, 15, 17) or samples from animal models (6, 7).The aim of this study was to develop a rapid and sensitive technique for the detection and identification of clinically important mucormycetes. We adopted primers from a qualitative method previously published by Bialek et al. (1) that is specific for members of the order Mucorales targeting 18S ribosomal DNA (rDNA). We modified it to seminested real-time PCR with EvaGreen dye, followed by species distinction by high-resolution melt (HRM) analysis. HRM analysis uses amplification of DNA in the presence of intercalation dye. Fluorescence is measured during a controlled melting of PCR product that results in a melt curve that depends mainly on GC content, length, and sequence of the PCR product. This simple method can be used for genotyping or mutation scanning without the need for time-consuming sequencing (4, 12).DNA was isolated from 50 μl of fungal culture (inoculum was prepared by covering sporulating colonies with approximately 2 ml of sterile 0.85% saline) or a piece of fresh tissue (2 by 1 mm) using the ZR fungal/bacterial DNA kit (Zymo Research). Tissue samples were incubated in lysis buffer overnight, and cultures were immediately processed according to the manufacturer''s protocol. Disruption was extended to 15 min (Disruptor Genie; Scientific Industries). DNA from formalin-fixed, paraffin wax-embedded (FFPE) tissue samples was isolated from 2 or 3 scrolls (5 to 10 μm each) of paraffin block using a DNeasy blood and tissue kit (Qiagen). Paraffin was dissolved in 1 ml of xylene, and then the tissue was washed two times using 1 ml of 96% ethanol and incubated in 180 μl of ATL buffer (Qiagen) and 20 μl of proteinase K (600 mAU/ml solution, where one mAU represents the activity of proteinase K that releases folin-positive amino acids and peptides corresponding to 1 μmol of tyrosine per min) at 55°C overnight and then at 90°C for 1 h. The next steps were done in accordance with the manufacturer''s protocol. DNA isolation from clinical samples was done in a biological safety cabinet. An aliquot of sterile water was processed with each set of samples as a control of potential contamination during the isolation process.Five microliters of DNA was amplified in 25 μl of amplification mixture that contained a 0.2 μM concentration each of primers ZM1 and ZM2 (1), 120 μM deoxynucleoside triphosphates (dNTPs; Roche, Germany), 2.5 mM MgCl₂, 1× GeneAmp PCR Gold buffer, and 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The cycling conditions were 10 min at 95°C, 16 cycles of 30 s at 94°C, 30 s at 50°C, and 60 s at 72°C, and 7 min at 72°C. One microliter of PCR product from the external round was then amplified in duplicate using Rotorgene 6000 (Corbett Research, Australia). Twenty-five microliters of the amplification mixture contained a 0.4 μM concentration each of primers ZM1 and ZM3 (1), 12.5 μl of SensiMix HRM, and 1 μl of EvaGreen (both from a SensiMix HRM kit; Quantace, United Kingdom). The cycling conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 20 s at 60°C, and 30 s at 72°C (acquired on the green channel), followed by HRM analysis (ramp from 74°C to 79.5°C, rising by 0.1°C each cycle, acquired on the HRM channel). Rotorgene 6000 series software (version 1.7) was used for analysis of the results. All positive results were confirmed by sequencing of the PCR product. DNA was purified using a QIAquick PCR purification kit (Qiagen, Germany) and sequenced using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) on an ABI Prism 310 genetic analyzer (Applied Biosystems). Sequences were analyzed using the BLAST alignment program of the GenBank database.We used DNA extracted from five mucormycete cultures diluted in Tris-EDTA (TE) buffer as positive controls in every run. A DNA isolation control (sterile water processed with clinical samples) and a negative control of PCR (sterile water) were added to each run as well.In this study, we tested 31 fungal isolates, comprising 10 mucormycete isolates and 21 isolates from other filamentous fungal groups (Department of Clinical Microbiology, University Hospital Brno and Czech Collection of Microorganisms, Czech Republic). All mucormycete isolates were correctly identified. The melting temperatures (T_m) for each species were as follows: for Rhizopus microsporus, 76.46°C; for Rhizopus oryzae, 76.59°C; for Mucor racemosus, 76.78°C; for Mucor circinelloides, 76.98°C; for Rhizomucor pusillus, 77.87°C; and for Lichtheimia corymbifera, 78.56°C. Representative HRM curves for six different mucormycetes are shown in Fig. ​Fig.1.1. All HRM analysis results were confirmed by sequencing. None of the nonmucormycete fungi were positively tested. The results are summarized in Table ​Table11.Open in a separate windowFIG. 1.Representative result of high-resolution melt (HRM) analysis. Shown are HRM curves for six mucormycete isolates (black curves) and one negative and one positive tissue sample (gray curves).

TABLE 1.

List of fungal isolates used in this study and results of HRM analysis^a

Expression and transport activity of breast cancer resistance protein (Bcrp/Abcg2) in dually perfused rat placenta and HRP-1 cell line

Breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette transporter family that recognizes a variety of chemically unrelated compounds. Its expression has been revealed in many mammal tissues, including placenta. The purpose of this study was to describe its role in transplacental pharmacokinetics using rat placental HRP-1 cell line and dually perfused rat placenta. In HRP-1 cells, expression of Bcrp, but not P-glycoprotein, was revealed at mRNA and protein levels. Cell accumulation studies confirmed Bcrp-dependent uptake of BODIPY FL prazosin. In the placental perfusion studies, a pharmacokinetic model was applied to distinguish between passive and Bcrp-mediated transplacental passage of cimetidine as a model substrate. Bcrp was shown to act in a concentration-dependent manner and to hinder maternal-to-fetal transport of the drug. Fetal-to-maternal clearance of cimetidine was found to be 25 times higher than that in the opposite direction; this asymmetry was partly eliminated by BCRP inhibitors fumitremorgin C (2 microM) or N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918; 2 microM) and abolished at high cimetidine concentrations (1000 microM). When fetal perfusate was recirculated, Bcrp was found to actively remove cimetidine from the fetal compartment to the maternal compartment even against a concentration gradient and to establish a 2-fold maternal-to-fetal concentration ratio. Based on our results, we propose a two-level defensive role of Bcrp in the rat placenta in which the transporter 1) reduces passage of its substrates from mother to fetus but also 2) removes the drug already present in the fetal circulation.