Postoperative serum pro-calcitonin and C-reactive protein levels in patients with orthopedic infections

QUESTIONS UNDER STUDY / PRINCIPLES: The value of postoperative pro-calcitonin (PCT) in the follow-up of patients with localised infections in the orthopaedic domain is unknown.

Protein disulfide isomerase blocks CEBPA translation and is up-regulated during the unfolded protein response in AML

Deregulation of the myeloid key transcription factor CEBPA is a common event in acute myeloid leukemia (AML). We previously reported that the chaperone calreticulin is activated in subgroups of AML patients and that calreticulin binds to the stem loop region of the CEBPA mRNA, thereby blocking CEBPA translation. In this study, we screened for additional CEBPA mRNA binding proteins and we identified protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) resident protein, to bind to the CEBPA mRNA stem loop region. We found that forced PDI expression in myeloid leukemic cells in fact blocked CEBPA translation, but not transcription, whereas abolishing PDI function restored CEBPA protein. In addition, PDI protein displayed direct physical interaction with calreticulin. Induction of ER stress in leukemic HL60 and U937 cells activated PDI expression, thereby decreasing CEBPA protein levels. Finally, leukemic cells from 25.4% of all AML patients displayed activation of the unfolded protein response as a marker for ER stress, and these patients also expressed significantly higher PDI levels. Our results indicate a novel role of PDI as a member of the ER stress-associated complex mediating blocked CEBPA translation and thereby suppressing myeloid differentiation in AML patients with activated unfolded protein response (UPR).

Activation of the unfolded protein response in human acute myeloid leukemia

There is accumulating evidence for the involvement of the unfolded protein response (UPR) in the pathogenesis of many tumor types in humans. This is particularly the case in rapidly growing solid tumors in which the demand for oxygen and nutrients can exceed the supply until new tumor-initiated blood vessels are formed. In contrast, the role of the UPR during leukemogenesis remains largely unknown. Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by the accumulation of somatic mutations in hematopoietic progenitor cells that alter the physiological regulation of self-renewal, survival, proliferation, or differentiation. The CCAAT/enhancer-binding protein alpha (CEBPA) gene is a key myeloid transcription factor and a frequent target for disruption in AML. In particular, translation of CEBPA mRNA can be specifically blocked by binding of the chaperone calreticulin (CALR), a well-established effector of the UPR, to a stem loop structure within the 5' region of the CEBPA mRNA. The relevance of this mechanism was first elucidated in certain AML subtypes carrying the gene rearrangements t(3;21) or inv(16). In our recent work, we could demonstrate the induction of key effectors of the UPR in leukemic cells of AML patients comprising all subtypes (according to the French-American-British (FAB) classification for human AML). The formation of the spliced variant of the X-box binding protein (XBP1s) was detectable in 17.4% (17 of 105) of AML patients. Consistent with an activated UPR, this group had significantly increased expression of the UPR target genes CALR, the 78 kDa glucose-regulated protein (GRP78), and the CCAAT/enhancer-binding protein homologous protein (CHOP). Consistently, in vitro studies confirmed that calreticulin expression was upregulated via activation of the ATF6 pathway in myeloid leukemic cells. As a consequence, CEBPA protein expression was inhibited in vitro as well as in leukemic cells from patients with activated UPR. We therefore propose a model of the UPR being involved in leukemogenesis through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation and cell-cycle deregulation which represent key features of the leukemic phenotype. From a more clinical point of view, the presence of activated UPR in AML patient samples was found to be associated with a favorable disease course.

Elevated systemic monocyte chemoattractrant protein-1 in hepatic steatosis without significant hepatic inflammation

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and the metabolic syndrome. It encompasses a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine monocyte chemoattractant protein 1 (MCP-1) plays an important role in the progression of hepatic inflammation and fibrosis, and both increased hepatic expression and circulating serum levels have been described in NASH. Here, we aimed to investigate MCP-1 expression in simple hepatic steatosis. Upon feeding a high-fat diet mice developed hepatic steatosis in the absence of significant hepatic inflammation, but elevated hepatic MCP-1 expression compared to control mice fed a standard chow. Interestingly, high-fat diet fed mice had significantly higher MCP-1 serum levels, and MCP-1 mRNA expression was significantly increased in visceral adipose tissue. Furthermore, MCP-1 serum levels were also elevated in patients with ultrasound-diagnosed NAFLD and correlated with the body-mass index and fasting glucose. In conclusion, our data indicate both the liver and adipose tissue as cellular sources of elevated circulating MCP-1 levels already in the early phase of hepatic steatosis. Since MCP-1 derived from visceral adipose tissue reaches the liver via portal circulation at high concentrations it may significantly contribute to the progression of simple steatosis to NASH.

A tool for the qualitative comparison of membrane-embedded and detergent-solubilized membrane protein structures in projection

The calculation of projection structures (PSs) from Protein Data Bank (PDB)-coordinate files of membrane proteins is not well-established. Reports on such attempts exist but are rare. In addition, the different procedures are barely described and thus difficult if not impossible to reproduce. Here we present a simple, fast and well-documented method for the calculation and visualization of PSs from PDB-coordinate files of membrane proteins: the projection structure visualization (PSV)-method. The PSV-method was successfully validated using the PS of aquaporin-1 (AQP1) from 2D crystals and cryo-transmission electron microscopy, and the PDB-coordinate file of AQP1 determined from 3D crystals and X-ray crystallography. Besides AQP1, which is a relatively rigid protein, we also studied a flexible membrane transport protein, i.e. the L-arginine/agmatine antiporter AdiC. Comparison of PSs calculated from the existing PDB-coordinate files of substrate-free and L-arginine-bound AdiC indicated that conformational changes are detected in projection. Importantly, structural differences were found between the PSV-method calculated PSs of the detergent-solubilized AdiC proteins and the PS from cryo-TEM of membrane-embedded AdiC. These differences are particularly exciting since they may reflect a different conformation of AdiC induced by the lateral pressure in the lipid bilayer.

Micropreparative isoelectric focusing protein separation in a suspended drop

IEF protein binary separations were performed in a 12-μL drop suspended between two palladium electrodes, using pH gradients created by electrolysis of simple buffers at low voltages (1.5-5 V). The dynamics of pH gradient formation and protein separation were investigated by computer simulation and experimentally via digital video microscope imaging in the presence and absence of pH indicator solution. Albumin, ferritin, myoglobin, and cytochrome c were used as model proteins. A drop containing 2.4 μg of each protein was applied, electrophoresed, and allowed to evaporate until it splits to produce two fractions that were recovered by rinsing the electrodes with a few microliters of buffer. Analysis by gel electrophoresis revealed that anode and cathode fractions were depleted from high pI and low pI proteins, respectively, whereas proteins with intermediate pI values were recovered in both fractions. Comparable data were obtained with diluted bovine serum that was fortified with myoglobin and cytochrome c.

Distinct proteinase K-resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak

Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrP(res)) in a highly scrapie-affected goat flock in Greece. The PrP(res) profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrP(res) fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrP(res) phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.

Protein synthesis in jejunum and liver of neonatal calves fed vitamin A and lactoferrin

Rates of protein synthesis (PS) and turnover are more rapid during the neonatal period than during any other stage of postnatal life. Vitamin A and lactoferrin (Lf) can stimulate PS in neonates. However, newborn calves are vitamin A deficient and have a low Lf status, but plasma vitamin A and Lf levels increase rapidly after ingestion of colostrum. Neonatal calves (n = 6 per group) were fed colostrum or a milk-based formula without or with vitamin A, Lf, or vitamin A plus Lf to study PS in the jejunum and liver. l-[(13)C]Valine was intravenously administered to determine isotopic enrichment of free (nonprotein-bound) Val (AP(Free)) in the protein precursor pool, atom percentage excess (APE) of protein-bound Val, fractional protein synthesis rate (FSR) in the jejunum and liver, and isotopic enrichment of Val in plasma (APE(Pla)) and in the CO(2) of exhaled air (APE(Ex)). The APE, AP(Free), and FSR in the jejunum and liver did not differ significantly among groups. The APE(Ex) increased, whereas APE(Pla) decreased over time, but there were no group differences. Correlations were calculated between FSR(Jej) and histomorphometrical and histochemical data of the jejunum, and between FSR(Liv) and blood metabolites. There were negative correlations between FSR(Liv) and plasma albumin concentrations and between FSR(Jej) and the ratio of villus height:crypt depth, and there was a positive correlation between FSR(Jej) and small intestinal cell proliferation in crypts. Hence, there were no effects of vitamin A and Lf and no interactions between vitamin A and Lf on intestinal and hepatic PS. However, FSR(Jej) was correlated with histomorphometrical traits of the jejunum and FSR(Liv) was correlated with plasma albumin concentrations.

Differential gene and protein expression in abluminal sprouting and intraluminal splitting forms of angiogenesis

In adult skeletal muscle, abluminal sprouting or longitudinal splitting of capillaries can be initiated separately by muscle overload and elevated microcirculation shear stress respectively. In the present study, gene and protein expression patterns associated with the different forms of angiogenesis were examined using a targeted gene array (Superarray), validated by quantitative RT (reverse transcription)-PCR and immunoblots. Sprouting angiogenesis induced large changes in expression levels in genes associated with extracellular matrix remodelling, such as MMP-2 (matrix metalloproteinase-2), TIMP (tissue inhibitor of metalloproteinases), SPARC (secreted protein, acidic and rich in cysteine) and thrombospondin. Changes in neuropilin, midkine and restin levels, which may underpin changes in endothelial morphology, were seen during splitting angiogenesis. Up-regulation of VEGF (vascular endothelial growth factor), Flk-1, angiopoietin-2 and PECAM-1 (platelet/endothelial cell adhesion molecule-1) was seen in both forms of angiogenesis, representing a common angiogenic response of endothelial cells. In conclusion, the present study demonstrates that general angiogenic signals from growth factors can be influenced by the local microenvironment resulting in differing forms of capillary growth to produce a co-ordinated expansion of the vascular bed.

Serum leptin and C-reactive protein levels in the physiological spontaneous menstrual cycle in reproductive age women

OBJECTIVE: Only a few studies have investigated variations of different markers for inflammatory processes during the physiological menstrual cycle. The results are conflicting, particularly concerning the correlation between the marker leptin and steroid hormones. The aim of the study was to investigate the inflammatory markers C-reactive protein (CRP) and leptin in the serum of healthy, normally ovulating women and to correlate these with each other and with the hormones of the gonadal axis. A cycle-dependence of the markers studied would imply an exact timing of the blood sampling for clinical needs. DESIGN: Observational study investigating the two inflammatory markers CRP and leptin in relation to the hormonal pattern of the gonadal axis during the normal cycle. METHODS: Ovulatory cycles of 36 healthy, young, normo-androgenic women, having a normal body mass index were evaluated. Serum concentrations of leptin and CRP, as well as of follicle-stimulating hormone, luteinising hormone, 17beta-oestradiol, progesterone, prolactin (PRL) and free testosterone were measured every 1-2 days during one full cycle. RESULTS: Serum levels of leptin and CRP behaved differently during ovulatory cycles, with higher concentrations for leptin only during certain phases. Significant correlations were found in the follicular phase between leptin and PRL and leptin and free testosterone. CONCLUSIONS: Leptin levels change during the menstrual cycle. Leptin levels are more stable on cycle days 1-5 than later in the cycle. For precise cycle-independent measurements, these fluctuations have to be taken into account. There is no similar cyclic pattern for CRP.

Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer

N-myc downstream-regulated gene-1 (NDRG1) is a recently described hypoxia-inducible protein that is upregulated in various human cancers. Pancreatic ductal adenocarcinoma, called pancreatic cancer, is a highly aggressive cancer that is characterised by its avascular structure, which results in a severe hypoxic environment. In this study, we investigated whether NDRG1 is upregulated in these tumours, thus providing a novel marker for malignant cells in the pancreas. By immunohistochemistry, we observed that NDRG1 was highly expressed in well-differentiated cells of pancreatic cancer, whereas the poorly differentiated tumour cells were negative. In addition, hyperplastic islets and ducts of nonquiescent pancreatic tissue were positive. To further explore its selective expression in tumours, two well-established pancreatic cancer cell lines of unequal differentiation status were exposed to 2% oxygen. NDRG1 mRNA and protein were upregulated by hypoxia in the moderately differentiated Capan-1 cells; however, its levels remained unchanged in the poorly differentiated Panc-1 cell line. Taken together, our data suggest that NDRG1 will not serve as a reliable marker of tumour cells in the pancreas, but may serve as a marker of differentiation. Furthermore, we present the novel finding that cellular differentiation may be an important factor that determines the hypoxia-induced regulation of NDRG1.

A randomized, blinded, multicenter trial of lipid-associated amphotericin B alone versus in combination with an antibody-based inhibitor of heat shock protein 90 in patients with invasive candidiasis

BACKGROUND: Mycograb (NeuTec Pharma) is a human recombinant monoclonal antibody against heat shock protein 90 that, in laboratory studies, was revealed to have synergy with amphotericin B against a broad spectrum of Candida species. METHODS: A double-blind, randomized study was conducted to determine whether lipid-associated amphotericin B plus Mycograb was superior to amphotericin B plus placebo in patients with culture-confirmed invasive candidiasis. Patients received a lipid-associated formulation of amphotericin B plus a 5-day course of Mycograb or placebo, having been stratified on the basis of Candida species (Candida albicans vs. non-albicans species of Candida). Inclusion criteria included clinical evidence of active infection at trial entry plus growth of Candida species on culture of a specimen from a clinically significant site within 3 days after initiation of study treatment. The primary efficacy variable was overall response to treatment (clinical and mycological resolution) by day 10. RESULTS: Of the 139 patients enrolled from Europe and the United States, 117 were included in the modified intention-to-treat population. A complete overall response by day 10 was obtained for 29 (48%) of 61 patients in the amphotericin B group, compared with 47 (84%) of 56 patients in the Mycograb combination therapy group (odds ratio [OR], 5.8; 95% confidence interval [CI], 2.41-13.79; P<.001). The following efficacy criteria were also met: clinical response (52% vs. 86%; OR, 5.4; 95% CI, 2.21-13.39; P<.001), mycological response (54% vs. 89%; OR, 7.1; 95% CI, 2.64-18.94; P<.001), Candida-attributable mortality (18% vs. 4%; OR, 0.2; 95% CI, 0.04-0.80; P = .025), and rate of culture-confirmed clearance of the infection (hazard ratio, 2.3; 95% CI, 1.4-3.8; P = .001). Mycograb was well tolerated. CONCLUSIONS: Mycograb plus lipid-associated amphotericin B produced significant clinical and culture-confirmed improvement in outcome for patients with invasive candidiasis.

C-reactive protein production in term human placental tissue

OBJECTIVE: C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. MATERIAL AND METHODS: Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. RESULTS: The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. CONCLUSIONS: Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.