Risk factors for community-associated Clostridioides difficile infection in young children.

The majority of paediatric Clostridioides difficile infections (CDI) are community-associated (CA), but few data exist regarding associated risk factors. We conducted a case-control study to evaluate CA-CDI risk factors in young children. Participants were enrolled from eight US sites during October 2014-February 2016. Case-patients were defined as children aged 1-5 years with a positive C. difficile specimen collected as an outpatient or ⩽3 days of hospital admission, who had no healthcare facility admission in the prior 12 weeks and no history of CDI. Each case-patient was matched to one control. Caregivers were interviewed regarding relevant exposures. Multivariable conditional logistic regression was performed. Of 68 pairs, 44.1% were female. More case-patients than controls had a comorbidity (33.3% vs. 12.1%; P = 0.01); recent higher-risk outpatient exposures (34.9% vs. 17.7%; P = 0.03); recent antibiotic use (54.4% vs. 19.4%; P < 0.0001); or recent exposure to a household member with diarrhoea (41.3% vs. 21.5%; P = 0.04). In multivariable analysis, antibiotic exposure in the preceding 12 weeks was significantly associated with CA-CDI (adjusted matched odds ratio, 6.25; 95% CI 2.18-17.96). Improved antibiotic prescribing might reduce CA-CDI in this population. Further evaluation of the potential role of outpatient healthcare and household exposures in C. difficile transmission is needed.

Enterovirus infections in early childhood and the risk of atopic disease--a nested case-control study.

BACKGROUND: Enterovirus infections in childhood have been associated with a reduced risk of atopy in cross-sectional studies. OBJECTIVE: To study the relation between enterovirus infections in the first 2 years of life and atopic disease with IgE sensitization in a prospective study setting. METHODS: This was a nested case-control study among children who had been followed from birth. Neutralizing antibodies against 12 enterovirus serotypes were analysed at the age of 2 years from 71 atopic children and 142 non-atopic control children. Atopy was defined as having an atopic disease and IgE antibodies against at least one aeroallergen by the age of 5 years. RESULTS: Cumulative exposure to different enterovirus serotypes was inversely associated with atopy [odds ratio (OR) 0.73; 95% confidence interval (CI): 0.56-0.96]. The most pronounced protection was seen when echoviruses were analysed as a separate group (OR 0.63; 95%CI: 0.46-0.88). CONCLUSIONS AND CLINICAL RELEVANCE: We propose that exposure to several different enteroviruses in early childhood is inversely associated with atopic diseases. Our results support the hypothesis that repeated microbial infections in early life may protect from atopic sensitization and atopic diseases.

Phenotypic and genotypic characterization of 10 Finnish patients with von Willebrand disease type 3: discovery of two main mutations.

Severe von Willebrand's disease (VWD) type 3 is a rare autosomal-recessively inherited bleeding disorder, showing considerable genotypic heterogeneity. We investigated the phenotype in correlation with the genotype in Finnish type 3 VWD patients. Ten patients previously diagnosed with VWD type 3 treated at the Coagulation Disorder Unit in Helsinki University Hospital were re-evaluated for bleeding tendency and treatment. Phenotypic characterization included coagulation and platelet function testing confirming the diagnosis. The genotype was assessed by initial screening for the common c.2435delC mutation and subsequently if needed, by analysing all 51 coding exons of the von Willebrand factor gene. Our result confirmed the diagnosis of type 3 VWD for all 10 patients. We discovered two common mutations: nine of the 20 alleles (45%) were found to carry the c.2435delC frameshift mutation, previously described to be frequent in countries surrounding the Baltic Sea. The nonsense mutation c.4975C>T (p.R1659X) was found on 8/20 (40%) of the alleles. In addition, three novel mutations, a potential splice site mutation (c.874+2T>C) and two frameshift mutations (c.1668delC and c.2072delCCinsG) were found. Seven patients were homozygous and three compound heterozygous for the reported mutations. This study indicates that mainly two mutations (c.2435delC and p.R1659X) cause the majority of type 3 VWD in Finland. This result sets future standards for the genetic testing among the Finnish type 3 VWD population.

Platelets significantly modify procoagulant activities in haemophilia A.

Haemophilia A replacement therapy is dosed according to patient's weight and plasma FVIII activity (FVIII:C). The FVIII interacts with platelet membrane but limited data on the impact of platelet procoagulant activity (PCA) are available in haemophilia A. Our aim was to characterize individual PCA in vitro in 20 adult haemophilia A patients at various FVIII:C levels. We detected thrombin generation in platelet-poor (PPP) and platelet-rich plasma (PRP) using: (i) calibrated automated thrombography (CAT) triggered with tissue factor, (ii) adhesion-induced PCA upon collagen and (iii) annexin V binding, expression of P-selectin and active glycoprotein (GP) IIbIIIa on platelets after stimulation of GPVI with collagen-related peptide. The FVIII:C levels varied between <1% and 37%. Thrombin generation was individual and strongly enforced by platelets and associated within the three methods. Range of thrombin generation was maximal (up to 30-fold) at FVIII:C levels 1-5%, underlining the impact of platelets in the presence of traces of replacement therapy. At FVIII:C > 5% platelet contribution in the variance faded. Platelet PCA and P-selectin exposure lead to a fivefold variation. Intriguingly, at FVIII:C < 1% thrombin generation in PPP associated negatively with platelet GPVI activation, suggestive of a regulatory interplay between plasma and platelets. In haemophilia A, the variability in thrombin generation is partially related to plasma FVIII:C, but mainly dependent on platelet procoagulant capacity. Annexin V binding and PCA in response to activation by collagen receptors contribute to this variability. In all, platelet PCA at least following collagen interaction significantly impacts thrombin generation in haemophilia A.

The Measurement Methods of Movement and Grip Strength in Children with a Previous Upper Extremity Fracture: A Comparative, Prospective Research.

BACKGROUND AND AIMS: Decreased range of motion of the elbow and forearm and decreased grip strength are potential findings following a childhood upper extremity fracture. Clinical follow-up is essential because spontaneous improvement is seen several months after the injury. Freehand measuring with a goniometer and hydraulic dynamometer is used to evaluate clinical result. The new methods are justified in avoiding human typewriting errors, thus improving patient safety. Nevertheless, their feasibility in child patients is unknown. This study aimed to evaluate congruence between the computer-assisted and the free-hand measuring methods. MATERIALS AND METHODS: A total of 59 children with a previous supracondylar humerus fracture were clinically examined by means of free-hand (transparent goniometer and hydraulic dynamometer; Jamar, Lafayette Ltd.) and computer-assisted (E-Link System Packages, Biometrics Ltd.) methods. The range of motion and grip strength were measured separately using both methods. Agreement between the measurements was evaluated using the Bland-Altman method. RESULTS: The results between the two methods were incongruent and the differences between measurements increased along with the mean of measurements in all categories except elbow extension. Rotational range of motions were smaller and grip strength was weaker while measuring with the computer-assisted method. The mean discrepancy was 0.97° (95% confidence interval = -2.46 to 0.53) for elbow extension and 7.97° (95% confidence interval = 6.60-9.33) for elbow flexion. CONCLUSIONS: Grip strength is used to evaluate impairment of hand function. The study method showed slightly lower results in grip strength. Range of motion is essential when evaluating the outcome of supracondylar humerus fracture, while >10° of change in elbow range of motion associate with impaired function. As compared with the gold-standard goniometer, the methods were not congruent. However, all differences were under 10° and probably beyond clinical importance. Because of its advantages in recording the outcomes to electronical charts, the computer-assisted method is recommended option in performing the follow-up of complicated pediatric supracondylar humerus fractures.

Expression of X-chromosome linked inhibitor of apoptosis protein in mature Purkinje cells and in retinal bipolar cells in transgenic mice induces neurodegeneration.

Transgenic mice with overexpression of the caspase-inhibitor, X-chromosome-linked inhibitor of apoptosis protein (XIAP) in Purkinje cell (PC) and in retinal bipolar cells (RBCs) were produced to study the regulation of cell death. Unexpectedly, an increased neurodegeneration was observed in the PCs in these L7-XIAP mice after the third postnatal week with the mice exhibiting severe ataxia. The loss of PCs was independent of Bax as shown by crossing the L7-XIAP mice with Bax gene-deleted mice. Electron microscopy revealed intact organelles in PCs but with the stacking of ER cisterns indicative of cell stress. Immunostaining for cell death proteins showed an increased phosphorylation of c-Jun in the PCs, suggesting an involvement in cell degeneration. Apart from PCs, the number of RBCs was decreased in adult retina in line with the expression pattern for the L7 promoter. The data show that overexpression of the anti-apoptotic protein XIAP in vulnerable neurons leads to enhanced cell death. The mechanisms underlying this neurodegeneration can be related to the effects of XIAP on cell stress and altered cell signaling.

Comprehensive re-evaluation of historical von Willebrand disease diagnosis in association with whole blood platelet aggregation and function.

INTRODUCTION: Diagnosis of von Willebrand disease (VWD) is challenging, particularly for type 1. The current diagnostic guidelines emphasize simultaneous bleeding symptoms and von Willebrand factor (VWF) levels of <30-40 IU/dL. Historical diagnoses require updated evaluation. We assessed the accuracy of past VWD diagnoses in our comprehensive care center with the standardized bleeding score (BS) and central laboratory analysis, focusing on VWF-dependent platelet functions in whole blood. METHODS: Our study comprised 83 adults with prior VWD who were diagnosed a median of 20 years ago. We assessed BS, VWF antigen and activity (minimum of 3 measurements), FVIII, PFA-100® , and platelet aggregation via Multiplate® . Genetic testing was targeted to types 3, 2N, 2B, and equivocal cases. RESULTS: All 13/13 (100%) type 3 and 29/32 (90%) type 2, but only 10/38 (26%) of type 1 (overall 52/83 (63%)) patients met the current criteria for VWD. All confirmed cases had abnormal BS, impaired PFA-100® , and decreased or absent ristocetin-induced platelet aggregation (RIPA), except subtype 2B. VWF, FVIII, RIPA, and PFA correlated with BS including all study subjects. Ten of the 38 patients with previous type 1 had low VWF (35-50 IU/dL) and variable VWF-dependent platelet function. Altogether, 21/83 patients (25%) had repeatedly normal VWF:RCo (>50 IU/dL). CONCLUSION: von Willebrand disease is associated with impaired VWF-dependent whole blood platelet functions that match traditional VWF measurements. We detected normal VWF in 25% of historically diagnosed patients, mainly type 1 patients, implying that there is a need to systematically re-evaluate historical VWD diagnoses.

ER stress and neurodegenerative diseases.

Endoplasmic reticulum (ER) stress is caused by disturbances in the structure and function of the ER with the accumulation of misfolded proteins and alterations in the calcium homeostasis. The ER response is characterized by changes in specific proteins, causing translational attenuation, induction of ER chaperones and degradation of misfolded proteins. In case of prolonged or aggravated ER stress, cellular signals leading to cell death are activated. ER stress has been suggested to be involved in some human neuronal diseases, such as Parkinson's disease, Alzheimer's and prion disease, as well as other disorders. The exact contributions to and casual effects of ER stress in the various disease processes, however, are not known. Here we will discuss the possible role of ER stress in neurodegenerative diseases, and highlight current knowledge in this field that may reveal novel insight into disease mechanisms and help to design better therapies for these disorders.

New potent and selective cytochrome P450 2B6 (CYP2B6) inhibitors based on three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis.

BACKGROUND AND PURPOSE: The cytochrome P450 2B6 (CYP2B6) enzyme metabolises a number of clinically important drugs. Drug-drug interactions resulting from inhibition or induction of CYP2B6 activity may cause serious adverse effects. The aims of this study were to construct a three-dimensional structure-activity relationship (3D-QSAR) model of the CYP2B6 protein and to identify novel potent and selective inhibitors of CYP2B6 for in vitro research purposes. EXPERIMENTAL APPROACH: The inhibition potencies (IC(50) values) of structurally diverse chemicals were determined with recombinant human CYP2B6 enzyme. Two successive models were constructed using Comparative Molecular Field Analysis (CoMFA). KEY RESULTS: Three compounds proved to be very potent and selective competitive inhibitors of CYP2B6 in vitro (IC(50)<1 microM): 4-(4-chlorobenzyl)pyridine (CBP), 4-(4-nitrobenzyl)pyridine (NBP), and 4-benzylpyridine (BP). A complete inhibition of CYP2B6 activity was achieved with 0.1 microM CBP, whereas other CYP-related activities were not affected. Forty-one compounds were selected for further testing and construction of the final CoMFA model. The created CoMFA model was of high quality and predicted accurately the inhibition potency of a test set (n=7) of structurally diverse compounds. CONCLUSIONS AND IMPLICATIONS: Two CoMFA models were created which revealed the key molecular characteristics of inhibitors of the CYP2B6 enzyme. The final model accurately predicted the inhibitory potencies of several structurally unrelated compounds. CBP, BP and NBP were identified as novel potent and selective inhibitors of CYP2B6 and CBP especially is a suitable inhibitor for in vitro screening studies.

Altered distribution and levels of cathepsinD and cystatins in amyotrophic lateral sclerosis transgenic mice: possible roles in motor neuron survival.

In amyotrophic lateral sclerosis (ALS) there is a selective degeneration of motor neurons leading to muscle paralysis and death. The mechanism underlying cell demise in ALS is not fully understood, but involves the activation of different proteolytic enzymes, including the caspase family of cysteine proteases. We have here studied whether other proteases, such as the cathepsins, residing in lysosomes, and the cathepsin inhibitors, cystatinB and -C are changed in ALS. The expression and protein levels of the cathepsinB, -L and -D all increased in the spinal cord in ALS mice, carrying the mutant copper/zinc superoxide dismutase (SOD1) gene. At the cellular level, cathepsinB and -L were present in ventral motor neurons in controls, but in the ALS mice cathepsinB was also expressed by glial fibrillary acidic protein (GFAP) positive astrocytes. The distribution of the aspartic protease, cathepsinD also changed in ALS with a loss of the lysosomal staining in motor neurons. Inhibition of caspases by means of X-chromosome-linked inhibitor of apoptosis protein (XIAP) overexpression did not inhibit cleavage of cathepsinD in ALS mice, suggesting a caspase-independent pathway. Expression of cystatinB and -C increased slightly in the ALS spinal cords. Immunostaining showed that in ALS, cystatinC was present in motor neurons and in GFAP positive astrocytes. CystatinB that is a neuroprotective factor decreased in motor neurons in ALS but was expressed by activated microglial cells. The observed changes in the levels and distributions of cathepsinD and cystatinB and-C indicate a role of these proteins in the degeneration of motor neurons in ALS.

Irradiation-induced progenitor cell death in the developing brain is resistant to erythropoietin treatment and caspase inhibition.

One hemisphere of postnatal day 8 (P8) rats or P10 mice was irradiated with a single dose of 4-12 Gy, and animals were killed from 2 h to 8 weeks after irradiation (IR). In the subventricular zone (SVZ) and the granular cell layer (GCL) of the dentate gyrus, harboring neural and other progenitor cells, nitrosylation and p53 peaked 2-12 h after IR, followed by markers for active caspase-3, apoptosis-inducing factor and TUNEL (6-24 h). Ki67-positive (proliferating) cells had disappeared by 12 h and partly reappeared by 7 days post-IR. The SVZ and GCL areas decreased approximately 50% 7 days after IR. The development of white matter was hampered, resulting in 50-70% less myelin basic protein staining. Pretreatment with erythropoietin did not confer protection against IR. Caspase inhibition by overexpression of XIAP prevented caspase-9 and caspase-3 activation but not cell death, presumably because of increased caspase-independent cell death.

Immunohistochemical localization of carbonic anhydrase isoenzyme C in the central and peripheral nervous system of the mouse.

The regional and cellular distribution of the high activity carbonic anhydrase isoenzyme (CA C or CA II) in the mouse nervous system was investigated by an indirect immunoperoxidase (peroxidase-antiperoxidase) method using cross-reactive antibodies prepared against human CA C. In the mature brain an overall strong CA C specific reactivity was revealed in the heavily myelinated nerve tracts, the main immunostaining originating from small, intensively reacting cells interpreted as oligodendrocytes and from the myelin sheaths. An obvious straining was also revealed in the choroid plexus cells, especially in their free borders, and in the erythrocytes of the blood vessels, while the glial cells of the retina similarly exhibited a strong reaction. In the developing brain, CA C was absent shortly after birth, but achieved almost the mature pattern of distribution within about 3 weeks. In the spinal cord most of the positive cells were found in the grey matter, their processes projecting towards the white matter. No reaction was obtained in the sciatic nerve fibers or in the neuronal or satellite cells of the coeliac ganglion.

Carbonic anhydrase in the type I skeletal muscle fibers of the rat. An immunohistochemical study.

The localization of carbonic anhydrase (CA) was studied in rat skeletal muscles with the use of immunohistochemical (peroxidase-antiperoxidase) method. CA was observed in all those fibers that also showed pH 4.3 stable actomyosin adenosine triphosphatase activity (type I fibers), but the reverse did not necessarily hold. More specifically, CA was apparently localized in I-bands, and a weak reaction was also observed in sarcolemma. The function of CA in muscle fibers is possibly connected with the greater demands on CO2 transport and buffer system in muscles adapted to long-lasting contractions.

Immunolabeling of carbonic anhydrase isoenzyme C and glial fibrillary acidic protein in paraffin-embedded tissue sections of human brain and retina.

The specificities of carbonic anhydrase isoenzyme C (CA C) and glial fibrillary acidic (GFA) protein as immunocytochemical markers for different glial cell populations in human brain and retina were studied using indirect immunofluorescence and peroxidase-antiperoxidase complex methods. With antibodies against CA C, only those cerebral cells that were morphologically oligodendrocytes and Müller cells of the retina showed positive immunostaining reaction, whereas antibodies against GFA protein selectively labeled cerebral astrocytes and a part of the glial cells and fibers in the inner layers of the retina. In double labeling, when both glial cell markers were successively localized in the same cerebral tissue sections, GFA protein immunofluorescence was never found in the immunoperoxidase-stained CA C-positive cells, which further supports the oligodendrocyte-specificity of CA C in human brain.

Brain derived neurotrophic factor is increased in cerebrospinal fluid of children suffering from asphyxia.

Brain derived neurotrophic factor (BDNF) is a neurotrophic factor that is relatively highly expressed in developing and adult brain. Whereas clinical determinations of nerve growth factor (NGF) in human serum and cerebrospinal fluid (CSF) in different conditions have been undertaken there are no reports on levels of BDNF in human CSF. Here we show that BDNF is increased in CSF of neonatal children suffering from asphyxia which is characterised by periods of brain hypoxic-ischemia. In contrast to BDNF, levels of CSF NGF were largely decreased in these children. The present results show that BDNF can be detected in human CSF and that the levels increase following hypoxic-ischemic brain injury. As suggested by animal studies the increased BDNF might counteract neuronal damage observed in these patients following asphyxia.

A single-step solid phase radioimmunoassay for quantifying human carbonic anhydrase I and II in cerebrospinal fluid.

A single-step solid phase radioimmunoassay was developed to detect human carbonic anhydrase (CA) isoenzymes I (CA I) and II (CA II) in cerebrospinal fluid (CSF). The assay is capable of routinely detecting both isoenzymes at ng levels compared to the microgram levels of the traditional catalytic methods, which failed to demonstrate any CA activity in CSF. When the values of immunoreactive CA II in CSF were corrected for blood contamination (the CA I/CA II ratio of blood was about 7.9), the amount of brain tissue originated CA II could be calculated. The CA II values in CSF samples from 13 patients with multiple sclerosis were higher than those in CSF samples from 11 patients with various peripheral neurological disorders. Since CA II has been specifically localized to oligodendrocytes and myelin, our preliminary results suggest the possibility of CA II leakage from oligodendrocytes and myelin into CSF in demyelinating disease.

Interleukin-6 and interleukin-1 receptor antagonist in cerebrospinal fluid from patients with recent tonic-clonic seizures.

We have previously reported increased concentrations of interleukin (1L)-6 in CSF from patients with tonic-clonic seizures, where increased cytokine production most likely is a consequence of neuronal epileptic activity associated with seizures. The biological effects of IL-6 are mediated by other cytokines, which are studied here in addition to IL-6. The purpose of this study was to analyze levels of soluble cytokines from plasma and CSF from patients with newly developed tonic-clonic seizures. The concentrations of IL-6, IL-1 receptor antagonist (IL-1RA), IL-1beta, tumor necrosis factor (TNFalpha) and nerve growth factor (NGF) were measured from plasma and CSF from 22 patients with newly developed tonic-clonic seizures within 24 h from the seizure and 18 controls. The mean concentrations of IL-6 were significantly increased in CSF (P<0.001) and plasma (P<0.01) after tonic-clonic seizures, there was some indication of increased concentrations of IL-1RA and no significant change in NGF, IL-1beta or TNFalpha. Our study shows that cytokine network is activated in patients after recent tonic-clonic seizures. We provide evidence of intrathecal production of IL-6 associated with electrical seizure activity.

Gas chromatographic-mass spectrometric detection of 2- and 3-hydroxy fatty acids as methyl esters from soil, sediment and biofilm.

Hydroxy fatty acids (OH-FAs) can be used in the characterization of microbial communities, especially Gram-negative bacteria. We prepared methyl esters of 2- and 3-OH-FAs from the lipid extraction residue of soil, sediment, and biofilm samples without further purification or derivatization of hydroxyl groups. OH-FA methyl esters were analyzed using a gas chromatograph equipped with a mass selective detector (GC-MS). The ions followed in MS were m/z 103 for 3-OH-FAs and m/z 90 and M-59 for 2-OH-FAs. The rapid determination of 3- and 2-OH-FAs concomitantly with phospholipid fatty acids provided more detailed information on the microbial communities present in soil, sediment, and drinking water biofilm.

Neurotrophic factors in cerebrospinal fluid and serum of patients with Rett syndrome.

Rett syndrome is now considered to be a neurodevelopmental disease. Its cause is unknown, but it has been suggested that neuronal growth factors and neurotransmitters play important roles. We measured levels of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in cerebrospinal fluid, and nerve growth factor and brain-derived neurotrophic factor in serum in child and adolescent patients with Rett syndrome. Levels of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in cerebrospinal fluid were below the limit of sensitivity of the methods used. Serum levels of nerve growth factor and brain-derived neurotrophic factor did not differ from control values. In Rett syndrome, the normal serum levels of nerve growth factor together and previously reported low levels of the factor in cerebrospinal fluid indicate that the latter may reflect low levels of nerve growth factor in the central nervous system.

Cerebrospinal nerve growth factor--a marker of asphyxia?

Asphyxia in neonates is characterized by different degrees of hypoxia-ischemia, with the outcome depending on the severity of the underlying brain cell damage. Neurotrophic factors rescue neurons from cell death after injury and promote neuronal survival during development. The authors have used enzyme-linked immunosorbent assay to study levels of nerve growth factor in the cerebrospinal fluid of children with asphyxia at birth (n = 10) and of controls (n = 23). Compared with reference groups the children who had had severe asphyxia had lower or negligible levels of cerebrospinal fluid nerve growth factor in the neonatal period or later. The level of cerebrospinal fluid nerve growth factor measured in the neonatal period was 3.76+/-4.13 pg/mL in children with asphyxia (n = 8), which is significantly lower than in children without asphyxia or infection (n = 10) 9.42+/-4.09 pg/mL or in those without asphyxia but with infection (n = 13) 17.63+/-11.48 pg/mL (P = 0.0186 and P = 0.0013, respectively). However, in some children with asphyxia the cerebrospinal fluid nerve growth factor levels were virtually normal, and most importantly these children subsequently had normal neurologic development. These results suggest that cerebrospinal fluid nerve growth factor might be used as a biochemical marker for early estimates of hypoxic-ischemic brain damage in asphyxiated neonates.