Faecal microbiota transplantation for eradication of co-infection with Clostridioides difficile and extensively drug-resistant KPC-producing Klebsiella pneumoniae.

Clostridioides difficile infection may be complicated by co-infection with other pathogens. We here describe the successful use of faecal microbiota transplantation to eradicate concomitant C. difficile and extensively drug-resistant (XDR) KPC-producing Klebsiella pneumoniae. Donor microbiota efficiently engrafted in the patient, and a donor-like microbial assemblage persisted in the patient during six months follow-up. The report explores the potential for the donor microbiota to eradicate and replace multi-resistant microorganisms.

Alcohol consumption in adolescence is associated with a lower risk of multiple sclerosis in a Danish cohort.

BACKGROUND AND OBJECTIVE: Due to the possible existence of a vulnerable period of multiple sclerosis (MS) susceptibility in adolescence and because Danish teenagers have a high alcohol consumption, we investigated the association between alcohol consumption at ages 15-19 and the risk of developing MS. METHODS: A total of 1717 patients with MS and 4685 healthy blood donors filled in a comprehensive environmental and lifestyle questionnaire. Data were analysed by logistic regression models and adjusted for selected confounders. RESULTS: We found an inverse association between alcohol consumption in adolescence and risk of developing MS in both women (p < 0.001) and men (p = 0.012). Women with low alcohol consumption had an odds ratio (OR) of 0.56 (95% confidence interval (CI): 0.47-0.66) compared with non-drinking women. The ORs were similar for women with moderate (OR = 0.49, 95% CI: 0.38-0.62) and high consumption (OR = 0.57, 95% CI: 0.38-0.84). Men with low alcohol consumption had an OR of 0.69 (95% CI: 0.53-0.89) compared with non-drinking men but no decreased risk was found for men with moderate and high consumption. CONCLUSION: Alcohol consumption in adolescence was associated with lower risk of developing MS among both sexes.

Heterologous immunological effects of early BCG vaccination in low-birth-weight infants in Guinea-Bissau: a randomized-controlled trial.

BACKGROUND: Bacillus Calmette-Guérin (BCG) seems to have beneficial nonspecific effects; early BCG vaccination of low-birth-weight (LBW) newborns reduces neonatal mortality by >40% due to prevention of primarily septicemia and pneumonia. METHODS: Within a randomized trial in LBW infants in Guinea-Bissau of early BCG vs the usual postponed BCG, a subgroup was bled 4 weeks after randomization. Levels of interleukin (IL)-1ß, IL-5, IL-6, IL-10, IL-17, interferon (IFN)-γ and tumor necrosis factor (TNF)-α were measured from whole-blood assays stimulated with innate agonists to Toll-like receptor (TLR)-2, -4 or -7/8, or purified protein derivative (PPD). RESULTS: Among 467 infants, BCG significantly increased the in vitro cytokine responses to purified protein derivative of Mycobacterium tuberculosis (PPD), as expected. BCG was also associated with increased responses to heterologous innate stimulation, particularly of the cytokines IL-1ß, IL-6, TNF-α, and IFN-γ. CONCLUSION: Four weeks after immunization, BCG-vaccinated infants have a significantly increased production of cytokines upon heterologous challenge, particularly T helper cell type 1 polarizing and typically monocyte-derived pro-inflammatory cytokines. BCG may accelerate the development of the neonatal immune system, mediating comprehensive protection against infections and mortality.

Interactions of oral permeation enhancers with lipid membranes in simulated intestinal environments.

The oral bioavailability of therapeutic peptides is generally low. To increase peptide transport across the gastrointestinal barrier, permeation enhancers are often used. Despite their widespread use, mechanistic knowledge of permeation enhancers is limited. To address this, we here investigate the interactions of six commonly used permeation enhancers with lipid membranes in simulated intestinal environments. Specifically, we study the interactions of the permeation enhancers sodium caprate, dodecyl maltoside, sodium cholate, sodium dodecyl sulfate, melittin, and penetratin with epithelial cell-like model membranes. To mimic the molecular composition of the real intestinal environment, the experiments are performed with two peptide drugs, salmon calcitonin and desB30 insulin, in fasted-state simulated intestinal fluid. Besides providing a comparison of the membrane interactions of the studied permeation enhancers, our results demonstrate that peptide drugs as well as intestinal-fluid components may substantially change the membrane activity of permeation enhancers. This highlights the importance of testing permeation enhancement in realistic physiological environments and carefully choosing a permeation enhancer for each individual peptide drug.

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism.

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.

Neonatal chemokine levels and risk of autism spectrum disorders: findings from a Danish historic birth cohort follow-up study.

A potential role of chemokines in the pathophysiology of Autism Spectrum Disorders (ASDs) has been previously suggested. In a recent study we examined levels of three inflammatory chemokines (MCP-1, MIP-1α and RANTES) in samples of amniotic fluid of children diagnosed later in life with ASD and controls frequency-matched to cases on gender and year of birth. In this follow-up study, levels of the same chemokines were analyzed postnatally in dried blood spot samples from the same subjects utilizing the Danish Newborn Screening Biobank. Crude estimates showed decreased levels of RANTES. In the adjusted estimates, no differences were found in levels of the three examined chemokines in ASD cases compared to controls. Our findings may cautiously suggest an altered cell-mediated immunity during the early neonatal period in ASD. Further research is needed to examine the relationship between maternal/fetal and neonatal chemokine levels and their role in ASD.

Biochemical screening of 504,049 newborns in Denmark, the Faroe Islands and Greenland--experience and development of a routine program for expanded newborn screening.

Expanded newborn screening for selected inborn errors of metabolism (IEM) in Denmark, the Faroe Islands and Greenland was introduced in 2002. We now present clinical, biochemical, and statistical results of expanded screening (excluding PKU) of 504,049 newborns during nine years as well as diagnoses and clinical findings in 82,930 unscreened newborns born in the same period. The frequencies of diagnoses made within the panel of disorders screened for are compared with the frequencies of the disorders in the decade preceding expanded newborn screening. The expanded screening was performed as a pilot study during the first seven years, and the experience obtained during these years was used in the development of the routine neonatal screening program introduced in 2009. Methods for screening included tandem mass spectrometry and an assay for determination of biotinidase activity. A total of 310 samples from 504,049 newborns gave positive screening results. Of the 310 results, 114 were true positive, including results from 12 newborns in which the disease in question was subsequently diagnosed in their mothers. Thus, the overall frequency of an IEM in the screening panel was 1:4942 (mothers excluded) or 1:4421 (mothers included). The false positive rate was 0.038% and positive predictive value 37%. Overall specificity was 99.99%. All patients with true positive results were followed in The Center for Inherited Metabolic Disorders in Copenhagen, and the mean follow-up period was 45 months (range 2109 months). There were no deaths among the 102 children, and 94% had no clinically significant sequelae at last follow-up. Our study confirms the higher frequency of selected IEM after implementation of expanded newborn screening and suggests an improved outcome for several disorders. We argue that newborn screening for these disorders should be standard of care, though unresolved issues remain, e.g. about newborns with a potential for remaining asymptomatic throughout life. Well organized logistics of the screening program from screening laboratory to centralized, clinical management is important.

Amniotic fluid chemokines and autism spectrum disorders: an exploratory study utilizing a Danish Historic Birth Cohort.

INTRODUCTION: Elevated levels of chemokines have been reported in plasma and brain tissue of individuals with Autism Spectrum Disorders (ASD). The aim of this study was to examine chemokine levels in amniotic fluid (AF) samples of individuals diagnosed with ASD and their controls. MATERIAL AND METHODS: A Danish Historic Birth Cohort (HBC) kept at Statens Serum Institute, Copenhagen was utilized. Using data from Danish nation-wide health registers, a case-control study design of 414 cases and 820 controls was adopted. Levels of MCP-1, MIP-1α and RANTES were analyzed using Luminex xMAP technology. Case-control differences were assessed as dichotomized at below the 10th percentile or above the 90th percentile cut-off points derived from the control biomarker distributions (logistic regression) or continuous measures (tobit regression). RESULTS AND CONCLUSION: AF volume for 331 cases and 698 controls was sufficient for Luminex analysis. Including all individuals in the cohort yielded no significant differences in chemokine levels in cases versus controls. Logistic regression analyses, performed on individuals diagnosed using ICD-10 only, showed increased risk for ASD with elevated MCP-1 (elevated 90th percentile adjusted OR: 2.32 [95% CI: 1.17-4.61]) compared to controls. An increased risk for infantile autism with elevated MCP-1 was also found (adjusted OR: 2.28 [95% CI: 1.16-4.48]). Elevated levels of MCP-1 may decipher an etiologic immunologic dysfunction or play rather an indirect role in the pathophysiology of ASD. Further studies to confirm its role and to identify the potential pathways through which MCP-1 may contribute to the development of ASD are necessary.

Cerebral autoregulation in the first day after preterm birth: no evidence of association with systemic inflammation.

INTRODUCTION: Both systemic inflammation and impaired cerebral autoregulation (CA) have been associated with brain injury in preterm infants. We hypothesized that impaired CA represents a hemodynamic link between inflammation and brain injury. RESULTS: Neither fetal vasculitis nor interleukin-6 (IL-6) affected CA significantly. A high level of IL-6 was associated with hypotension (P = 0.03) irrespective of dopamine therapy. The magnitude of impairment in CA increased with decreasing mean arterial blood pressure (MAP) (P = 0.02). No significant associations were found between these parameters and either intraventricular hemorrhage (IVH) (n = 10) or neonatal mortality (n = 8). DISCUSSION: In conclusion, postnatal inflammation was weakly associated with arterial hypotension, and hypotension was weakly associated with impaired autoregulation. There was no direct association, however, between autoregulation and antenatal or postnatal signs of inflammation. METHODS: In our study, 60 infants (mean (±SD) of gestational age (GA) 27 (±1.3) wk) underwent continuous recording of MAP and cerebral oxygenation index (OI) by means of near-infrared spectroscopy (NIRS) for 2.3 ± 0.5 h, starting 18 ± 9 h after birth. Coherence and transfer function gain between MAP and OI represented the presence and degree of impairment of CA, respectively. We considered fetal vasculitis (placenta histology) to be an antenatal marker of inflammation, and used the level of IL-6 in blood, measured at 18 ± 10 h after birth, as a postnatal marker of inflammation. Definition of hypotension was MAP (mm Hg) ≤ GA (wk).

Imaging therapeutic peptide transport across intestinal barriers.

Oral delivery is a highly preferred method for drug administration due to high patient compliance. However, oral administration is intrinsically challenging for pharmacologically interesting drug classes, in particular pharmaceutical peptides, due to the biological barriers associated with the gastrointestinal tract. In this review, we start by summarizing the pharmacological performance of several clinically relevant orally administrated therapeutic peptides, highlighting their low bioavailabilities. Thus, there is a strong need to increase the transport of peptide drugs across the intestinal barrier to realize future treatment needs and further development in the field. Currently, progress is hampered by a lack of understanding of transport mechanisms that govern intestinal absorption and transport of peptide drugs, including the effects of the permeability enhancers commonly used to mediate uptake. We describe how, for the past decades, mechanistic insights have predominantly been gained using functional assays with end-point read-out capabilities, which only allow indirect study of peptide transport mechanisms. We then focus on fluorescence imaging that, on the other hand, provides opportunities to directly visualize and thus follow peptide transport at high spatiotemporal resolution. Consequently, it may provide new and detailed mechanistic understanding of the interplay between the physicochemical properties of peptides and cellular processes; an interplay that determines the efficiency of transport. We review current methodology and state of the art in the field of fluorescence imaging to study intestinal barrier transport of peptides, and provide a comprehensive overview of the imaging-compatible in vitro, ex vivo, and in vivo platforms that currently are being developed to accelerate this emerging field of research.

Small angle X-ray scattering study of calreticulin reveals conformational plasticity.

Calreticulin plays a central role in vital cell processes such as protein folding, Ca(2+) homeostasis and immunogenicity. Even so, only limited three-dimensional structural information is presently available. We present a series of Small-Angle X-ray Scattering data on human placenta calreticulin. The data from the calreticulin monomer reveal the shape of calreticulin in solution: The previously structurally un-described C-terminal is seen as a globular domain, and the P-domain beta-hairpin extends from the N-domain in a spiral like conformation. In the calreticulin solution dimer, the N-, C-, and P-domains are easily identified, and the P-domain is in an extended conformation connecting to the second calreticulin molecule. The SAXS solution data enables the construction of a medium-resolution model of calreticulin. In the light of the unresolved chaperone mechanism of calreticulin and calnexin, we discuss the functional consequences of the conformational plasticity of the calreticulin P-domain.

Impaired Cytokine Responses to Epstein-Barr Virus Antigens in Systemic Lupus Erythematosus Patients.

We analyzed cytokine responses against latent and lytic Epstein-Barr virus (EBV) antigens in systemic lupus erythematosus (SLE) patients and healthy controls (HCs) to obtain an overview of the distinctive immune regulatory response in SLE patients and to expand the previously determined impaired EBV-directed T-cell response. The concentrations of 14 cytokines (IL2, IL4, IL5, IL6, IL10, IL12, IL17, IL18, IL1ß, IFNγ, TNFα, TNFß, TGFß, and GM-CSF) were quantified upon stimulation of whole blood with latent state antigen EBNA1, lytic cycle antigen EBV-EA/D, and the superantigen SEB. To avoid results affected by lack of lymphocytes, we focused on SLE patients with normal levels. Decreased induction of IL12, IFNγ, IL17, and IL6 upon EBNA1 stimulation and that of IFNγ, IL6, TNFß, IL1ß, and GM-CSF upon EBV-EA/D stimulation were detected in SLE patients compared to HCs. IFNγ responses, especially, were shown to be reduced. Induction of several cytokines was furthermore impaired in SLE patients upon SEB stimulation, but no difference was observed in basic levels. Results substantiate the previously proposed impaired regulation of the immune response against latent and lytic cycle EBV infection in SLE patients without lymphopenia. Furthermore, results indicate general dysfunction of leukocytes and their cytokine regulations in SLE patients.

Conformational stability of calreticulin.

The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (Tm) increased from 31 degrees C at pH 5 to 51 degrees C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C-terminal alpha-helix was of major importance to the conformational stability of calreticulin.

Aquaporin-Based Biomimetic Polymeric Membranes: Approaches and Challenges.

In recent years, aquaporin biomimetic membranes (ABMs) for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs: aquaporin proteins (AQPs), block copolymers for AQP reconstitution, and polymer-based supporting structures. First, we briefly cover challenges and review recent developments in understanding the interplay between AQP and block copolymers. Second, we review some experimental characterization methods for investigating AQP incorporation including freeze-fracture transmission electron microscopy, fluorescence correlation spectroscopy, stopped-flow light scattering, and small-angle X-ray scattering. Third, we focus on recent efforts in embedding reconstituted AQPs in membrane designs that are based on conventional thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes.

Fetal gender and several cytokines are associated with the number of fetal cells in maternal blood--an observational study.

OBJECTIVE: To identify factors influencing the number of fetal cells in maternal blood. METHODS: A total of 57 pregnant women at a gestational age of weeks 11-14 were included. The number of fetal cells in maternal blood was assessed in 30 ml of blood using specific markers for both enrichment and subsequent identification. RESULTS: Participants carrying male fetuses had a higher median number of fetal cells in maternal blood than those carrying female fetuses (5 vs. 3, p = 0.04). Certain cytokines (RANTES, IL-2 and IL-5) were significantly associated with the number of fetal cells in maternal blood. CONCLUSION: The number of fetal cells in maternal blood is associated with certain cytokines and fetal gender.

Amniotic fluid inflammatory cytokines: potential markers of immunologic dysfunction in autism spectrum disorders.

OBJECTIVES: The aim of the study was to analyze cytokine profiles in amniotic fluid (AF) samples of children developing autism spectrum disorders (ASD) and controls, adjusting for maternal autoimmune disorders and maternal infections during pregnancy. METHODS: AF samples of 331 ASD cases and 698 controls were analyzed for inflammatory cytokines using Luminex xMAP technology utilizing a historic birth cohort. Clinical data were retrieved from nationwide registers, and case-control differences in AF cytokine levels were assessed using chi-square tests, logistic and tobit regression models. RESULTS: Overall, individuals with ASD had significantly elevated AF levels of TNF-α and TNF-ß compared to controls. Analyzing individuals diagnosed only with ICD-10 codes yielded significantly elevated levels of IL-4, IL-10, TNF-α and TNF-ß in ASD patients. Restricting analysis to infantile autism cases showed significantly elevated levels of IL-4, TNF-α and TNF-ß compared to controls with no psychiatric comorbidities. Elevated levels of IL-6 and IL-5 were found in individuals with other childhood psychiatric disorders (OCPD) when compared to controls with no psychiatric comorbidities. CONCLUSIONS: AF samples of individuals with ASD or OCPD showed differential cytokine profiles compared to frequency-matched controls. Further studies to examine the specificity of the reported cytokine profiles in ASD and OCPD are required.

Amniotic fluid MMP-9 and neurotrophins in autism spectrum disorders: an exploratory study.

Evidence suggests that some developmental disorders, such as autism spectrum disorders (ASDs), are caused by errors in brain plasticity. Given the important role of matrix metalloproteinases (MMPs) and neurotrophins (NTs) in neuroplasticity, amniotic fluid samples for 331 ASD cases and 698 frequency-matched controls were analyzed for levels of MMP-9, brain-derived neurotrophic factor, NT-4 and transforming growth factor-ß utilizing a Danish historic birth cohort and Danish nationwide health registers. Laboratory measurements were performed using an in-house multiplex sandwich immunoassay Luminex xMAP method, and measurements were analyzed using tobit and logistic regression. Results showed elevated levels of MMP-9 in ASD cases compared with controls (crude and adjusted tobit regression P-values: 0.01 and 0.06). Our results highlight the importance of exploring the biologic impact of MMP-9 and potential therapeutic roles of its inhibitors in ASD and may indicate that neuroplastic impairments in ASD may present during pregnancy.

Neonatal levels of cytokines and risk of autism spectrum disorders: an exploratory register-based historic birth cohort study utilizing the Danish Newborn Screening Biobank.

The aim of the study was to analyze cytokine profiles in neonatal dried blood samples (n-DBSS) retrieved from The Danish Newborn Screening Biobank of children developing Autism Spectrum Disorders (ASD) later in life and controls. Samples of 359 ASD cases and 741 controls were analyzed using Luminex xMAP technology and clinical data were retrieved from nationwide registers. Findings showed that children developing ASD were more likely to have decreased levels of both T helper-1(Th-1)-like cytokines (i.e. IFN-γ) and Th-2like cytokines (i.e. IL-4, IL-10) which may suggest a depressed or hypoactive immune cell activity during neonatal period in ASD.

The effects of detomidine, romifidine or acepromazine on echocardiographic measurements and cardiac function in normal horses.

OBJECTIVE: To evaluate by echo- and electrocardiography the cardiac effects of sedation with detomidine hydrochloride, romifidine hydrochloride or acepromazine maleate in horses. STUDY DESIGN: An experimental study using a cross-over design without randomization. ANIMALS: Eight clinically normal Standardbred trotters. MATERIALS AND METHODS: Echocardiographic examinations (two-dimensional, guided M-mode and colour Doppler) were recorded on five different days. Heart rate (HR) and standard limb lead electrocardiograms were also obtained. Subsequently, horses were sedated with detomidine (0.01 mg kg(-1)), romifidine (0.04 mg kg(-1)) or acepromazine (0.1 mg kg(-1)) administered intravenously and all examinations repeated. RESULTS: Heart rate before treatment with the three drugs did not differ significantly (p = 0.98). Both detomidine and romifidine induced a significant decrease (p < 0.001) in HR during the first 25 minutes after sedation; while acepromazine had a varying effect on HR. For detomidine, there was a significant increase in LVIDd (left ventricular internal diameter in diastole; p = 0.034) and LVIDs (left ventricular internal diameter in systole; p < 0.001). In addition, a significant decrease was found in IVSs (the interventricular septum in systole; p < 0.001), LVFWs (the left ventricular free wall in systole; p = 0.002) and FS% (fractional shortening; p < 0.001). The frequency of pulmonary regurgitation was increased significantly (p < 0.001). Romifidine induced a significant increase in LVIDs (p < 0.001) and a significant decrease in IVSs (p < 0.001) and FS% (p = 0.002). Acepromazine had no significant effect upon any of the measured values. CONCLUSIONS: and clinical relevance The results indicate that sedation of horses with detomidine and to a lesser extent romifidine at the doses given in this study has a significant effect on heart function, echocardiographic measurements of heart dimensions and the occurrence of valvular regurgitation. Although the clinical significance of these results may be minimal, the potential effects of sedative drugs should be taken into account when echocardiographic variables are interpreted in clinical cases.