Unfractionated heparin dosing requirements in the presence of inflammation during the first six months of life.

BACKGROUND: Critically ill neonates with inflammation secondary to SIRS or sepsis often develop an acquired pro-thrombotic state. Unfractionated heparin (UFH) is commonly prescribed in this population, but these subjects often remain sub-therapeutic or require very high doses of UFH to achieve and sustain therapeutic anti-Xa activity. This is due, in part, to the unique pharmacokinetics/dynamics of this population but may also be influenced by the degree of inflammation. OBJECTIVE: To evaluate UFH dosing requirements in neonates and infants <6 months of age with variable degrees of systemic inflammation. Clinical outcomes of bleeding and clotting will also be examined. SUBJECTS/METHODS: A retrospective chart review was performed in infants <6 months of age treated with intravenous UFH for at least 24 h with intent to reach a goal anti-Xa of 0.3-0.7 U/mL at Children's Hospital Colorado between October 2008 and August 2014. Subjects were divided into two groups, based on their ability to achieve and maintain anti-Xa concentrations between 0.3 and 0.7 U/mL. The relationship between UFH dose (U/kg/h) and inflammatory status (using pediatric age-specific definitions for SIRS, sepsis, severe sepsis, or septic shock) was examined. RESULTS: Seventy-three subjects were included in the analysis. Twenty-three subjects (mean age = 41.2 days ±â€¯standard deviation [SD] 52.3) achieved therapeutic anti-Xa concentrations while fifty subjects (mean age = 43.4 days ±â€¯SD 53) did not. The median UFH dose needed in subjects who achieved goal anti-Xa concentrations in the absence of SIRS or sepsis criteria was 24.5 U/kg/h (interquartile range [IQR] = 23.6-25.9) while the median dose of UFH in subjects who achieved goal anti-Xa level in the setting of infection, SIRS, or sepsis of any type was 36.1 U/kg/h (IQR = 34-43.5) (p < 0.0001). In subjects who maintained therapeutic anticoagulation, there was a direct relationship between UFH dose and the severity of inflammation as determined by pediatric SIRS/sepsis criteria. CONCLUSIONS: Maintenance of therapeutic UFH levels remains a challenge in infants, especially in those with concomitant inflammatory processes. Infection, SIRS, and sepsis of any type were collectively associated with a 32% increase in unfractionated heparin dose required to achieve and maintain therapeutic anti-Xa serum concentrations.

A Cross-Sectional Study of Antibiotic Prescribing for Childhood Upper Respiratory Tract Infections in Irish General Practice

Introduction This study aimed to analyse antibiotic prescribing in cases of upper respiratory tract infection (URTI) in children under 6 years attending Irish daytime and out-of-hours General Practice (GP) services. There have been large scale changes in entitlements for free GP care for this group in recent years. Methods A cross-sectional study of children under 6 years with URTI presentations was performed, over a two-week period for three years from 2015 to 2017. Factors associated with antibiotic prescription and preferred antibiotic compliance were examined using multivariate logistic regression. Results 1,007 Under-6 patients presented with an URTI in our sample over the study period. Following introduction of free GP care, patients were 50% less likely to receive an antibiotic prescription. Overall antibiotic prescribing fell from 70% to 50% in daytime services and from 72% to 60% in the out-of-hours setting. Patients presenting to out-of-hours services were more likely to receive an antibiotic (OR: 1.42) and less likely to receive a deferred antibiotic (OR: 0.53). One quarter to one third of all prescriptions were for deferred antibiotics. Year-on-year trends showed a 13% decrease in prescriptions and 13% increase in preferred antibiotic use. Conclusion The introduction of free GP care led to significant reductions in antibiotic prescribing, which may be due to changes in health seeking behaviour by parents or other reasons. Antibiotic prescribing was more commonplace in the out-of-hours setting, and rates remains high by international standards. This study underlines the importance of ongoing work around GP antimicrobial stewardship, particularly in the out-of-hours setting.

Gender differences in tuberculosis treatment outcomes: a post hoc analysis of the REMoxTB study.

BACKGROUND: In the REMoxTB study of 4-month treatment-shortening regimens containing moxifloxacin compared to the standard 6-month regimen for tuberculosis, the proportion of unfavourable outcomes for women was similar in all study arms, but men had more frequent unfavourable outcomes (bacteriologically or clinically defined failure or relapse within 18 months after randomisation) on the shortened moxifloxacin-containing regimens. The reason for this gender disparity in treatment outcome is poorly understood. METHODS: The gender differences in baseline variables were calculated, as was time to smear and culture conversion and Kaplan-Meier plots were constructed. In post hoc exploratory analyses, multivariable logistic regression modelling and an observed case analysis were used to explore factors associated with both gender and unfavourable treatment outcome. RESULTS: The per-protocol population included 472/1548 (30%) women. Women were younger and had lower rates of cavitation, smoking and weight (all p < 0.05) and higher prevalence of HIV (10% vs 6%, p = 0.001). They received higher doses (mg/kg) than men of rifampicin, isoniazid, pyrazinamide and moxifloxacin (p ≤ 0.005). There was no difference in baseline smear grading or mycobacterial growth indicator tube (MGIT) time to positivity. Women converted to negative cultures more quickly than men on Lowenstein-Jensen (HR 1.14, p = 0.008) and MGIT media (HR 1.19, p < 0.001). In men, the presence of cavitation, positive HIV status, higher age, lower BMI and 'ever smoked' were independently associated with unfavourable treatment outcome. In women, only 'ever smoked' was independently associated with unfavourable treatment outcome. Only for cavitation was there a gender difference in treatment outcomes by regimen; their outcome in the 4-month arms was significantly poorer compared to the 6-month treatment arm (p < 0.001). Women, with or without cavities, and men without cavities had a similar outcome on all treatment arms (p = 0.218, 0.224 and 0.689 respectively). For all other covariate subgroups, there were no differences in treatment effects for men or women. CONCLUSIONS: Gender differences in TB treatment responses for the shorter regimens in the REMoxTB study may be explained by poor outcomes in men with cavitation on the moxifloxacin-containing regimens. We observed that women with cavities, or without, on the 4-month moxifloxacin regimens had similar outcomes to all patients on the standard 6-month treatment. The biological reasons for this difference are poorly understood and require further exploration.

Real-world use of fidaxomicin in a large UK tertiary hospital: how effective is it for treating recurrent disease?

All courses of fidaxomicin use in the study hospital were reviewed. It was used for first recurrence (six times), second recurrence (eight times) and one case of third recurrence. One patients received fidaxomicin as first-line treatment. Eight patients initially responded to therapy; of these, three patients were asymptomatic at 90 days, three patients remained asymptomatic at 30 days, and two patients had recurrences five and nine days after stopping therapy. Four patients failed to respond; of these, two patients required faecal transplantation and one patient required a colectomy. Two patients deteriorated and two patients died. Fidaxomicin was well tolerated. These findings suggest that the utility of fidaxomicin at this stage of infection is unclear.

Pretreatment chest x-ray severity and its relation to bacterial burden in smear positive pulmonary tuberculosis.

BACKGROUND: Chest radiographs are used for diagnosis and severity assessment in tuberculosis (TB). The extent of disease as determined by smear grade and cavitation as a binary measure can predict 2-month smear results, but little has been done to determine whether radiological severity reflects the bacterial burden at diagnosis. METHODS: Pre-treatment chest x-rays from 1837 participants with smear-positive pulmonary TB enrolled into the REMoxTB trial (Gillespie et al., N Engl J Med 371:1577-87, 2014) were retrospectively reviewed. Two clinicians blinded to clinical details using the Ralph scoring system performed separate readings. An independent reader reviewed discrepant results for quality assessment and cavity presence. Cavitation presence was plotted against time to positivity (TTP) of sputum liquid cultures (MGIT 960). The Wilcoxon rank sum test was performed to calculate the difference in average TTP for these groups. The average lung field affected was compared to log 10 TTP by linear regression. Baseline markers of disease severity and patient characteristics were added in univariable regression analysis against radiological severity and a multivariable regression model was created to explore their relationship. RESULTS: For 1354 participants, the median TTP was 117 h (4.88 days), being 26 h longer (95% CI 16-30, p < 0.001) in patients without cavitation compared to those with cavitation. The median percentage of lung-field affected was 18.1% (IQR 11.3-28.8%). For every 10-fold increase in TTP, the area of lung field affected decreased by 11.4%. Multivariable models showed that serum albumin decreased significantly as the percentage of lung field area increased in both those with and without cavitation. In addition, BMI and logged TTP had a small but significant effect in those with cavitation and the number of severe TB symptoms in the non-cavitation group also had a small effect, whilst other factors found to be significant on univariable analysis lost this effect in the model. CONCLUSIONS: The radiological severity of disease on chest x-ray prior to treatment in smear positive pulmonary TB patients is weakly associated with the bacterial burden. When compared against other variables at diagnosis, this effect is lost in those without cavitation. Radiological severity does reflect the overall disease severity in smear positive pulmonary TB, but we suggest that clinicians should be cautious in over-interpreting the significance of radiological disease extent at diagnosis.

Managing malaria in tuberculosis patients on fluoroquinolone-containing regimens: assessing the risk of QT prolongation.

The role of fluoroquinolones (FQs) in the management of drug-susceptible and drug-resistant tuberculosis (TB) is under investigation. They are currently being used off-licence to treat TB patients who develop hepatotoxicity on standard therapy, and in patients with drug-resistant disease. Prolongation of ventricular repolarisation, recorded as lengthening of the QT interval on an electrocardiogram, is a recognised adverse effect associated with FQs. Significant prolongation of the QT interval may precipitate torsades de pointe, a potentially fatal tachyarrhythmia. Currently licensed FQs are considered safe, and there are very few reports of associated arrhythmias, but most labels contraindicate concomitant administration of other agents that prolong QT. In many high TB burden countries, malaria is also endemic. Many antimalarials, and possibly malaria infection itself, may prolong QT; under current licence, co-administration of FQs with these antimalarials is contraindicated due to potential risk of additive QT prolongation. This poses significant challenges in planning future policy on FQ use for first-line anti-tuberculosis treatment; the duration of TB treatment makes concomitant malaria treatment inevitable, and options without FQ contraindications are limited. Furthermore, malaria diagnosis is often poor and access to treatment uncontrolled, with many patients buying 'over-the-counter' and/or 'traditional' remedies; concomitant use with anti-tuberculosis treatment is thus likely to be unregulated. Drug interaction studies are urgently required to assess the safety of managing patients with TB and malaria within endemic, resource-poor settings where programmatic management and low-cost monitoring are essential for effective implementation of public health strategies.

The p53 family and programmed cell death.

The p53 tumor suppressor continues to hold distinction as the most frequently mutated gene in human cancer. The ability of p53 to induce programmed cell death, or apoptosis, of cells exposed to environmental or oncogenic stress constitutes a major pathway whereby p53 exerts its tumor suppressor function. In the past decade, we have discovered that p53 is not alone in its mission to destroy damaged or aberrantly proliferating cells: it has two homologs, p63 and p73, that in various cellular contexts and stresses contribute to this process. In this review, the mechanisms whereby p53, and in some cases p63 and p73, induce apoptosis are discussed. Other reviews have focused more extensively on the contribution of individual p53-regulated genes to apoptosis induction by this protein, whereas in this review, we focus more on those factors that mediate the decision between growth arrest and apoptosis by p53, p63 and p73, and on the post-translational modifications and protein-protein interactions that influence this decision.

Common tumour p53 mutations in immortalized cells from Hupki mice heterozygous at codon 72.

Codon 72 of human p53 gene is polymorphic, encoding arginine or proline. Here we report construction of a human p53 knock-in (Hupki) mouse encoding the codon 72(pro) variant. The new strain was crossed with the original Hupki mice (codon 72(arg/arg)) to obtain primary embryonic fibroblasts polymorphic at codon 72 or homozygous for codon 72(pro). The fibroblasts, cultured under standard conditions, immortalized within 12 weeks and acquired p53 mutations similarly to Hupki codon 72(arg/arg) cells investigated previously. Sequencing of human p53 exons 4-9 in immortalized cultures revealed missense mutations found repeatedly in human tumours. In cell lines ensuing from benzo(a)pyrene-treated cultures the combined p53 mutation pattern from experiments with the 3 codon 72 genotypes showed a predominance of strand-biased G to T transversions (18 of 36 mutations), and mutations recurring at smokers' lung tumour hotspot codons 157 and 273, supporting involvement of tobacco carcinogens in shaping the mutation signature in lung cancers of smokers. Mutations in cell lines from unexposed cultures did not cluster at these codons and G to T transversions were uncommon (2 of 52 mutations) (Fisher's exact test P<0.0001). Most mutations (13/16) in cell lines derived from cells polymorphic at codon 72 were found on the proline allele, with loss of the arginine allele.

Dietary fish oil and pectin enhance colonocyte apoptosis in part through suppression of PPARdelta/PGE2 and elevation of PGE3.

We have shown that dietary fish oil and pectin (FP) protects against radiation-enhanced colon cancer by upregulating apoptosis in colonic mucosa. To investigate the mechanism of action, we provided rats (n = 40) with diets containing the combination of FP or corn oil and cellulose (CC) prior to exposure to 1 Gy, 1 GeV/nucleon Fe-ion. All rats were injected with a colon-specific carcinogen, azoxymethane (AOM; 15 mg/kg), 10 and 17 days after irradiation. Levels of colonocyte apoptosis, prostaglandin E(2) (PGE(2)), PGE(3), microsomal prostaglandin E synthase-2 (mPGES-2), total beta-catenin, nuclear beta-catenin staining (%) and peroxisome proliferator-activated receptor delta (PPARdelta) expression were quantified 31 weeks after the last AOM injection. FP induced a higher (P < 0.01) apoptotic index in both treatment groups, which was associated with suppression (P < 0.05) of antiapoptotic mediators in the cyclooxygenase (COX) pathway (mPGES-2 and PGE(2)) and the Wnt/beta-catenin pathway [total beta-catenin and nuclear beta-catenin staining (%); P < 0.01] compared with the CC diet. Downregulation of COX and Wnt/beta-catenin pathways was associated with a concurrent suppression (P < 0.05) of PPARdelta levels in FP-fed rats. In addition, colonic mucosa from FP animals contained (P < 0.05) a proapoptotic, eicosapentaenoic acid-derived COX metabolite, PGE(3). These results indicate that FP enhances colonocyte apoptosis in AOM-alone and irradiated AOM rats, in part through the suppression of PPARdelta and PGE(2) and elevation of PGE(3). These data suggest that the dietary FP combination may be used as a possible countermeasure to colon carcinogenesis, as apoptosis is enhanced even when colonocytes are exposed to radiation and/or an alkylating agent.

Type-2 copper-containing enzymes.

Type-2 Cu sites are found in all the major branches of life and are often involved in the catalysis of oxygen species. Four type-2 Cu protein families are selected as model systems for review: amine oxidases, Cu monooxygenases, nitrite reductase/multicopper oxidase, and CuZn superoxide dismutase. For each model protein, the availability of multiple crystal structures and detailed enzymological studies provides a detailed molecular view of the type-2 Cu site and delineation of the mechanistic role of the Cu in biological function. Comparison of these model proteins leads to the identification of common properties of the Cu sites and insight into the evolution of the trinuclear active site found in multicopper oxidases.

Polymorphisms in the p53 pathway.

The p53 tumor suppressor gene continues to be distinguished as the most frequently mutated gene in human cancer; this gene can be found mutated in up to 50% of human tumors of diverse histological type. It is generally accepted that the ability of p53 to induce either growth arrest or programmed cell death in response to diverse stimuli underlies the powerful selection against this protein in the development of cancer. It is somewhat surprising, then, to find p53 and several target genes in this pathway containing polymorphisms that impair their function. The nature of these polymorphic variants, and the mechanism whereby they impair the function of the p53 pathway, are reviewed here-in. The impact of these polymorphisms on cancer risk and the efficacy of therapy are only now becoming unraveled. Of particular relevance in these efforts will be the generation of mouse models of polymorphic variants in p53 and its target genes. Equally important will be better-controlled human studies, where-in haplotypes for p53 (that is, combinations of different polymorphisms in the p53 gene) and for p53-target genes are taken into account, instead of analyses of single gene variants, which have largely predominated to date. Studies in both regards should shed light on an emerging area in cancer biology, the significance of inter-individual differences in genotype on cancer risk, prognosis, and the efficacy of cancer therapy.

Epizootiological survey of hantavirus among rodent species in Ningxia Hui Autonomous Province, China.

Hantaviral antibodies were detected in the sera from Apodemus (A.) agrarius and A. peninsulae captured in Ningxia province, China by several different serological diagnostic methods. A total of 409 sera from rodent and insectivore species were collected in 1999 and examined by indirect immunofluorescent antibody assay (IFA). Among them, 19 of 191 (9.9%) sera of A. agrarius and 1 of 13 (7.7%) sera of A. peninsulae were positive for hantaviral antibodies. The other species (Rattus norvegicus, Mus musculus, Cricetulus triton, and Sorex cylindricauda) were negative. The reaction pattern of positive serum was characterized as scattered and granular virus antigens in the cytoplasm of hantavirus infected Vero E6 cells. Some of the A. agrarius sera positive for hantavirus were further examined by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and the focus reduction neutralization test (FRNT). By WB, positive sera showed the same specific reaction pattern of baculovirus-expressed recombinant hantaviral nucleocapsid protein, as shown in hantavirus-immune serum. By ELISA, IFA-positive sera showed significantly higher optical densities (around 1.0) than the negative A. agrarius sera. Hantaan type hantavirus was neutralized with the positive sera. These results suggest that A. agrarius have hantavirus infection and may play a role as a reservoir animal for hantavirus in Ningxia Hui Autonomous Province, China.

Alternate substrate binding modes to two mutant (D98N and H255N) forms of nitrite reductase from Alcaligenes faecalis S-6: structural model of a transient catalytic intermediate.

High-resolution nitrite soaked oxidized and reduced crystal structures of two active site mutants, D98N and H255N, of nitrite reductase (NIR) from Alcaligenes faecalis S-6 were determined to better than 2.0 A resolution. In the oxidized D98N nitrite-soaked structures, nitrite is coordinated to the type II copper via its oxygen atoms in an asymmetric bidentate manner; however, elevated B-factors and weak electron density indicate that both nitrite and Asn98 are less ordered than in the native enzyme. This disorder likely results from the inability of the N delta 2 atom of Asn98 to form a hydrogen bond with the bound protonated nitrite, indicating that the hydrogen bond between Asp98 and nitrite in the native NIR structure is essential in anchoring nitrite in the active site for catalysis. In the oxidized nitrite soaked H255N crystal structure, nitrite does not displace the ligand water and is instead coordinated in an alternative mode via a single oxygen to the type II copper. His255 is clearly essential in defining the nitrite binding site despite the lack of direct interaction with the substrate in the native enzyme. The resulting pentacoordinate copper site in the H255N structure also serves as a model for a proposed transient intermediate in the catalytic mechanism consisting of a hydroxyl and nitric oxide molecule coordinated to the copper. The formation of an unusual dinuclear type I copper site in the reduced nitrite soaked D98N and H255N crystal structures may represent an evolutionary link between the mononuclear type I copper centers and dinuclear Cu(A) sites.

Characterization of in vitro and in vivo antiviral activity of lactoferrin and ribavirin upon hantavirus.

Mechanisms of anti-hantaviral activities of bovine lactoferrin (LF) and ribavirin (Rbv) were investigated. Hantavirus focus formation at 48 hr was 15% of the control in cells treated with 400 microg/ml LF for 1 hr at 37 degrees C prior to viral infection. Post infection treatment with 100 microg/ml Rbv also inhibited the focus formation to 2.5% of the control. Combined LF pre- and Rbv post-infection treatment completely inhibited focus formation. Viral glycoprotein (G2) and nucleocapsid protein (NP) syntheses were delayed in LF pretreated cells up to 24 hr post infection (hpi) but became comparable to the control by 48 hpi. Further, LF inhibited viral shedding at 24 hpi but did not inhibit shedding after 48 hpi. However, Rbv was able to inhibit synthesis of viral proteins, (+) and (-) strand RNAs also inhibited viral shedding after 24 hr. These results suggest that LF inhibits viral adsorption to cells, while Rbv inhibits viral RNA synthesis. For in vivo trials of LF and Rbv, LF pre- and Rbv post-treatment were evaluated in suckling mice infected with hantavirus, of which 7% survived. LF concentrations of 40 and 160 mg/kg administered prior to viral challenge improved survival rates to 15% and 70%, respectively for single administration and 85% and 94%, respectively, for double administration. Rbv concentrations of 25 and 50 mg/kg gave survival rates of 68% and 81%, respectively. This suggests that both LF and Rbv are efficacious in hantavirus infection in vivo.

Efficacy of an energy conservation course for persons with multiple sclerosis.

OBJECTIVE: To evaluate the efficacy of an energy conservation course on fatigue impact, self-efficacy, and quality of life (QOL) for persons with multiple sclerosis (MS). DESIGN: Repeated measures with control and experimental interventions conducted during a 19-week study. SETTING: Community-based treatment center. PARTICIPANTS: A convenience sample of 54 individuals from 79 community-dwelling volunteers with fatigue secondary to MS. INTERVENTION: A 6-session, 2-hr/wk energy conservation course taught by occupational therapists for groups of 8 to 10 participants. MAIN OUTCOME MEASURES: Fatigue Impact Scale (self-report measure of fatigue impact on cognitive, physical, social functions), Self-Efficacy Gauge (self-report measure of confidence in ability to perform specific behaviors), and Medical Outcomes Study Short-Form Health Survey (QOL measure). RESULTS: Participants reported, as predicted, significantly less fatigue impact, increased self-efficacy, and improved QOL (ie, 3 of 4 subscales expected to improve). There were no significant differences, as predicted, in any of the dependent variables after the control (ie, support group) and no intervention periods. CONCLUSION: Results provide strong evidence for the efficacy of this energy conservation course for persons with MS.

Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution.

The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi flavin reductase and the NADPH-dependent nitro/flavin reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.