Identification of N-homocysteinylated apolipoprotein AI in normal human serum

Background: In human serum, a portion of homocysteine (Hcy) exists as an N-linked form to the {varepsilon}-amino group of protein lysine residues. N-homocysteinylated proteins differ structurally and functionally from native proteins. The present study strives to develop detection and potential semi-quantification methods for N-homocysteinylated apolipoprotein AI (N-Hcy-apoAI) in human serum.Methods: Serum treated with or without cysteamine was supplied to isoelectric focusing (IEF) followed by an immunoblot using an anti-apoAI antibody. Cysteamine treatment increased the isoelectric point for N-Hcy-apoAI, but not for unmodified apoAI, due to the presence of -SH group(s) derived from Hcy and the absence of a cysteine residue in the apoAI molecule. N-Hcy-apoAI was semi-quantified from the scanned immunoblot pattern via a computer.Results: After cysteamine treatment, N-Hcy-apoAI in the serum was identified by IEF at the position with a higher pI value compared with intact apoAI. The reproducibility (between assays) of the semi-quantification method was 19.1% CV (coefficient of variation) for an average ratio 5.9% of N-Hcy-apoAI to the whole apoAI in the serum. Approximately 1.0–7.4% of apoAI was N-homocysteinylated in the serum obtained from 27 healthy subjects. Neither the ratio of N-Hcy-apoAI nor its concentration, calculated by total apoAI concentration, indicated correlation with the so-called total (free and S-linked) Hcy concentration.Conclusions: We directly found that a portion of apoAI in the serum undergoes homocysteinylation in an N-linkage manner, and used this to develop a potential semi-quantification method for N-Hcy-apoAI

Self-reported pediatricians' management of the well-appearing young child with fever without a source: first survey in an European country in the anti-pneumococcal vaccine era

<p>Abstract</p> <p>Background</p> <p>Recent studies suggest a substantially reduced risk of invasive bacterial infection in children vaccinated with heptavalent pneumococcal conjugate vaccine (PCV). To investigate whether the introduction of PCV might affect clinical decision making, we conducted a cross-sectional survey aimed at Italian Pediatric physicians.</p> <p>Results</p> <p>The study included 348 (46.5%) primary care pediatricians; 251 (36.4%) hospital pediatricians, and 139 (20.1%) pediatric residents. In an hypothetical scenario, a well-appearing 12-month-old child with fever without source would be sent home with no therapy by 60.7% (419/690) of physicians if the child was not vaccinated with PCV. The proportion increased to 74.2% (512/690) if the child had received PCV (P < 0.0001). Also, physicians would obtain blood tests less frequently in the vaccinated than in unvaccinated children (139/690 [20.1%] <it>vs</it>. 205/690 [29.7%]; P < 0.0001), and started empiric antibiotic therapy less frequently (3.0% <it>vs</it>. 7.5%; P < 0.0001). In the hypothetical event that white blood cell count was 17,500/μL, a significantly lower proportion of physicians would ask for erythrocyte sedimentation rate (P < 0.017), C reactive protein (P < 0.0001), blood culture (P = 0.022), and urine analysis or dipstick (P = 0.028), if the child had received PCV. Only one third of participants routinely recommended PCV.</p> <p>Conclusion</p> <p>Our data suggest that implementation of educational programs regarding the proper management of the febrile child is needed.</p

Criteria for Pediatric Sepsis-A Systematic Review and Meta-Analysis by the Pediatric Sepsis Definition Taskforce

OBJECTIVE: To determine the associations of demographic, clinical, laboratory, organ dysfunction, and illness severity variable values with: 1) sepsis, severe sepsis, or septic shock in children with infection and 2) multiple organ dysfunction or death in children with sepsis, severe sepsis, or septic shock. DATA SOURCES: MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials were searched from January 1, 2004, and November 16, 2020. STUDY SELECTION: Case-control studies, cohort studies, and randomized controlled trials in children greater than or equal to 37-week-old postconception to 18 years with suspected or confirmed infection, which included the terms "sepsis," "septicemia," or "septic shock" in the title or abstract. DATA EXTRACTION: Study characteristics, patient demographics, clinical signs or interventions, laboratory values, organ dysfunction measures, and illness severity scores were extracted from eligible articles. Random-effects meta-analysis was performed. DATA SYNTHESIS: One hundred and six studies met eligibility criteria of which 81 were included in the meta-analysis. Sixteen studies (9,629 patients) provided data for the sepsis, severe sepsis, or septic shock outcome and 71 studies (154,674 patients) for the mortality outcome. In children with infection, decreased level of consciousness and higher Pediatric Risk of Mortality scores were associated with sepsis/severe sepsis. In children with sepsis/severe sepsis/septic shock, chronic conditions, oncologic diagnosis, use of vasoactive/inotropic agents, mechanical ventilation, serum lactate, platelet count, fibrinogen, procalcitonin, multi-organ dysfunction syndrome, Pediatric Logistic Organ Dysfunction score, Pediatric Index of Mortality-3, and Pediatric Risk of Mortality score each demonstrated significant and consistent associations with mortality. Pooled mortality rates varied among high-, upper middle-, and lower middle-income countries for patients with sepsis, severe sepsis, and septic shock (p < 0.0001). CONCLUSIONS: Strong associations of several markers of organ dysfunction with the outcomes of interest among infected and septic children support their inclusion in the data validation phase of the Pediatric Sepsis Definition Taskforce

Pandemic A/H1N1v influenza 2009 in hospitalized children: a multicenter Belgian survey

<p>Abstract</p> <p>Background</p> <p>During the 2009 influenza A/H1N1v pandemic, children were identified as a specific "at risk" group. We conducted a multicentric study to describe pattern of influenza A/H1N1v infection among hospitalized children in Brussels, Belgium.</p> <p>Methods</p> <p>From July 1, 2009, to January 31, 2010, we collected epidemiological and clinical data of all proven (positive H1N1v PCR) and probable (positive influenza A antigen or culture) pediatric cases of influenza A/H1N1v infections, hospitalized in four tertiary centers.</p> <p>Results</p> <p>During the epidemic period, an excess of 18% of pediatric outpatients and emergency department visits was registered. 215 children were hospitalized with proven/probable influenza A/H1N1v infection. Median age was 31 months. 47% had ≥ 1 comorbid conditions. Febrile respiratory illness was the most common presentation. 36% presented with initial gastrointestinal symptoms and 10% with neurological manifestations. 34% had pneumonia. Only 24% of the patients received oseltamivir but 57% received antibiotics. 10% of children were admitted to PICU, seven of whom with ARDS. Case fatality-rate was 5/215 (2%), concerning only children suffering from chronic neurological disorders. Children over 2 years of age showed a higher propensity to be admitted to PICU (16% vs 1%, p = 0.002) and a higher mortality rate (4% vs 0%, p = 0.06). Infants less than 3 months old showed a milder course of infection, with few respiratory and neurological complications.</p> <p>Conclusion</p> <p>Although influenza A/H1N1v infections were generally self-limited, pediatric burden of disease was significant. Compared to other countries experiencing different health care systems, our Belgian cohort was younger and received less frequently antiviral therapy; disease course and mortality were however similar.</p

Macrophage populations and expressions of regulatory proinflammatory factors in the rat meninx under lipopolysaccharide treatment in vivo and in vitro

Macrophages play important roles in host defense mechanisms. In the brain, besides microglial cells, meningeal macrophages are present. However, the pathobiological characteristics of meningeal macrophages in rats remain to be investigated. In normal meninx, immunohistochemically, macrophages reacting to CD163 (macrophage scavenger receptor) and major histocompatibility complex (MHC) class II-expressing cells (involving activated macrophages or dendritic cells) were sporadically seen without age-dependent changes. Injection of lipoplysaccharide (LPS) (5 μg; Escherichia coli) into the cerebrum increased the number of anti-CD68-positive macrophages (with greater phagocytic activity) in the meninx, with a peak at 12 h during observation period until 48 h; MHC class IIexpressing cells showed a gradual increase in number from 3 h after injection; however, anti-CD163-positive macrophages did not show significant change. In in vitro studies, LPS (0, 0.02, 0.05, 0.5, 5, 50 and 100 μg/ml) was added to KMY-1 or KMY-2 cells, both of which had been established from a rat malignant meningioma. KMY-1 originally reacted to CD163, but LPS addition at 0.5 μg/ml and greater concentrations decreased the anti- CD163-positive cell number and instead increased the anti-CD68-positive cell number. LPS-treated KMY-2 increased the anti-CD163-positive cell number at 0.05 and 0.5 μg/ml. By RT-PCR methods, LPS (0, 0.5, 5, 50, and 100 μg/ml)-treated KMY-1 and KMY-2 showed an increase in mRNA of monocyte chemoattractant protein- 1 (MCP-1, a chemokine), and LPS-treated KMY-2 increased mRNA of nerve growth factor (NGF, an immunological effecter). Collectively, under LPS treatment, macrophages with heterogeneous functions appear in rat meninx; rat meninx-forming cells may be involved in pathogenesis of meningeal inflammation by expressing different immunophenotypes and by producing regulatory proinflammatory factors such as MCP-1 and NGF