From 1985 to 1987, Streptococcus pneumoniae isolates were collected from children under 5 years of age in the Asaro Valley, Papua New Guinea as part of a study on bacterial colonization and respiratory tract infections. Data on serogroup and colony morphology were collected to survey serogroups and associated colony morphologies present in the area and to assess whether colony morphology can be indicative of serogroup. In total, 5989 colonies were examined; serogroups 6, 10, 14, 15, 19, 23, 33, 34, 35 and nonserotypeable strains were the most common and accounted for 77% of all the colonies, with serogroups 6, 19 and 23 accounting for 48%. The majority of colonies displayed the typical draughtsman morphology, though serogroup 10 and non-serotypeable isolates most often displayed a raised colony morphology. Of the 15 mucoid colonies identified 73% were serotype 3, though only 29% of serotype 3 isolates were mucoid. Thus colony morphology is of limited value in identifying the pneumococcal serogroup/serotype apart from mucoid colonies, which are likely to be serotype 3.

We have previously described a novel artificial NFEV β-secretase (BACE1) cleavage site, which when introduced into the amyloid-β precursor protein (APP), significantly enhances APP cleavage by BACE1 in in vitro and cellular assays. In this study, we describe the identification and characterization of a single chain fragment of variable region (scFv), specific to the EV neo-epitope derived from BACE1 cleavage of the NFEV-containing peptide, and its conversion to IgG1. Both the scFv displayed on phage and EV-IgG1 show exquisite specificity for binding to the EV neoepitope without cross-reactivity to other NFEV containing peptides or WT-APP KMDA cleavage products. EV-IgG1 can detect as little as 0.3 nmol/L of the EV peptide. EV-IgG1 antibody was purified, conjugated with alkaline phosphatase and utilized in various biological assays. In the BACE1 enzymatic assay using NFEV substrate, a BACE1 inhibitor MRK-3 inhibited cleavage with an IC(50) of 2.4 nmol/L with excellent reproducibility. In an APP_NFEV stable SH-SY5Y cellular assay, the EC(50) for inhibition of EV-Aβ peptide secretion with MRK-3 was 236 nmol/L, consistent with values derived using an EV polyclonal antibody. In an APP_NFEV knock-in mouse model, both Aβ_EV40 and Aβ_EV42 peptides in brain homogenate showed excellent gene dosage dependence. In conclusion, the EV neoepitope specific monoclonal antibody is a novel reagent for BACE1 inhibitor discovery for both in vitro, cellular screening assays and in vivo biochemical studies. The methods described herein are generally applicable to novel synthetic substrates and enzyme targets to enable robust screening platforms for enzyme inhibitors.
Amino Acid Sequence ; Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; chemistry ; genetics ; Amyloid beta-Protein Precursor ; Animals ; Antibodies ; pharmacology ; Brain Chemistry ; drug effects ; Disease Models, Animal ; Enzyme Inhibitors ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Gene Knock-In Techniques ; Humans ; Inhibitory Concentration 50 ; Mice ; Molecular Sequence Data ; Single-Chain Antibodies ; pharmacology

OBJECTIVETo determine if vitamin D intake is associated with acute lower respiratory infections (ALRI) in children.

METHODSThe vitamin D intakes of children younger than 5 years of age admitted to hospital with either bronchiolitis or pneumonia were compared to an unmatched control group of the same age without respiratory infection. Caregivers of 197 children completed a questionnaire collecting information on demographic variables, ALRI risk factors and diet. Associations of ALRI with vitamin D intake and other ALRI risk factors were determined.

RESULTSThe mean vitamin D intake of children with ALRI was 48 IU/kg/d compared to 60 IU/kg/d in the control group. When controlling for age, ethnicity, socio-economic status, northern residence, breastfeeding, immunizations and smoking contact, children with a vitamin D intake of less than 80 IU/kg/d were greater than 4 times more likely to have ALRI compared to children with a vitamin D intake exceeding 80 IU/kg/d (OR=4.9; 95%CI: 1.5-16.4).

CONCLUSIONSA higher vitamin D intake than currently recommended might be needed to offer protection against diseases such as ALRI. Increased vitamin D supplementation could have important public health consequences, as bronchiolitis and pneumonia are the most common reasons for hospitalization in young children. (Full English version will be available online at www.amepc.org/tp.).

Acute Disease ; Bronchiolitis ; etiology ; Child, Preschool ; Female ; Humans ; Hydrogen-Ion Concentration ; Infant ; Infant, Newborn ; Male ; Pneumonia ; etiology ; Respiratory Tract Infections ; etiology ; prevention & control ; Vitamin D ; administration & dosage