An Open Sandwich Immunochromatography for Non-competitive Detection of Small Antigens.

Immunochromatography assay is an easy and rapid on-site detection method. However, conventional sandwich immunochromatographies using two antibodies can only detect target molecules above a threshold size. Small molecules below 1000 in molecular weight are usually detected using competitive immunoassay. However, competitive immunoassay is not suitable for visual detection of low concentration samples. Based on the principles of open sandwich immunoassay, which detects small molecules via interchain interaction of separated variable region fragments (VH and VL) from a single antibody, we developed non-competitive open sandwich immunochromatography. Bone Gla protein (BGP)-C7, a peptide containing the seven C-terminal amino acids of human osteocalcin, was selected as the target. By using VL fragments fixed on a nitrocellulose membrane, and colored cellulose bead-labeled VH fragments, we specifically detected 10 ng/mL of BGP-C7. This is the first report of open sandwich immunochromatography, which is an easy and rapid method for on-site, signal-on detection of small molecules.

Does respiratory virus coinfection increases the clinical severity of acute respiratory infection among children infected with respiratory syncytial virus?

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory infection in children less than 5 years of age. The impact of non-RSV respiratory virus coinfection on the severity of RSV disease is unknown. METHODS: This hospital-based prospective study was conducted in Nagasaki, Japan, on all children less than 5 years of age with acute respiratory infection (ARI) who had undergone a rapid RSV diagnostic test between April 2009 and March 2010. Thirteen respiratory viruses were identified from nasopharyngeal swab samples using a multiplex polymerase chain reaction; polymerase chain reaction-positive samples were considered as confirmed respiratory virus infections. The cases were classified into 3 categories (pneumonia, moderate-to-severe nonpneumonic ARI and mild ARI) according to the findings of the chest radiograph and the hospitalization records. RESULTS: Among 384 cases enrolled, 371 were eligible for analysis, of whom 85 (23%) were classified as pneumonia cases; 137 (37%) as moderate-to-severe nonpneumonic ARI cases and 162 (40%) as mild ARI cases. RSV was detected in 172 cases (61.6%), and 31 cases (18.0%) had double or triple infections with other respiratory viruses. RSV infection was more frequently observed in pneumonia cases (odds ratio [OR]: 2.3; 95% confidence interval [CI]: 1.31-3.9) and moderate-to-severe nonpneumonic ARI cases (OR: 2.95; 95% CI: 1.82-4.78) than in mild ARI cases. The association with moderate-to-severe nonpneumonic ARI cases was stronger with RSV/non-RSV respiratory virus coinfection (adjusted OR: 4.91; 95% CI: 1.9-12.7) than with RSV single infection (adjusted OR: 2.77; 95% CI: 1.64-4.7). CONCLUSIONS: Non-RSV respiratory virus coinfection is not uncommon in RSV-infected children and may increase the severity of RSV disease.

Estimating the influenza vaccine effectiveness against medically attended influenza in clinical settings: a hospital-based case-control study with a rapid diagnostic test in Japan.

BACKGROUND: Influenza vaccine effectiveness (VE) studies are usually conducted by specialized agencies and require time and resources. The objective of this study was to estimate the influenza VE against medically attended influenza using a test-negative case-control design with rapid influenza diagnostic tests (RIDT) in a clinical setting. METHODS: A prospective study was conducted at a community hospital in Nagasaki, western Japan during the 2010/11 influenza season. All outpatients aged 15 years and older with influenza-like illnesses (ILI) who had undergone RIDT were enrolled. A test-negative case-control design was applied to estimate the VEs: the cases were ILI patients with positive RIDT results and the controls were ILI patients with negative RIDT results. Information on patient characteristics, including vaccination histories, was collected using questionnaires and medical records. RESULTS: Between December 2010 and April 2011, 526 ILI patients were tested with RIDT, and 476 were eligible for the analysis. The overall VE estimate against medically attended influenza was 47.6%, after adjusting for the patients' age groups, presence of chronic conditions, month of visit, and smoking and alcohol use. The seasonal influenza vaccine reduced the risk of medically attended influenza by 60.9% for patients less than 50 years of age, but a significant reduction was not observed for patients 50 years of age and older. A sensitivity analysis provided similar figures. CONCLUSION: The test-negative case-control study using RIDT provided moderate influenza VE consistent with other reports. Utilizing the commonly used RIDT to estimate VE provides rapid assessment of VE; however, it may require validation with more specific endpoint.

Engineering unusual amino acids into peptides using lantibiotic synthetase.

Alteration of protein structure and function by introducing unusual amino acids has great potential to develop new biological tool and to produce novel therapeutic agents. Lantibiotics produced by Gram-positive bacteria are ribosomally synthesized and post-translationally modified antimicrobial peptides. The modification enzyme involved in lantibiotic biosynthesis can catalyze the formation of unusual amino acids in the nascent lantibiotic prepeptide. Here, a novel methodology on the lantibiotic nukacin ISK-1 is described for engineering unusual amino acid residues into hexa-histidine-tagged (His-tagged) prepeptide NukA by the modification enzyme NukM in Escherichia coli. Co-expression of His-tagged NukA and NukM, purification of the resulting His-tagged prepeptide by affinity chromatography, and subsequent mass spectrometry analysis show that the prepeptide is converted into a postulated peptide with decrease in mass which results from the formation of unusual amino acids such as dehydrated amino acid, lanthionine, or 3-methyl lanthionine at the expected positions. The modified prepeptide can be readily obtained by one-step purification. This strategy will thus be a simple and powerful tool for introducing unusual amino acid residues aimed at peptide engineering.

Effectiveness of pneumococcal polysaccharide vaccine against pneumonia and cost analysis for the elderly who receive seasonal influenza vaccine in Japan.

To determine the clinical efficacy and cost-saving effect of pneumococcal polysaccharide vaccine (PPV) against community-acquired pneumonia (CAP), an open-label, randomized clinical trial was conducted involving 786 Japanese subjects older than 65 years of age receiving a routine influenza vaccine during the 2-year period. Study subjects were randomly assigned to either a PPV group (n=394) or to a non-PPV group (n=392). The incidence, admission and the medical cost for all-cause pneumonia were compared between these two groups. PPV vaccination significantly reduced the incidence of admission for all-cause pneumonia for subjects older than 75 years of age (41.5%, P=0.039) and for those who had difficulty walking (62.7%, P=0.005), but not for all study subjects older than 65 years of age (P=0.183), for the 2-year period. The Kaplan-Meier survival curves for subjects who had difficulty walking free from all-cause pneumonia demonstrated a significant difference (P=0.0146) between the two groups. PPV vaccination significantly reduced medical costs for all study subjects during the first year period (P=0.027). Our present data demonstrated that PPV was effective for all-cause pneumonia for study subjects older than 75 years of age, although the effect was not significant for all study subjects older than 65 years of age.

Characterization of modification enzyme NukM and engineering of a novel thioether bridge in lantibiotic nukacin ISK-1.

The lantibiotic nukacin ISK-1 is an antimicrobial peptide containing unusual amino acids such as lanthionine and dehydrobutyrine. The nukacin ISK-1 prepeptide (NukA) undergoes posttranslational modifications, such as the dehydration and cyclization reactions required to form the unusual amino acids by the modification enzyme NukM. We have previously constructed a system for the introduction of unusual amino acids into NukA by coexpression of NukM in Escherichia coli. Using this system, we describe the substrate specificity of NukM by the coexpression of a series of NukA mutants. Our results revealed the following characteristics of NukM: (1) its dehydration activity is not coupled to its cyclization activity; (2) its dehydration activity is site-specific; (3) the length of the substrate is important for its dehydration activity. Furthermore, we succeeded in introducing a novel thioether bridge in NukA by replacing an unmodified Ser at position 27 with a Cys residue.

Binding specificity of the lantibiotic-binding immunity protein NukH.

NukH is a lantibiotic-binding immunity protein that shows strong binding activity against type A(II) lantibiotics. In this study, the binding specificity of NukH was analyzed by using derivatives of nukacin ISK-1, which is a type A(II) lantibiotic produced by Staphylococcus warneri ISK-1. Interactions between cells of Lactococcus lactis transformants expressing nukH and nukacin ISK-1 derivatives were analyzed by using a quantitative peptide-binding assay. Differences in the cell-binding rates of each nukacin ISK-1 derivative suggested that three lysine residues at positions 1 to 3 of nukacin ISK-1 contribute to the effective binding of nukacin ISK-1 to nukH-expressing cells. The binding levels of mutants with lanthionine and dehydrobutyrine substitutions (S11A nukacin(4-27) and T24A nukacin(4-27), respectively) to nukH-expressing cells were considerably lower than those of nukacin(4-27), suggesting that unusual amino acids play a decisive role in NukH recognition. Additionally, it was suggested that T9A nukacin(4-27), a mutant with a 3-methyllanthionine substitution, binds to NukH via an intermolecular disulfide bond after it is weakly recognized by NukH. We succeeded in the detection of specific type A(II) lantibiotics from the culture supernatants of various bacteriocin producers by using the binding specificity of nukH-expressing cells.

[A case of pulmonary nocardiosis with Nocardia beijingensis].

A 60-year-old woman seen at the National Hospital Organization Nagasaki Medical Center of Neurology with a cough and abnormal chest radiography was found in CT to have interstitial shadows in the bilateral lower lung fields. She was diagnosed with interstitial pneumonia and treated with steroids. Treatment was effective, and the predonisolone dosage was gradually tapered. When dosage was 17.5 mg/day, her chest Xray showed exacerbation. Cyclophosphamide at 50mg/day was added, and chest radiography improved. Two months later, her chest radiography showed infiltration with cavities in the left lung field. Although several antibiotics (sulbactam/cefoperazone, levofloxacin) were administered, no improvement was seen. Sputa on hospital day 60 showed the presence of gram-positive branched rods, identified as Nocardia beijingensis. We administered sulfamethoxazole/trimethoprim, meropenem and levofloxacin together, and shadows improved. With recurrent aggravation of interstitial pneumonia, however, new cavity shadows occurred in the bilateral lung due to Aspergillus fumigatus. Shadows worsened and she died of respiratory failure. Testing for pulmonary nocardiosis should be added to differential diagnosis procedures as an opportunistic infection in immune-compromised hosts.

[Case of fatal liver failure due to anti-tuberculous therapy].

A 65-year-old female was started anti-tuberculous therapy for her pulmonary tuberculosis on admission. Liver dysfunction had occurred on 33rd day after starting treatment. AST was elevated to 301 IU/L, and ALT was also elevated to 141 IU/L. Therefore, all medicated drugs were stopped. She had jaundice on 42nd day and liver failure deteriorated. She was medicated with steroids, but she died by liver failure on 64th day. This is a rare case of fatal liver failure due to antituberculous therapy.

[Case of pulmonary multi-drug resistant tuberculosis with left destroyed lung, treated with pneumonectomy].

A 31-year-old woman complained of cough and fever for 2 months. She was admitted to a hospital and was diagnosed as pulmonary tuberculosis. She received combination therapy with isoniazid, rifampicin, ethambutol, and pyrazinamide. As the drug susceptibility test revealed that the isolated strain was multi-drug resistant, the regimen was changed to pyrazinamide, ethionamide, cycloserine, enviomycin, and levofloxacin. The chemotherapy was not effective, so she received pneumonectomy for left destroyed lung. After surgical treatment, her sputa converted to negative for tubercle bacilli. Surgical treatment such as pneumonectomy is considered to be useful in a case of multi-drug resistant pulmonary tuberculosis.

Lanthionine introduction into nukacin ISK-1 prepeptide by co-expression with modification enzyme NukM in Escherichia coli.

We demonstrated lanthionine introduction into hexa-histidine-tagged (His-tagged) nukacin ISK-1 prepeptide NukA by modification enzyme NukM in Escherichia coli. Co-expression of nukA and nukM, purification of the resulting His-tagged prepeptide by affinity chromatography, and subsequent mass spectrometry analysis showed that the prepeptide was converted into a postulated peptide with decrease in mass of 72Da which resulted from dehydration of four amino acids. Characterization of the resultant prepeptide indicated the presence of unusual amino acids, such as dehydrated amino acid, lanthionine or 3-methyllanthionine, in its C-terminal propeptide moiety. The modified prepeptide encompassing the leader peptide attached to the post-translationally modified propeptide moiety was readily obtained by one-step purification. Our findings will thus be a powerful tool for introducing unusual amino acids aimed at peptide engineering and also helpful to provide new insight for further understanding of lanthionine-forming enzymes for lantibiotics.