First-line treatment and overall survival in EGFR mutation-positive advanced non-small cell lung cancer: a national cohort study.

OBJECTIVE: Non-squamous non-small cell lung cancer (NSCLC) is the first leading cause of cancer-related deaths in Taiwan. This study aimed at evaluating the effectiveness of first-line targeted therapy for advanced epidermal growth factor receptor (EGFR) mutation-positive non-squamous NSCLC in Taiwan. PATIENTS AND METHODS: This was a real-world, retrospective, observational study of patients diagnosed with advanced non-squamous NSCLC (N=63,248). Between 2011 and 2019, 19,458 patients received targeted therapy and 22,994 patients received chemotherapy alone; between 2002 and 2010, 20,796 patients received chemotherapy alone. Overall survival (OS) was determined. RESULTS: The median OS for patients treated with first-line targeted therapy (22.9 months) was longer than that of patients receiving chemotherapy alone (11.7 months). HR: 0.521, log-rank test, p<0.001. CONCLUSIONS: These data represent the potential survival outcomes of Taiwanese patients with advanced EGFR mutation-positive non-squamous NSCLC in clinical practice.

Real-world results of immune checkpoint inhibitors from the Taiwan National Health Insurance Registration System.

OBJECTIVE: Immune checkpoint inhibitors (ICIs) are a major advance in cancer treatment, but their payment benefits are unclear, resulting in financial risk. In Taiwan, the National Health Insurance Administration (NHIA) has adapted risk-sharing mechanisms to cover ICIs by collecting and assessing real-world evidence, such as case registration data, to adjust benefit packages for each medication, increase payment benefits of ICIs, and enable national health insurance sustainability. PATIENTS AND METHODS: This nationwide, multicenter, retrospective cohort study assessed the real-world use, effectiveness, and safety of ICIs reimbursed by the NHIA for treating multiple advanced cancers in Taiwan. We obtained data mainly from the NHIA Immune Checkpoint Inhibitor Registry Database. RESULTS: Between April 1, 2019, and March 31, 2020, 1644 patients received at least one dose of ICIs. The overall response rate (RR) was 29.1%. The metastatic urothelial carcinoma of patients ineligible for chemotherapy showed the highest RR. The estimated median progression-free survival (PFS) was 2.8 months (95% confidence interval [CI]=2.7-3 months), and renal cell carcinoma showed the longest PFS. The median PFS was reached in patients with most cancers except classic Hodgkin's lymphoma, which had a small sample size. The estimated survival probability was 50%. CONCLUSIONS: Under the national registration tracking system, Taiwan's high-cost drug policy has enabled access to new medicines and maximized patient benefits.

Asp159 is a critical core amino acid of an IgE-binding and cross-reactive epitope of a dust mite allergen Der f 7.

Der f 7 and Der p 7 are important house dust mite allergens with known structure and suggested biological function recently. However, their IgE-binding determinants remain unknown. The purpose of this study is to identify the IgE-reactive epitopes of Der f 7 and the determinants of IgE-mediated cross-reactivity between Der f 7 and Der p 7. IgE-reactive determinants were identified by immunodot blot inhibition using synthetic overlapping peptides, allergen mutants, and a Der f 7 structural model. Our results showed that synthetic peptides with sequence (156)SILDP(160) on Der f 7 bind IgE in two of the 30 asthmatic serum samples tested. Recombinant Der f 7 I157A, L158A, or D159A mutants have reduced IgE-binding activity. Inhibition experiments confirmed Asp159 as a critical core residue for IgE-binding. Among Der p 7, Der f 7 and Der f 7 mutants with single substitution between residues 156 and 160, only the D159A mutant cannot inhibit significantly IgE-binding against Der p 7. Therefore, Asp159 contributes to IgE-mediated cross-reactivity between Der f 7 and Der p 7. The structural model constructed for Der f 7 suggests that the IgE-binding epitope forms a loop-like structure on the surface of the molecule. In conclusion, Asp 159 is a critical core residue of an IgE-binding and IgE-mediated cross-reactive epitope (156)SILDP(160) of Der f 7. Results obtained from this study provide more information on molecular and structural features related to allergenicity, underlying basis of IgE cross-reactivity between allergens, and in designing safer immunotherapy.

Transaldolases are novel and immunoglobulin E cross-reacting fungal allergens.

BACKGROUND: Mould-induced atopic respiratory diseases are a worldwide problem. Characterization of fungal allergens is of major clinical importance. OBJECTIVE: We identified a novel transaldolase family allergen of Cladosporium and Penicillium species. METHODS: Fungal allergens were identified by immunoblotting, peptide mass mapping and partial sequencing, cDNA cloning and IgE epitope mapping. RESULTS: A 36.5 kDa IgE-binding component in a partially purified C. cladosporioides preparation was identified. Mass spectrometric analyses suggest that this novel IgE-reacting allergen is a transaldolase. A corresponding full-length 1246 bp cDNA encoding a polypeptide of 325 residues was isolated. The newly identified transaldolase allergen has been designated as Cla c 14.0101. The cDNA encoding the Pencillium chrysogenum transaldolase was isolated by RT-PCR according to the cDNA sequence encoding a P. chrysogenum Wisconsin 54-1255 hypothetical protein. The purified rCla c 14.0101 protein reacted with IgE antibodies in 10 (38%) of 26 Cladosporium cladosporioides-sensitized asthmatic patients. Nine of the 10 rCla c 14.0101-positive sera have IgE binding against the recombinant Penicillium transaldolase (rPen ch 35.0101). Among the eight fungal transaldolase-positive sera tested, three showed IgE binding against the recombinant human transaldolase. To determine cross-reactivity between the Cladosporium and Penicillium fungi, IgE cross-reactivity was detected between these two fungal transaldolase allergens by inhibition assays. Both the N- and the C-terminal fragments of Cla c 14.0101 were recognized by IgE antibodies. The C-terminal IgE-reacting determinant was narrowed down to a region encompassing Thr257 to Ser278 of Cla c 14.0101. It was mapped onto a loop-like structure of a 3D model constructed for Cla c 14.0101. CONCLUSION AND CLINICAL RELEVANCE: We identified transaldolase as a novel and IgE cross-reactive allergen family of C. cladosporioides and P. chrysogenum. In addition, an IgE-reacting fragment (Thr257 to Ser278) was pinpointed to a loop-like structure on Cla c 14.0101. Results obtained provide important information in clinical mould allergy.

Pen ch 13 allergen induces secretion of mediators and degradation of occludin protein of human lung epithelial cells.

BACKGROUND: Alkaline serine proteases from six prevalent airborne Penicillium and Aspergillus species have been identified as a group of major allergens (group 13). After entering human airways, the allergens are in initial contacts with respiratory epithelial cells. The purpose of this study is to investigate interactions between the Pen ch 13 allergen from P. chrysogenum and human lung epithelial cells. METHODS: A549 cells, 16HBE14o- cells and primary cultures of human bronchial epithelial cells (HBEpC) were exposed to purified Pen ch 13 and mediators released into culture supernatants were assayed with enzyme-linked immunosorbent assay (ELISA) kits. Cleavage of occludin in 16HBE14o- cells was analysed by immunofluorescent staining of whole cells and immunoblot analysis of whole cell extracts. Fragments generated by incubating Pen ch 13 and a synthetic peptide carrying the occludin sequence were analysed by mass spectrometry. RESULTS: Pen ch 13 induced productions of prostaglandin-E2 (PGE2), interleukin (IL)-8 and transforming growth factor (TGF)-beta1 by A549 cells, 16HBE14o- cells and primary cultures of HBEpC. The protease activity of Pen ch 13 is needed for the induction of PGE2 IL-8, TGF-beta1 and cyclo-oxygenase (COX)-2 expression. A tight junction protein occludin of 16HBE14o- cells can be cleaved by Pen ch 13 at Gln202 and Gln211 which are within the second extracellular domain of the protein. CONCLUSION: Pen ch 13 may contribute to asthma by damaging the barrier formed by the airway epithelium and stimulating the release of mediators that orchestrate local immune responses and inflammatory process from HBEpC.

cDNA cloning and immunologic characterization of a novel EF-1beta allergen from Penicillium citrinum.

BACKGROUND: We have identified previously that Penicillium citrinum is the most prevalent Penicillium species in the Taipei area. It is important to delineate the whole spectrum of allergenic components of this prevalent airborne fungus. The purpose of this study was to identify novel P. citrinum allergens through molecular cloning of allergen genes using a cDNA library of P. citrinum and sera from patients with bronchial asthma. METHODS: A lambda-Uni-ZAP XR-based cDNA library of P. citrinum was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and heterologously expressed in Escherichia coli. The frequency of IgE-binding to the expressed protein and the IgE reactivity to allergen subunits were analyzed by immunoblotting. RESULTS: An IgE-reactive cDNA clone (clone B) was isolated by plaque immunoassay. The cDNA insert is 876-bp long and encodes a 228-amino acid polypeptide with a calculated molecular mass of 25 035 Da. Protein database search with the deduced clone B sequence revealed that 121 (53%) and 82 (36%) of the 228 amino acids were identical to those of the elongation factor 1-beta (EF-1beta) proteins from the yeast Saccharomyces cerevisiae and the parasite Echinococcus granulosus, respectively. His-tagged recombinant clone B proteins were constructed and expressed in E. coli. Seven (8%) of the 92 serum samples from patients with bronchial asthma showed IgE-binding to the recombinant clone B protein. Among these seven positive sera, five demonstrated IgE-binding to the C-terminal fragment (aa 119-228) while the other two sera showed IgE reactivity to the N-terminal fragment (aa 1-118) of this newly identified EF-1betaPenicillium allergen. CONCLUSIONS: A novel P. citrinum allergen (Pen c 24) was identified and characterized in the present study. Results obtained provide more information about allergens of prevalent airborne fungi and a basis to understand more about the IgE responses in human atopic disorders and in parasitic infections.

Molecular and structural analysis of immunoglobulin E-binding epitopes of Pen ch 13, an alkaline serine protease major allergen from Penicillium chrysogenum.

BACKGROUND: Through proteomic and genomic approaches we have previously identified and characterized an alkaline serine protease that is a major allergen (88% frequency of IgE binding) of Penicillium chrysogenum (Pen ch 13). OBJECTIVE: The aim of the present study is to identify the linear IgE-binding epitopes of Pen ch 13. METHODS: IgE-binding regions were identified by dot-blot immunoassay using 11 phage-displayed peptide fragments spanning the whole molecule of Pen ch 13. The minimal epitope requirements for IgE binding were further defined with overlapping peptides synthesized on derivatized cellulose membranes using SPOTs technology. The critical residues on the immunodominant epitopes were mapped through site-directed mutagenesis. The locations of the IgE epitopes identified were correlated with a three-dimensional structure of Pen ch 13. RESULTS: IgE antibodies in 35 serum samples reacted with at least one of the 11 peptide fragments of Pen ch 13. Peptide f-2n (residues 31-61) showed a high-intensity and the highest frequency (77%) of IgE binding. The frequencies of IgE binding to peptide f-4 (residues 93-133), f-1 (residues 1-37) and f-7 (residues 168-206) were 51%, 34% and 31%, respectively. SPOTs assay narrowed down the region of IgE binding of f-2n to residues 48-55 (GHADFGGR). Three, two and one epitope(s) that are four to nine amino acids in length, within f-4, f-1 and f-7, respectively, were found. Site-directed mutagenesis of Pen ch 13 revealed that substitution of His49 and/or Phe52 on Pen ch 13 with methionine resulted in proteins with drastic loss of IgE binding in seven sera tested. Proteins with amino acid replacements at residues 15-18 (RISS), or at residues 112 (I) and 116 (D) have lower IgE-binding reactivity in one of the two patient's sera tested. Substituting residues 117 (W), 119 (V) and 120 (K) also block most of the IgE binding in one of the two patient's sera tested. In addition, replacing residues 203 (V) and 204 (D) along with a deletion at residue 206 (Y) diminished the IgE binding in two serum samples tested. A model was constructed based on the structure of P. cyclopium subtilisin protease that has >90% (256 out of 283 amino acids) sequence identity with Pen ch 13. The major epitope (GHADFGGR) on Pen ch 13 formed a loop-like structure and was located at the surface of the allergen. CONCLUSIONS: Several linear IgE-reactive epitopes and their critical core amino acid residues were identified for the Pen ch 13 allergen. The major linear IgE-binding epitope, 48GHADFGGR55, formed a loop-like structure at the surface of the allergen. Substitution of His49 and/or Phe52 with methionine significantly reduced IgE-binding to Pen ch 13. Mapping of these results on a 3D model of the allergen provides valuable information about the molecular basis of allergenicity for Pen ch 13 and for designing specific immunotherapeutics.

Causes for the evolution of case management and the development of a working model in an acute care hospital in Singapore.

Case management is a collaborative process which assesses, plans, implements, co-ordinates, monitors and evaluates options and services to meet an individual's health needs. The case manager performs a key role in ensuring that collaboration and communication between the multi-disciplinary healthcare team and patient is maintained, and that quality clinical outcomes are achieved and the patients reach an optimal level of wellness and function. Patients that can benefit from case management in an acute hospital setting are frequently elderly and have complex medical and care issues arising out of changes in functional status. The components of a successful case management system include identifying a suitable model for implementation and recruiting the correct persons to function as case managers. A defined assessment and recruitment guideline will ensure that appropriate patients are placed on case management. Communication and collaboration with all stakeholders in formulating a patient's plan of care are essential. The subsequent implementation and monitoring of the plan is crucial, as variances that occur must be adequately managed in order to ensure that a satisfactory clinical outcome is achieved without inappropriate expenditure of resources. The barriers to a successful case management programme can result from inappropriate case loads and failure of the other healthcare team members to understand and appreciate the case manager's role and functions. However, this can be overcome by education and using outcome data to demonstrate the effectiveness of case management.

A quantitative assay for measuring human foamy virus using an established indicator cell line.

In order to improve the accuracy for detecting human foamy virus (HFV), an indicator cell line was established by co-transfecting baby hamster kidney-21 cells with two plasmids: one containing a G418 antibiotic resistance marker and the other including the luc gene which was placed downstream of the inducible HFV long terminal repeat promoter (from -533 to +20). Among 11 independent subclones, IdB14 was found to be stable with a low basal level of luciferase activity. Although the changes in luciferase activity in infected clones showed time-dependency and peaked at day 8, it is possible to differentiate infected and uninfected cells on day 2. The sensitivity of the foamy virus activated luciferase (FAL) assay was 400 times higher than the end-point syncytium formation by TCID(50). The HFV LTR promoter in the IdB14 cell line was specific for this virus. Moreover, a linear relationship was found between the MOI and the activated intensity of luciferase expression. These findings suggest that the FAL assay using the IdB14 indicator cell line is a simple and useful technique for rapid diagnosis and quantitation of active HFV infection.

Prophylactic esmolol infusion for the control of cardiovascular responses to extubation after intracranial surgery.

INTRODUCTION: Emergence from general anaesthesia and extubation are often accompanied by significant surges in heart rate and blood pressure. To document these changes and the efficacy of low-dose beta-blocker infusions in ameliorating these rises, we undertook a descriptive dose-ranging study comparing the use of esmolol to placebo in patients emerging from neuro-anaesthesia. MATERIALS AND METHODS: Thirty-six patients undergoing intracranial surgery were randomised to receive saline, esmolol 100 micrograms/kg/min or 200 micrograms/kg/min infusions. The number of patients developing severe hypertension or tachycardia in each group was compared using Fisher's exact test. RESULTS: Systolic blood pressure (SBP) and heart rate (HR) increased in all 3 groups during emergence and peaked at extubation. The proportion of patients with severe tachycardia or hypertension was reduced from 92% in the placebo group to 40% (P = 0.02) and 8% (P = 0.001) in the low and intermediate dose esmolol groups, respectively. Results were better in the intermediate dose group but the difference was not statistically significant. Two patients from the esmolol infusion groups required supplemental medication for bradycardia. CONCLUSION: Severe hypertension or tachycardia occurs in 92% of patients during extubation following neuro-anaesthesia and warrants the consideration of routine prophylaxis. Prophylactic esmolol infusion for the control of haemodynamic disturbances during extubation is feasible and safe. A modest level of obtundation is evident at 100 micrograms/kg/min but a rate of 200 micrograms/kg/min may prove to be more effective.

Intravenous sedation for upper gastrointestinal endoscopy: Midazolam versus propofol.

Propofol was compared with Midazolam for sedation during upper gastrointestinal endoscopy in a randomised, double blind study. Both drugs were equally acceptable to endoscopists and patients. There was significant oxygen desaturation after sedation and during endoscopy (p < 10(-6)). Significantly more patients in the propofol group could remember the diagnosis which was revealed to them immediately after the gastroscopy (p < 0.001).