Yen-Ta Lu

Clinical and Laboratory Features of Severe Acute Respiratory Syndrome Vis-A ` -Vis Onset of Fever*

Study objectives: Severe acute respiratory syndrome (SARS) is a rapidly progressive disease caused by a novel coronavirus (CoV) infection. However, the disease presentation is nonspecific. The aim of this study was to define clearly the presentation, clinical progression, and laboratory data in a group of patients who had SARS. Design: Retrospective observational study. Setting: A tertiary care medical center with 51 negative-pressure isolation rooms in Taipei, Taiwan. Patients: Fifty-three patients with SARS seen between April 27 and June 16, 2003. Results: Fever (ie, temperature > 38°C) was the most common symptom (98%) and the earliest. When admitted to the isolation unit of the hospital for observation, most patients reported nonspecific symptoms associated with their fever. Only two patients with preexisting illnesses had cough on the same day the fever began. Eventually, 39 patients (74%) developed cough, beginning at a mean (± SD) time of 4.5 ± 1.9 days after fever onset, and 35 patients (66%) had diarrhea beginning at a mean time of 6.0 ± 3.3 days after fever onset. Thirty-one patients (59%) had abnormal findings on chest radiographs on hospital admission, and all but 1 patient (98%) eventually developed lung infiltrates that were consistent with pneumonia. The majority of patients (63%) first developed unifocal infiltrates at a mean time of 4.5 ± 2.1 days after fever onset, while in 37% of patients the initial infiltrates were multifocal, appearing at a mean time of 5.8 ± 1.3 days after fever onset. Common laboratory findings included lymphopenia (on hospital admission, 70%; during hospitalization, 95%), thrombocytopenia (on hospital admission, 28%; during hospitalization, 40%), elevated lactate dehydrogenase (on hospital admission, 58%; during hospitalization, 88%), creatine kinase (on hospital admission, 18%; during hospitalization, 32%), and aspartate aminotransferase or alanine aminotransferase levels (on hospital admission, 27%; during hospitalization, 62%). Throat or nasopharyngeal swab for SARS-CoV by reverse transcriptase polymerase chain reaction (PCR) and real-time PCR was positive in 40 of the 47 patients (85%) in whom the test was performed. Conclusions: None of the presenting symptoms or laboratory findings are pathognomonic for SARS. Even though cough developed in a majority of patients, it did not occur until later in the disease course, suggesting that a cough preceding or concurrent with the onset of fever is less likely to indicate SARS. While PCR for SARS-CoV appears to be the best early diagnostic test currently available, it is clear that better methods are needed to differentiate between SARS and non-SARS illness on initial presentation.

Immune impairment in patients with terminal cancers: influence of cancer treatments and cytomegalovirus infection

Although immunodeficiency is usually considered a prerequisite of oncogenesis, a detailed immune profile in cancer has not yet been described. Without such profiling, it is not surprising that there is a vast discrepancy in the responses of cancer patients to immunotherapy. Our results show that the integrity of the immune system deteriorates with cancer progression by displaying a trend toward decreasing levels of functional T cells, including CD4, naïve, and central memory T cells, and an expansion of hyporesponsive populations such as CD28− and CMV-specific T cells. One hundred and one patients constitute the study group for the observational study reported in this paper. Forty-eight patients with newly diagnosed stages III and IV and 53 patients with extensively treated stage IV disease. The costimulatory molecules CD27 and CD28 were downregulated in all patients. Among the proinflammatory cytokines (IL-6, TNF-α, IFN-γ), only IL-6 differed significantly among the groups, increasing as the cancer stage progressed. Plasma IL-7 did not differ among the participants. The relative deficits of naïve T cells in cancer patients may be associated with the downregulation of IL-7Rα expression rather than changes in the circulating levels of IL-7. The downregulation of IL-7Rα expression was shown to be associated with increased levels of intracellular CMV. The present study suggests that the immune impairment in patients with cancer is associated with multiple factors, such as the stage of cancer, consequence of CMV infection and impact of treatment.

Identification of differentially expressed proteins in human malignant pleural effusions

A high protein concentration in a pleural effusion makes it more likely to be a malignant than a transudative effusion. However, the variability in protein composition between these two forms of pleural effusion is not well understood. In order to compare their protein compositions, the proteomic profiles of 14 malignant and 13 transudative pleural effusions were studied using two-dimensional gel electrophoresis. Protein spots with differential expression were identified by matrix-assisted laser desorption/ionisation quadrupole time-of-flight mass spectrometry and liquid chromatography/tandem mass spectrometry. Targeted proteins were further examined by ELISA and Western immunoassay in all samples. Two-dimensional gel electrophoresis revealed seven spots whose expression was reduced in malignant pleural effusions. Four of the abnormal spots were identified as fibrinogen γ-chain precursor, two as fibrinogen β-chain precursor and one as pigment epithelium-derived factor. ELISA and Western immunoassay showed that pigment epithelium-derived factor levels were significantly lower in malignant than in transudative pleural effusions. It has been demonstrated that proteomic technologies may help in the elucidation of variable expression of proteins with particular functions. By applying these technologies, the level of pigment epithelium-derived factor, a potent anti-angiogenic factor, was found to be significantly lower in malignant than in transudative pleural effusions. This finding allows for further exploration regarding how underexpression of pigment epithelium-derived factor may relate to the pathogenesis of malignant pleural effusions.A high protein concentration in a pleural effusion makes it more likely to be a malignant than a transudative effusion. However, the variability in protein composition between these two forms of pleural effusion is not well understood. In order to compare their protein compositions, the proteomic profiles of 14 malignant and 13 transudative pleural effusions were studied using two-dimensional gel electrophoresis. Protein spots with differential expression were identified by matrix-assisted laser desorption/ionisation quadrupole time-of-flight mass spectrometry and liquid chromatography/tandem mass spectrometry. Targeted proteins were further examined by ELISA and Western immunoassay in all samples. Two-dimensional gel electrophoresis revealed seven spots whose expression was reduced in malignant pleural effusions. Four of the abnormal spots were identified as fibrinogen γ-chain precursor, two as fibrinogen β-chain precursor and one as pigment epithelium-derived factor. ELISA and Western immunoassay showed that pigment epithelium-derived factor levels were significantly lower in malignant than in transudative pleural effusions. It has been demonstrated that proteomic technologies may help in the elucidation of variable expression of proteins with particular functions. By applying these technologies, the level of pigment epithelium-derived factor, a potent anti-angiogenic factor, was found to be significantly lower in malignant than in transudative pleural effusions. This finding allows for further exploration regarding how underexpression of pigment epithelium-derived factor may relate to the pathogenesis of malignant pleural effusions.

Silencing of p29 Affects DNA Damage Responses with UV Irradiation

Human p29 is a newly identified nuclear protein whose function is largely undetermined. We found that p29 associated with chromatin, interacted with MCM3, and localized with proliferating cell nuclear antigen foci in the S phase. Silencing of p29 using small interfering RNA duplexes reduced DNA synthesis and increased the expression of p107, a member of the RB family, and of cyclin-dependent kinase inhibitor p21, accompanied with a decreased expression of DNA polymerase alpha. Lethal events consisting of premature chromatin condensation with a reduced Chk1 phosphorylation were observed in p29-depleted cells in response to UV irradiation. Intriguingly, the phosphorylation of ataxia telangectasia-mutated kinases at S1981 was suppressed in p29-depleted HeLa cells with UV irradiation, but not in hydroxyurea- and ionizing radiation-treated cells. Taken together, these results reveal a novel function of p29 in the regulation of DNA replication checkpoint responses.

Prognostic value of dynamic soluble triggering receptor expressed on myeloid cells in bronchoalveolar lavage fluid of patients with ventilator‐associated pneumonia

Background and objective:  The aim of this study was to investigate the time course, and correlation with prognosis, of BAL fluid concentrations of soluble triggering receptor expressed on myeloid cells (sTREM‐1) in patients with ventilator‐associated pneumonia (VAP).

Involvement of p29 in DNA damage responses and Fanconi anemia pathway

Human p29 is a chromatin-associated protein and the silencing of p29 expression increases cell population in G(1) phase and decreases phosphorylation levels of Chk1 and Chk2 in response to UV treatment. To further characterize the function of p29, U2OS and Fanconi anemia complementation group G (FA-G) cells with constitutive p29 expression have been established. Analyses of these cells identified increased phosphorylation levels of Chk1 and Chk2, which were accompanied by elevated amounts of chromatin-associated Mre11-Rad50-Nbs1 complex and ATR-IP. Monoubiquitination of the FA ID complex was restored in p29 stably expressing FA-G cells. Moreover, lower tumor incidence was observed in mp29 transgenic mice after UV irradiation. These results suggest the involvement of p29 in the DNA damage responses and Fanconi anemia pathway.

Amplification of immune responses against a DNA-delivered idiotypic lymphoma antigen by fusion to the B subunit of E. coli heat labile toxin

The pentameric B-subunit of Escherichia coli heat-labile enterotoxin (EtxB) is not only highly immunogenic itself, but can also act as a potent adjuvant or carrier to increase immune responses to other antigens. In this study, we investigated the ability of EtxB to promote anti-tumor immune responses using a fusion DNA vaccine design. EtxB was genetically linked to a single chain Fv sequence derived from the idiotypic immunoglobulin antigen (Id) of the mouse BCL1 B-cell lymphoma. We found that the EtxB-BCL1scFv fusion protein with a specifically selected linker retained the ability to pentamerize and to bind the GM1 ganglioside. Immunization of mice with the pEtxB-BCL1scFv DNA construct generated high levels of Id-specific antibody and protected against lethal tumor challenge. The immuno-enhancing activities of EtxB were highly dependent on GM1 binding, since fusion constructs unable to pentamerize or to bind to GM1 were less effective. Thus, the EtxB fusion vaccine approach may be an attractive strategy to increase the potency of tumor vaccines.

Identification of the novel allele HLA‐B*51:84 by sequence‐based typing method in a Taiwanese individual

A new human leukocyte antigen-B allele, B*51:84 (B*5184), has been found in Taiwan. It differs from B*51:01:01(B*510101) by one nucleotide substitution at codon 145 (CGC →GGC), resulting to one amino acid change (Arginine to Glycine).

Analysis of the immune response of human dendritic cells to Mycobacterium tuberculosis by quantitative proteomics

BackgroundThe cellular immune response for Mycobacterium tuberculosis (M. tuberculosis) infection remained incompletely understood. To uncover membrane proteins involved in this infection mechanism, an integrated approach consisting of an organic solvent-assisted membrane protein digestion, stable-isotope dimethyl labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to comparatively profile the membrane protein expression of human dendritic cells upon heat-killed M. tuberculosis (HKTB) treatment.ResultsOrganic solvent-assisted trypsin digestion coupled with stable-isotope labeling and LC-MS/MS analysis was applied to quantitatively analyze the membrane protein expression of THP-1 derived dendritic cells. We evaluated proteins that were upregulated in response to HKTB treatment, and applied STRING website database to analyze the correlations between these proteins. Of the investigated proteins, aminopeptidase N (CD13) was found to be largely expressed after HKTB treatment.By using confocal microscopy and flow cytometry, we found that membranous CD13 expression was upregulated and was capable of binding to live mycobacteria. Treatment dendritic cell with anti-CD13 antibody during M. tuberculosis infection enhanced the ability of T cell activation.ConclusionsVia proteomics data and STRING analysis, we demonstrated that the highly-expressed CD13 is also associated with proteins involved in the antigen presenting process, especially with CD1 proteins. Increasing expression of CD13 on dendritic cells while M. tuberculosis infection and enhancement of T cell activation after CD13 treated with anti-CD13 antibody indicates CD13 positively involved in the pathogenesis of M. tuberculosis.

A microfluidic approach towards hybridoma generation for cancer immunotherapy

Dendritic cells/tumor fusions have shown to elicit anti-cancer immunity in different cancer types. However, the application of these vaccines for human cancer immunotherapy are limited by the instable quality and insufficient quanity of fusion cells. We present a cell electrofusion chip fabricated using soft lithography technique, which combines the rapid and precise cell pairing microstructures and the high yield electrofusion micro-electrodes to improve the cell fusion. The design uses hydrodynamic trapping in combination with positive dielectrophoretic force (pDEP) to achieve cell fusion. The chip consists of total 960 pairs of trapping channels, which are capable of pairing and fusing both homogeneous and heterogeneous types of cells. The fused cells can be easily taken out of the chip that makes this device a distinguishable from other designs. We observe pairing efficiency of 68% with fusion efficiency of 64%.