Development of a microsphere-based fluorescent immunoassay and its comparison to an enzyme immunoassay for the detection of antibodies to three antigen preparations from Candida albicans

A sensitive assay for the simultaneous detection of multiple serum antibodies by flow cytometry was developed. Polystyrene microspheres of 5, 7 and 9.3 micron in diameter were used as solid supports for the attachment of three different antigen preparations from Candida albicans. These antigens were a whole cell extract; a cytoplasmic protein extract and a cell wall polysaccharide. Microsphere-associated fluorescence was quantitated by flow cytometry, with the different sized microspheres analyzed separately using electronic volume gating. This procedure allowed for different antigen-coated microspheres with discrete sizes to be analyzed independently for immunofluorescence. The assay detected antibody levels in human serum at dilutions up to 10(-6) and provided complete discrimination, using all three antigen preparations, between antibody levels seen in healthy subjects and those seen in patients suspected of having a systemic Candida infection. A standard enzyme immunoassay (EIA) failed to provide complete discrimination between healthy subjects and patient samples: at least 17% of patient values fell within the healthy subject range using all three antigen preparations. The microsphere assay which allowed for the simultaneous detection of multiple antibodies, has increased dynamic range over EIA and provides for better discrimination of patients from healthy subjects in comparison to EIA. Precise quantitation of antibodies is possible and the rapid analysis of thousands of microspheres markedly enhances the statistical accuracy of the assay. We suggest this assay is likely to have many other important applications in immunologic testing.     

The antigen-presenting environment in normal and human papillomavirus (HPV)-related premalignant cervical epithelium

The activation of HPV-specific T cells within the cervical microenvironment is likely to play an important part in the natural history of cervical intraepithelial neoplasia (CIN). The extent and the type of T cell activation will depend critically on the expression of MHC, costimulatory cell surface molecules and cytokines by keratinocytes and Langerhans cells within the cervical lesion. Expression of MHC class II (HLA-A-DR and -DQ), costimulatory/adhesion molecules (CD11a/18, CD50, CD54, CD58 and CD86) and cytokines (tumour necrosis factor-alpha (TNF-alpha) and IL-10) was therefore investigated by immunohistochemistry in normal squamous epithelium (n = 12), low-grade (n = 23) and high-grade (n = 18) squamous intraepithelial lesions of the cervix. CIN progression was associated with de novo expression of HLA-DR and CD54, and increased expression of CD58 by keratinocytes. However, significantly, there was no expression of any adhesion/costimulation molecule by epithelial Langerhans cells in any cervical biopsy studied. Furthermore, TNF-alpha, a potent activator of Langerhans cells, was expressed constitutively by basal keratinocytes in normal cervix (12+/12). but expression of this cytokine was absent in a number of CIN samples (20+/23 for low-grade, 12+/18 for high-grade CIN). Conversely, the suppressive cytokine IL-10 was absent in normal epithelium (0+/12), but was up-regulated in a number of CIN lesions (12+/23 for low-grade; 8+/18 for high-grade CIN). The restricted expression of costimulation/adhesion molecules and the nature of the cytokine microenvironment within the epithelium may act to limit effective immune responses in some CIN lesions.     

动态脑电图在昏迷病人中的应用

目的：探讨动态脑电图监测对昏迷病人的应用价值。方法：对107例昏迷患者采用多次动态脑电图监测，分析脑电图改变与临床结局的关系。结果：平坦波型昏迷死亡率较高(94．7％)，慢波型昏迷死亡率较低(30．3％)，且预后较好。结论：对昏迷病人实施动态脑电图监测，可提高对预后评估及脑死亡的正确判断率。     

Clinical features and risk factors for mortality in Aeromonas bacteremic adults with hematologic malignancies.

BACKGROUND AND PURPOSE: Aeromonas spp. often cause infections in immunocompromised patients. To specifically understand the clinical features of Aeromonas bacteremic adults with hematologic malignancies, we investigated the demographic, clinical and microbiologic characteristics of Aeromonas bacteremia in this patient population. METHODS: Retrospective study performed in a tertiary medical center in southern Taiwan, in which adults with hematologic malignancies suffered from Aeromonas bacteremia admitted between 1995 and 2003 were included for study. RESULTS: There were 45 episodes of Aeromonas bacteremia in 41 adults with hematologic malignancies. Episodes of Aeromonas bacteremia which occurred at least 2 months apart were counted as separate cases in the analysis. A total of 30 men and 15 women (mean age: 53.2 years), with 4 patients experiencing 2 episodes, was included. The 3 leading underlying hematologic malignancies were acute myelogenous leukemia (37.8%), myelodysplastic syndrome (26.7%) and non-Hodgkin's lymphoma (17.8%). No cluster of Aeromonas bacteremia was found during the study period. Twenty nine (64.4%) of the 31 patients with nosocomial Aeromonas bacteremia had received recent antineoplastic chemotherapy. The 3 leading clinical manifestations were fever (88.9%), septic shock (40%), and altered consciousness (26.7%). Eleven (24.4%) episodes of bacteremia were polymicrobial. Sixteen (35.6%) patients died within 14 days of onset of bacteremia. The mean duration from sampling blood for culture to death was 3.81 days. Altered consciousness (odds ratio, 8.999; 95% confidence interval, 1.787-45.33; p=0.008) was the only independent prognostic factor for mortality. High resistance rates (11.1% to piperacillin and 35.6% to imipenem) among Aeromonas isolates were also noted. CONCLUSION: In febrile patients with hematologic malignancies and suspected Aeromonas infections, particular attention to the development of alteration of consciousness is needed as it is an independent risk factor for mortality.     

Fibrillary inclusions in neoplastic and fetal acinar cells of the pancreas

We report a case of pancreatic acinar cell carcinoma which contained a large number of pleomorphic inclusions with fibrillary internal structures and mature zymogen granules. To clarify the significance of fibrillary inclusions in the differentiation of acinar cells of the pancreas, we further investigated fetal pancreases (gestational weeks 16, 17, 19, 20 and 28). We found two types of inclusions: type A, corresponding to fibrillary inclusion of neoplastic acinar cells, was observed only in a 19-week fetus; type B showed a homogeneous density similar to that of zymogen granules. Type B was observed in all the fetuses after the 17th gestational week. Although the type A inclusion might be generated throught a different mechanism than the type B inclusion, the appearance of a large number of fibrillary inclusions in neoplastic acinar cells may represent a transient form of zymogen granule.     

Persistent monocytosis after intravenous immunoglobulin therapy correlated with the development of coronary artery lesions in patients with Kawasaki disease.

BACKGROUND AND PURPOSE: This study was conducted to investigate whether changes in the complete blood count (CBC)/differential count (DC) and C-reactive protein (CRP) were correlated to Kawasaki disease (KD) with coronary artery lesions (CALs). METHODS: A retrospective analysis was performed of all children with KD at Chang Gung Memorial Hospital at Kaohsiung from 2001 to 2006. KD patients were divided into those with and without CALs for testing of correlations with changes in CBC/DC and CRP levels. RESULTS: A total of 147 patients were enrolled for this analysis. Serial CBC/DC and CRP measurements and echocardiographic data for determination of CAL formation were obtained before and after intravenous immunoglobulin (IVIG) treatment. There were 44 (29%) KD patients having CAL formation (>3 mm in diameter of internal lumen). There was no significant difference in terms of age distribution and major diagnostic criteria between KD patients with and without CALs. Male KD patients, however, had a significantly higher rate of CAL formation (p=0.009). In multivariate logistical regression analysis, persistent monocytosis after IVIG treatment was the only factor significantly correlated to CAL formation (p=0.003). CONCLUSIONS: Of the febrile routine measurements of CBC/DC and CRP in KD, persistent monocytosis after IVIG treatment was correlated to CAL formation. Further studies to clarify the mechanism of monocytosis may help prevent the CALs of KD.     

Power spectral analysis of the electroencephalographic and hemodynamic correlates of propofol anesthesia in the rat: Intravenous infusion

Based on the tail-flick response to noxious thermal stimuli, we determined in the present study that effective antinociception could be achieved in adult male Sprague-Dawley rats 15 min after intravenous infusion of propofol at 60 mg/kg/h. Simultaneous power spectral analysis of the electroencephalographic (EEG) and systemic arterial pressure signals further revealed a concomitant depression of the activity of all EEG frequency bands (δ, θ, , β), alongside hypotension, negative inotropic and chronotropic actions, and attenuated baroreceptor reflex and vasomotor activity. These effects were congruent with a plasma concentration of propofol in the arterial blood of 1.70 ± 0.13 μg/ml, as determined by high-performance liquid chromatography.     

适用于医学动态监测数据的稳健参数估计方法

目的 :介绍并分析适用于医学动态监测数据的稳健参数估计方法。方法 :结合分布假设和稳健统计给出了参数估计方法。首次提出了一种迭代求取形状参数稳健估计的算法。结果 :该方法对医学动态监测中某种生理量异常所占百分比值统计量的参数估计优于临床普遍采用的方法。结论 :通过对分布拟合、估计优效性和可实现性、估计稳健性等的理论分析和模拟实验表明该方法是一种高效、可靠的稳健参数估计方法     

Analytical sensitivity, reproducibility of results, and clinical performance of five PCR assays for detecting Chlamydia pneumoniae DNA in peripheral blood mononuclear cells

Chlamydia pneumoniae has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of C. pneumoniae in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned PstI fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting ompA; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of C. pneumoniae; the ompA nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of C. pneumoniae DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of C. pneumoniae IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0, P<0.001), with the PstI fragment (P<0.001) and 16S rRNA (P = 0.002) assays having lower reproducibility than the nested ompA and TETR assays. To further analyze assay sensitivity, C. pneumoniae-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for C. pneumoniae DNA. The ompA nested PCR detected C. pneumoniae DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (P<0.001), and the other 3 assays detected no positive specimens (P<0.001, compared with the ompA assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detect C. pneumoniae in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of C. pneumoniae in clinical material.