Yield of hepatitis C virus nucleic acid testing among antibody-reactive or confirmed-positive samples.
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Antibodies, Viral↗
Biomedical subjects
Michael Schmidt
Publications and source records attributed to Michael Schmidt.
Nucleic acid test screening of blood donors for orthopoxviruses can potentially prevent dispersion of viral agents in case of bioterrorism.
BACKGROUND: Microbiologic agents such as variola virus (VAR) are very attractive for terrorism. As a result of international collaboration under the WHO eradication campaign, smallpox was declared eradicated in 1980. Therefore, the immunization programs were discontinued worldwide. Because most people are now immunologically naive, VAR is considered to be a potential threat agent or bioterrorist weapon. Real-time polymerase chain reaction (PCR) followed by melting analysis was developed for fast and safe analysis and allows differentiation of VAR from other orthopoxviruses (OPVs) like vaccinia or camelpox virus. STUDY DESIGN AND METHODS: A RealArt Orthopox LC PCR kit (Artus GmbH) was used to amplify OPV sequences from blood donor samples. A total of 31,500 blood donor samples were tested in minipools of up to 96 samples. To evaluate the sensitivity of the assay, routine donor minipools (90 +/- 6 samples per pool) were spiked with vaccinia virus used as positive control. RESULTS: Specificity was 100 percent because none of 31,500 blood donors was positive for the presence OPV. The detection limit of the assay was 10.6 copies per PCR procedure. Therefore, a sensitivity of 1590 copies per mL was calculated. Overall, 0.28 percent of test results had to be considered invalid owing to negative internal controls. CONCLUSION: The RealArt Orthopox LC PCR kit enables reliable detection of OPV DNA in viremic blood donor samples, even at the beginning of the disease when patients present minor clinical symptoms, and could be implemented in our routine screening procedure immediately. Thus, the assay could potentially help to prevent dispersion of viral agents by blood transfusion in case of bioterrorism.
Bioterrorism↗
High-throughput purification of viral RNA based on novel aqueous chemistry for nucleic acid isolation.
BACKGROUND: Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive. METHODS: We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch. RESULTS: The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated. CONCLUSIONS: Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.
Autoanalysis↗
An image of mitral valve repair.
Mitral regurgitation is a severe problem of the mitral valve mechanism caused by many factors, including ischemic and nonischemic cardiomyopathy, inflammation, degeneration and others. Regardless of the cause of valve dysfunction, many changes in surgical approach have been undertaken. The pioneering work of Carpentier led to the era of mitral valve reconstruction a few decades ago. Understanding the different techniques applied in several pathoanatomic settings is of major interest. This review summarizes the indications and results of this specific surgical procedure in the setting of ventricular dysfunction by different causes.
Cardiac Surgical Procedures↗
Thalidomide induces apoptosis in human monocytes by using a cytochrome c-dependent pathway.
Thalidomide has been shown to be an effective treatment in various immunologic diseases such as Crohn's disease and rheumatoid arthritis. Its major effect is thought to be mediated by the inhibition of TNF-alpha, but the exact mechanism of action is still uncertain. Recent observations could demonstrate that the induction of monocyte apoptosis is a common feature of a variety of anti-inflammatory agents. Therefore, we investigated the role of thalidomide on monocyte apoptosis. Treatment with thalidomide resulted in apoptosis of human peripheral blood monocytes in a time- and dose-dependent manner as demonstrated by annexin V staining. Monocyte apoptosis required the activation of caspases, as combined stimulation by thalidomide together with the broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone markedly prevented monocyte cell death. Apoptosis was triggered by a CD95/CD95 ligand, TNF-RI, and TRAIL-R1 independent pathway with an inhibition of AKT-1 kinase and consecutive mitochondrial release of cytochrome c, followed by the proteolytic activation of initiator caspase-9 and effector caspase-3. Our data suggest that thalidomide-induced monocyte apoptosis is at least partially mediated by a mitochondrial signaling pathway and might contribute to the complex immunomodulatory properties of the drug.
Apoptosis↗
Prognostic value of depressive coping and depression in survival of lung cancer patients.
The aim of this investigation was to determine whether depressive coping and depression predict shorter survival among lung cancer patients. We conducted a prospective study using an inception cohort with a 3-5-year follow-up. The sample consisted of n = 59 (of n = 69 invited to participate) patients (mean age 65 years, S.D. = 9.7; 81% male) newly diagnosed with small cell lung cancer or non-small cell lung cancer Stage III or IV who were scheduled for later chemotherapy and/or radiotherapy at a tertiary care centre. Patients were investigated after their diagnosis and before the beginning of treatment. Depressive coping and depression were assessed using standardized self-report questionnaires (Freiburg Questionnaire of Coping with Illness; Hospital Anxiety and Depression Scale). Depressive coping was associated with shorter survival (hazard ratio 1.75, 95% confidence interval 1.04-2.93, p = 0.034) after adjusting for age, sex, stage, histological classification, and Karnofsky performance status but not treatment type, using the Cox proportional hazards regression. Depression, however, was not linked with survival (hazard ratio 1.05, 95% confidence interval 0.98-1.13, p = 0.18). To conclude, the prognostic value of depressive coping was partially confirmed, warranting further examination of the robustness of this relationship.
Adaptation, Psychological↗
BMP4 and CNTF are neuroprotective and suppress damage-induced proliferation of Müller glia in the retina.
In response to acute damage, Müller glia in the chicken retina have been shown to be a source of proliferating progenitor-like cells. The secreted factors and signaling pathways that regulate this process remain unknown. The purpose of this study was to test whether secreted factors, which are known to promote glial differentiation during development, regulate the ability of Müller glia to proliferate and become retinal progenitors in response to acute damage in mature retina. We made intraocular injections of BMP4, BMP7, EGF, NGF, BDNF, or CNTF before or after a single, toxic dose of N-methyl-d-aspartate (NMDA) and assayed for proliferating progenitor-like cells within the retina. We found that injections of BMP4, BMP7, or CNTF, but not EGF, NGF, or BDNF, before NMDA treatment reduced the number of Müller glia that proliferated and gave rise to progenitor-like cells. CNTF and BMP4, but not NGF or BDNF, greatly reduced the number of cells destroyed by toxin treatment indicating that these factors protect retinal neurons from a severe excitotoxic insult. Injections of CNTF 5 days before NMDA treatment prevented neurotoxin-induced cell death and Müller glial proliferation, while injections of BMP4 had no protective effect. In addition, CNTF injected after NMDA treatment suppressed glial proliferation, while BMP4 did not. We conclude that BMP4 and CNTF, when applied before a toxic insult, act as neuroprotective agents and likely suppress the proliferative response of Müller glia to retinal damage by attenuating the retinal damage; protecting bipolar and amacrine neurons from NMDA-induced cell death. When applied after a toxic insult, CNTF suppressed glial proliferation independent of levels of retinal damage.
Animals↗
Development of an unbiased method for the estimation of liver steatosis.
BACKGROUND: Steatosis significantly contributes to an organ's transplantability. Livers with >30% fat content have a 25% chance of developing primary non-function (PNF). The current practice of evaluating a hematoxylin and eosin (H&E) stained donor biopsy by visual interpretation is subjective. We hypothesized that H&E staining of frozen sections fails to accurately estimate the degree of steatosis present within a given liver biopsy. To address this problem of evaluating steatosis in prospective donor organs, we developed a fast, user friendly computer methodology to objectively assess fat content based on the differential quantification of color pixels in Oil Red O (ORO) stained liver biopsies. METHODS: The accuracy of human visual estimation of fat content by H&E and ORO stains was compared with computer-based measurements of the same slides from 25 frozen sections of donor biopsies. RESULTS: Samples with a fat content >20% showed marked variation between human interpretation and computer analysis. There was also a significant difference in the human interpretation of fat based on the method of staining. This difference ranged from 3 to 37% with H&E. DISCUSSION: Use of ORO resulted in a more consistent estimation of liver steatosis compared with H&E, but human interpretations failed to correlate with computer measurements. Such differences in fat content estimations might result in the rejection of a potentially transplantable organ or the acceptance of a marginal one. Ideally, our protocol can rapidly be applied to clinical practice for accurate and consistent measurement of fat in liver sections for the ultimate purpose of increasing the number of successful transplantable organs.
Algorithms↗
NAT screening of blood donors for severe acute respiratory syndrome coronavirus can potentially prevent transfusion associated transmissions.
BACKGROUND: The severe acute respiratory syndrome (SARS) was first described in February 2003. Close contact with symptomatic patients appears to be the main route of transmission, whereas blood transfusion transmission could not be ruled out. STUDY DESIGN AND METHODS: A SARS coronavirus (SARS-CoV) detection kit developed by C. Drosten (Bernhard Nocht Institute, BNI) was used to amplify SARS-CoV sequences from blood donor samples. We tested 31,151 blood donor samples in minipools of up to 96 samples. To validate the sensitivity of the assay, routine donor minipools (88 +/- 8 samples per pool) were spiked with plasma of an imported case of SARS or of a subsequently infected contact person, respectively. Gamma-irradiated cell culture supernatants of Vero E6 cells, infected with SARS-CoV, were used as positive controls. RESULTS: None of 31,151 blood donors were positive for the presence of SARS. Two 96-member plasma pools that were each spiked with 100 microL of plasma of the German index patient or his wife, respectively, were positive. Overall, 0.85 percent of test results had to be considered invalid owing to negative internal controls. CONCLUSION: A real-time CoV PCR test is able to detect SARS-CoV in viremic blood donor samples even in the beginning of the disease when patients present minor clinical symptoms. Thus the assay could potentially help to prevent transfusion-associated SARS-CoV transmissions.
Blood Donors↗
Comparison of two real-time quantitative assays for detection of severe acute respiratory syndrome coronavirus.
The new severe acute respiratory syndrome (SARS) coronavirus (CoV), described in February 2003, infected a total of 8,439 people. A total of 812 people died due to respiratory insufficiency. Close contact with symptomatic patients appeared to be the main route of transmission. However, potential transmission by blood transfusion could not be definitely excluded. Two real-time SARS-specific PCR assays were assessed for their sensitivities, agreement of test results, and intra-assay variabilities. Both assays rely on reverse transcription and amplification of extracted RNA. Dilutions of gamma-irradiated cell culture supernatants of SARS CoV-infected Vero E6 cells were prepared to determine the precisions, linear ranges, and accuracies of the assays. The linear range for the Artus RealArt HPA-Coronavirus assay (Artus assay) was 1 x 10(2) to 1 x 10(7) copies/ml, and that for the Roche LightCycler SARS CoV Quantification kit (Roche assay) was 1 x 10(4) to 2 x 10(8) copies/ml. The detection limit of the Roche assay was 3,982.1 copies/ml, whereas that of the Artus assay was 37.8 copies/ml. Detection limits were calculated with a standard preparation that was recommended for use by the World Health Organization. However, quantification of CoV in this preparation may be imprecise. In summary, both assays are suitable for quantitative measurement of SARS CoV at the high concentrations expected in sputum samples. The Artus assay is also suitable for detection of SARS CoV at the low concentrations found in serum samples.
Humans↗
Cloning and characterization of a bovine adeno-associated virus.
To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications.
Amino Acid Sequence↗
Identification of adeno-associated virus contamination in cell and virus stocks by PCR.
To further understand the biology of adeno-associated virus (AAV) and identify the presence of AAV in laboratory samples, we have developed a sensitive PCR-based assay using degenerate primers based on the sequence of seven diverse AAV isolates. Using these primers, we can detect free virus in viral stocks, cleared cell lysate, as well as in latently infected cells. The method can detect as little as 10 viral copies/microL of sample and can be adapted for high-throughput screening technology. With this method, we have also detected a new AAV isolate from a stock of bovine adenovirus.
Base Sequence↗
Local adeno-associated virus-mediated interleukin 10 gene transfer has disease-modifying effects in a murine model of Sjögren's syndrome.
Female nonobese diabetic (NOD) mice develop spontaneous autoimmune sialadenitis and loss of salivary flow, and are a widely used model of Sjögren's syndrome. We examined the feasibility of local salivary gland immunomodulatory gene delivery to alter these sequelae in NOD mice. We constructed recombinant adeno-associated virus (rAAV) vectors encoding either human interleukin 10 (rAAVhIL-10) or beta-galactosidase (rAAVLacZ, control vector). Mice received rAAVhIL-10 or rAAVLacZ by retrograde submandibular ductal instillation either at age 8 weeks (early, before onset of sialadenitis), or at 16 weeks (late, after onset of sialadenitis). As a systemic treatment control, separate mice received intramuscular delivery of rAAVhIL-10 at each time point. Both submandibular and intramuscular delivery of vector led to low circulating levels of hIL-10. After submandibular administration of rAAVhIL-10, salivary flow rates at 20 weeks for both the early and late treatment groups were significantly higher than for both rAAVLacZ-administered and untreated mice. Systemic delivery of rAAVhIL-10 led to improved salivary flow in the late treatment group. Inflammatory infiltrates in submandibular glands, however, were significantly reduced only in mice receiving rAAVhIL-10 locally in the salivary gland compared with mice receiving this vector intramuscularly, or rAAVLacZ or no treatment. In addition, after submandibular rAAVhIL-10 delivery, NOD mice exhibited significantly lower blood glucose, and higher serum insulin, levels than all other groups, indicating some systemic benefit of this treatment. These studies show that expression of hIL-10 by rAAV vectors can have disease-modifying effects in the salivary glands of NOD mice, and suggest that local immunomodulatory gene transfer may be useful for managing the salivary gland pathology in Sjögren's syndrome.
Animals↗
Adeno-associated virus type 5: transduction efficiency and cell-type specificity in the primate retina.
Gene transfer using adeno-associated viruses (AAVs) has been effective for treating inherited retinal diseases in animal models. Further evaluation in primates must be performed prior to clinical application, however, because of the difference between the retina of the primate and those of other animals. Prior work has shown that AAV2 can transduce rod-photoreceptor and RPE cells in the non-human primate retina and that AAV5 is more efficient at transducing photoreceptor cells than AAV2 in the rodent retina. In this study, we evaluated the efficiency of AAV5 in the non-human primate retina after subretinal injections of the vector to distinct anatomic retinal regions (superior, inferior, nasal, macula, temporal). rAAV5 led to a rapid onset of transgene expression (within 2 weeks), with expression persisting up to 10 months. Postoperative electrophysiology studies showed that global retinal function was preserved following gene transfer. Quantitative analysis of gene transfer demonstrated a maximum transduction efficiency of 22% in the injected areas. Evaluation of cell types using confocal microscopy and cone-specific antibodies revealed that AAV5, expressing reporter genes from the cytomegalovirus (CMV) promoter/enhancer, preferentially transduced rods. No significant differences were found in the regional tropism of AAV5 among the five areas injected despite variation in retinal topography. Immunohistochemical studies revealed that the AAV5 receptor, PDGFR-A, is localized to the outer segments of rods but not cones providing a basis for the observed tropism. Our results support the utility of AAV5 for rod photoreceptor degeneration therapies.
Adenoviridae↗
Secretory activity and cell cycle alteration of alveolar type II cells in the early and late phase after irradiation.
PURPOSE: Type II cells and the surfactant system have been proposed to play a central role in pathogenesis of radiation pneumonitis. We analyzed the secretory function and proliferation parameters of alveolar type II cells in the early (until 24 h) and late phase (1-5 weeks) after irradiation (RT) in vitro and in vivo. METHODS AND MATERIALS: Type II cells were isolated from rats according to the method of Dobbs. Stimulation of secretion was induced with terbutaline, adenosine triphosphate (ATP), and 12-O-tetradecanoylphorbol-13-acetate (TPA) for a 2-h period. Determination of secretion was performed using (3)H-labeled phosphatidylcholine. For the early-phase analysis, freshly isolated and adherent type II cells were irradiated in vitro with 9-21 Gy (stepwise increase of 3 Gy). Secretion stimulation was initiated 1, 6, 24, and 48 h after RT. For late-phase analysis, type II cells were isolated 1-5 weeks after 18 Gy whole lung or sham RT. Each experiment was repeated at least fivefold. Flow cytometry was used to determine cell cycle distribution and proliferating cell nuclear antigen index. RESULTS: During the early-phase (in vitro) analysis, we found a normal stimulation of surfactant secretion in irradiated, as well as unirradiated, cells. No change in basal secretion and no dose effect were seen. During the late phase, 1-5 weeks after whole lung RT, we observed enhanced secretory activity for all secretagogues and a small increase in basal secretion in Weeks 3 and 4 (pneumonitis phase) compared with controls. The total number of isolated type II cells, as well as the rate of viable cells, decreased after the second post-RT week. Cell cycle alterations suggesting an irreversible G(2)/M block occurred in the second post-RT week and did not resolve during the observation period. The proliferating cell nuclear antigen index of type II cells from irradiated rats did not differ from that of controls. CONCLUSION: In contrast to literature data, we observed no direct effect of radiation on secretory activity in the early phase after RT. In our study of isolated type II cells, as well as in intact animals, RT did not result in an impaired surfactant secretion up to 5 weeks after RT. Our in vivo experiments even showed an increased response of phosphatidylcholine secretion to all known secretagogues at Weeks 3 and 4 after whole lung RT, possibly due to inflammatory cytokines. Cell cycle alterations with G(2)/M block and cell loss in the late post-RT period may contribute more to the manifestation of radiation-induced lung damage than functional impairment in type II cells.
Animals↗
Exposure to high stress in the intensive care unit may have negative effects on health-related quality-of-life outcomes after cardiac surgery.
OBJECTIVE: Up to 20% of patients do not show improvements in health-related quality of life (HRQL) after cardiac surgery, despite apparently successful surgical procedures. We sought to determine whether failed improvements in HRQL after cardiac surgery are associated with the development of traumatic memories and chronic stress states as a result of high perioperative stress exposure. DESIGN: Prospective cohort study. SETTING: A 10-bed cardiovascular intensive care unit of a tertiary care university hospital. PATIENTS: A total of 148 cardiac surgical patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The patients were evaluated for traumatic memories from postoperative treatment in the cardiovascular intensive care unit (defined as the subjective recollection of pain, respiratory distress, anxiety/panic, and nightmares), symptoms of chronic stress, including those of posttraumatic stress disorder, and HRQL preoperatively (at baseline) and at 6 months after cardiac surgery. A state of chronic stress was defined as the development of posttraumatic stress disorder at 6 months after surgery. Factors predicting the decline in HRQL were determined by multivariable linear regression. Twenty-seven patients (18.2%) had posttraumatic stress disorder at 6 months after cardiac surgery; seven of these patients (4.8%) had evidence of preexisting posttraumatic stress disorder before undergoing cardiac surgery. Patients with new posttraumatic stress disorder at 6 months after cardiac surgery had a significantly higher number of traumatic memories from postoperative treatment in the cardiovascular intensive care unit (p =.01). A multiple regression model included the number of traumatic memories from the intensive care unit and stress symptom scores at 6 months after heart surgery as predictors for variations in physical HRQL outcome scores (R2 =.30, p <.04). Stress symptom scores were the most significant predictors of mental health HRQL outcomes (R2 =.52, p <.01). CONCLUSIONS: Exposure to high stress in the cardiovascular intensive care unit can have negative effects on HRQL outcomes of cardiac surgery.
Aged↗
Acoustical analysis of posed prosodic expressions: effects of emotion and sex.
Prosodic expression is an important channel of emotional communication and can be assessed through computerized acoustical analysis. Fundamental frequency (F0) is the most commonly studied acoustic measure and considered the best index of perceived pitch. In this study, we examined two factors that can influence prosodic expression, sex and emotion type. A special feature is the use of a neutral expression as a control measure. We also described procedures for eliciting posed prosodic expression via an experimental task from the New York Emotion Battery. Subjects were healthy men (n=10) and women (n=9), matched for age (M=29.2 yr.) and education (M=15.6 yr.). Subjects were asked to intone neutral-content sentences with happy, sad, and neutral prosody. F0 mean and standard deviation were measured using the Computerized Speech Lab program. Initial findings indicated that women produced significantly higher F0 values than did men and that happy sentences were produced with significantly higher F0 values than were sad sentences. When semitone conversions were applied and neutral prosody was subtracted out, differences remained for emotion type but not for sex. Findings are discussed in terms of implications for the assessment and treatment of prosody in clinical populations.
Adult↗
Forecasting the impact of a clinical practice guideline for perioperative beta-blockers to reduce cardiovascular morbidity and mortality.
BACKGROUND: Beta-blockers reduce morbidity and mortality when administered to high-risk patients undergoing major noncardiac surgery, yet little is known about how often they are being prescribed. Clinical practice guidelines are tools that can be used to speed the translation of research into practice and may be one method to improve the use of beta-blockers. Before implementing any guideline, it is important to forecast its potential clinical and financial impact. METHODS: We conducted a retrospective cohort study, using administrative and medical record review data, of all adult patients undergoing major noncardiac surgery at Baystate Medical Center, Springfield, Mass, during a 1-month period in 1999. Patients with 2 or more cardiac risk factors or with documented coronary artery disease were classified as high risk and were considered eligible for treatment with a beta-blocker if they had no obvious contraindications to its use. We estimated the potential clinical benefit of treating eligible patients with a beta-blocker by extrapolating the treatment effect observed in a previously reported randomized clinical trial. RESULTS: Of 158 patients undergoing major noncardiac surgery, 67 (42.4%) seemed to be ideal candidates for treatment with perioperative beta-blockers. Of these 67 patients, 25 (37%) received a beta-blocker at some time perioperatively. During the course of a year, we estimate that between 560 and 801 patients who do not receive beta-blockers might benefit from treatment with these medications. Full use of beta-blockers among eligible patients at our institution could result in 62 to 89 fewer deaths each year at an overall cost of $33 661 to $40 210. CONCLUSIONS: There seems to be a large opportunity to improve the quality of care of patients undergoing major noncardiac surgery by increasing the use of beta-blockers in the perioperative period. A clinical practice guideline may be one method to achieve these goals at little cost.
Adrenergic beta-Antagonists↗