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PubMed1985

Comparison of cold air, ultrasonic mist, and methacholine inhalations as tests of bronchial reactivity in normal and asthmatic children.

The sensitivity and specificity of cold air, ultrasonically nebulized distilled water mist (USM), and standard methacholine (MCH) challenges were studied in 21 children with asthma (mean age 11.5 years) and 12 normal children (mean age 14.2 years). The cold air challenge consisted of successive 3-minute periods of hyperventilation during which incremental volumes of subfreezing air (mean temperature -16 degrees C) were inhaled. To perform the USM challenge, subjects inhaled increasingly larger volumes of nebulized distilled water while breathing tidally. The specificity of both nonpharmacologic challenges was found to be 100%, whereas that of MCH was only 83%. The sensitivity of the cold air and USM tests was 57% and 71%, respectively, compared with 95% obtained with MCH challenge. We conclude that cold air and USM challenges are promising alternatives to the MCH challenge, and may be superior to it if optimal standard testing protocols are defined.

Adolescent↗
PubMed1998

Purification of novel kinesins from embryonic systems.

Several kinesin holoenzymes, including the heterotrimeric kinesin-II and bipolar KLP61F complexes described here, are being purified in our laboratory using microtubule affinity precipitation and conventional biochemical fractionation procedures. These protocols have been optimized by using pan-kinesin peptide antibodies and subunit-specific antibodies to monitor the enrichment of kinesin-related polypeptides in particular fractions by immunoblotting. Protein purification represents the most direct route available for determining the oligomeric state and subunit composition of a kinesin holoenzyme, for identifying tightly associated accessory subunits such as SpKAP115, and for determining the molecular architecture and functional properties of native kinesin motors. Protein purification methods therefore represent an important complementary approach to molecular genetic approaches that are being pursued in many other laboratories.

Animals↗
PubMed1999

Abnormal chromosome 8 copy number in stage I to stage IV breast cancer studied by fluorescence in situ hybridization.

To test the hypothesis that the frequency of abnormal chromosome 8 copy number increases with the severity of the disease as defined by an increase in clinical stage, we conducted a fluorescence in situ hybridization (FISH) study of a sample of 42 breast cancer specimens utilizing a protocol that was optimized by our laboratory. Cytogenetic results, obtained from blinded analyses of archival specimens, demonstrated that the higher clinical stages (i.e., stages III and IV) yield higher frequencies of abnormal chromosome 8 copy number. Specifically, 45.45% and 50% of the stage I and stage II cases, respectively, were abnormal, whereas 63.64% and 60% of the stage III and stage IV cases, respectively, were abnormal for chromosome 8 copy number. The overall frequency of abnormal chromosome 8 copy number was 54.76% (23 of 42 tumors studied). When the results of a control probe were taken into account, 34.78% (8 of 23) of the abnormal cases were trisomic, whereas the remaining cases were likely triploid. Thus, the present data not only established that chromosome 8 trisomy is a recurrent finding in breast cancer, but also confirmed a higher frequency of occurrence of abnormal chromosome 8 copy number with the higher clinical stages. Future experiments utilizing additional specimens in this laboratory and from other laboratories are necessary to confirm and extend the findings of the present study.

Aneuploidy↗
PubMed1999

A comparison of in situ hybridization and immunohistochemistry for the detection of a new porcine circovirus in formalin-fixed tissues from pigs with post-weaning multisystemic wasting syndrome (PMWS).

Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified condition affecting pigs in North America and Europe. Porcine circovirus antigen and nucleic acid have been demonstrated associated with lesions, and a new porcine circovirus designated PCV2 has been recovered from tissues of these animals. In this study, in situ hybridisation and immunohistochemical protocols were developed, optimized and compared for their relative sensitivity in detecting PCV2 antigens and nucleic acid in tissues from cases of PMWS that had been fixed for up to 6 months in formalin. For both immunohistochemistry and in situ hybridization, an increase in specific signal was observed following increased exposure to both protease XIV and proteinase K. Maximum signal and minimal loss of tissue morphology was seen after 40 min treatment with protease XIV (0.5 mg/ml). After optimisation, a comparison of these techniques on sequential sections demonstrated that both techniques successfully detected antigen or nucleic acid in all of the tissues examined. More positive cells, with increased signal intensity, were detected following immunohistochemistry.

Animals↗
PubMed1999

Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota.

The porcine gastrointestinal tract (GIT) microbiota has been studied to increase production efficiency, improve product quality, and help attempt to reduce disease. During the developmental period from birth through weaning, the intestinal microbiota undergoes a rapid ecological succession. There is interest in developing a monitoring technique that allows for analysis of bacterial population levels and shifts within the pig intestine. The objective of this study was to determine if denaturant gradient gel electrophoresis (DGGE) could be effectively applied to measure changes in bacterial populations of the pig GIT, as influenced by age, diet or compartment. Bacterial genetic diversity was determined using DGGE analysis of the V3 region of 16S rDNA PCR products (approximately 200 bp) obtained from primers specific for the domain Bacteria. Protocol development included optimization of: DNA extraction procedures, PCR amplification, removal of PCR artifacts, and optimization of gel preparation and image capture. DGGE analysis revealed diverse bacterial populations between pigs of different ages and among individual gut compartments. Comparison of fecal DNA from different aged pigs revealed several unique PCR product bands indicating the presence of unique bacterial populations. Comparison of different gut compartments demonstrated that bacterial populations were most similar (C, value > 50%) within a single compartment and between adjacent ones. Thus, DGGE can be used to examine bacterial diversity and population shifts in the pig GIT.

Aging↗
PubMed1999

Biodistribution of free and N-(2-hydroxypropyl)methacrylamide copolymer-bound mesochlorin e(6) and adriamycin in nude mice bearing human ovarian carcinoma OVCAR-3 xenografts.

The purpose of this study was to examine the biodistribution of the photosensitizing drug, mesochlorin e(6) monoethylenediamine (Mce(6)), and the antineoplastic agent, adriamycin (ADR), as well as their N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates in female nu/nu athymic mice bearing human ovarian carcinoma OVCAR-3 xenografts. The levels of Mce(6) and HPMA copolymer-bound Mce(6) in tissues were assayed spectrophotometrically, while the levels of ADR and HPMA copolymer-bound ADR were determined using high-performance liquid chromatography. It appeared that the circulation lifetimes of HPMA copolymer-bound Mce(6) and ADR were three times more than those of the drugs in the free form. The concentrations of the HPMA copolymer-conjugated drugs in tumor reached maximum levels 18 h post injection. Intravenous injection routinely gave higher tissue levels of the drugs than intraperitoneal administration at time intervals less than 24 h. The biodistribution of the HPMA copolymer-bound drugs in tumor-bearing mice was significantly different from that of the free drugs, which is important in optimizing the treatment protocols. In particular, the HPMA copolymer-conjugated drugs accumulated at significantly higher levels in tumor tissues. This effect is attributed to the increased vascular permeability and reduced lymphatic drainage characteristic of tumor tissues [enhanced permeability and retention (EPR) effect].

Animals↗
PubMedDate not supplied

[Applications of PCR techniques for molecular epidemiology of infectious diseases].

The development of new PCR-based typing methods in the last years have supposed an important advance in the study of infectious diseases. Arbitrarily primed PCR (AP-PCR) and repetitive element sequence-based PCR (rep-PCR) are the most widely used PCR-based fingerprinting methods for bacteria and fungi. Major advantages of these methods are flexibility, technical simplicity and high discriminatory power. The AP-PCR presents problems of low inter-run and inter-laboratory reproducibility which make necessary the optimization of the protocol and reagents. PCR-RFLP is based in the enzymatic digestion of polymorphic genes amplified by PCR. This method is easy to perform and discriminatory, although less than AP-PCR or rep-PCR. Amplified fragment length polymorphism (AFLP) is a highly reproducible and discriminatory typing method based on the amplification by PCR of restriction fragments obtained from chromosomic DNA. This method is more discriminative and reproducible than AP-PCR, rep-PCR and PCR-RFLP, but it is more time-consuming and expensive, and requires specialised personnel. Most of these PCR-based typing methods are less time-consuming, rapid and easy to perform and of interpretation than pulsed-field gel electrophoresis (PFGE; gold standard method for typing most bacterium and fungi), but they usually are less discriminative and reproducible than PFGE, depending on the species studied and the method of PCR used. In summary, there are several PCR-based methods which are useful as a primary approach to the study of the clonal relationship among microbial isolates. The selection of the method to be used depend on technical (rapid, low time-consuming, easy to perform and to interpret, reproducible and discriminatory) and economical (low cost) factors.

Bacterial Typing Techniques↗
PubMed2005

[Helical CT of abdominal aorta].

The purpose was to illustrate that single or multirow spiral CT angiography has become the main modality for the examination of the abdominal aorta. Our multidisciplinary group investigates weekly 15 abdominal aortas, with a single slice spiral CT since 1995, and a 4 row detector spiral CT since 1999. CT scans protocols and parameters optimization for the 2 CT systems are described. Indications and results of CT to investigate the various abdominal aortic diseases are illustrated and discussed: aneurisms (pretherapeutic staging, selection of indications of endovascular stent-grafting); obstructive aorto-arteriopathies (pretherapeutic staging, follow up after endovascular revascularization); dissection (diagnosis, pretherapeutic staging, follow up); inflammatory diseases; normal patterns and principal complications after surgery. Situations where multislice CT is superior to single slice CT and compared advantages of MRI are discussed. CT is at the present time the main technique to image abdominal aorta. Multislice CT is especially interesting to explore the whole aorta, poly traumatisms, and to plan and follow up endovascular treatments.

Aortic Dissection↗
PubMed1997

DNA vaccination followed by macromolecular multicomponent peptide vaccination against HIV-1 induces strong antigen-specific immunity.

The induction of a strong and long-lasting immunity characterized by both a humoral and cell-mediated immune (CMI) response is one of the most important considerations in developing an effective HIV vaccine. In previous studies, we have independently developed both DNA vaccine and macromolecular multicomponent peptide vaccine (VC1) candidates. In the present study, we attempted to optimize the vaccination protocol using mice, guinea pigs, rabbits and Macaca fuscata monkeys. Repeated vaccination with VC1 induced a substantial level of multivalent antibodies which neutralized various HIV-1 strains, as determined using a p24 inhibition assay. On the other hand, repeated immunization with DNA vaccine induced and sustained high levels of cytotoxic T lymphocytes (CTLs). In addition, when DNA vaccination was followed by multicomponent peptide vaccination, levels of both humoral immunity and CMI increased, and this effect continued for at least 10 months. These data clearly demonstrate that for inducing HIV-1 specific immunity, immunization with DNA vaccine followed by VC1 boosting produces better results than immunizing with either vaccine alone.

AIDS Vaccines↗
PubMed2000

Theoretical modeling of the effects of shock duration, frequency, and strength on the degree of electroporation.

Electroporation is becoming an increasingly important tool for introducing biologically active compounds into living cells, yet the effectiveness of this technique can be low, particularly in vivo. One way to improve the success rate is to optimize the shock protocols, but experimental studies are costly, time consuming, and yield only an indirect measurement of pore creation. Alternatively, this study models electroporation in two geometries, a space-clamped membrane and a single cell, and investigates the effects of pulse duration, frequency, shape, and strength. The creation of pores is described by a first order differential equation derived from the Smoluchowski equation. Both the membrane and the cell are exposed to monophasic and biphasic shocks of varying duration (membrane, 10 micros-100 s; cell, 0.1 micros-200 ms) and to trains of monophasic and biphasic pulses of varying frequency (membrane, 50 Hz-4 kHz; cell, 200 kHz-6 MHz). The effectiveness of each shock is measured by the fractional pore area (FPA). The results indicate that FPA is sensitive to shock duration only in a very narrow range (membrane, approximately 1 ms; cell, approximately 0.25 micros). In contrast, FPA is sensitive to shock strength and frequency of the pulse train, increasing linearly with shock strength and decreasing slowly with frequency. In all cases, monophasic shocks were at least as effective as biphasic shocks, implying that varying the strength and frequency of a monophasic pulse train is the most effective way to control the creation of pores.

Electroporation↗
PubMed2003

3-D imaging with MDCT.

Without doubt, the greatest challenge of multidetector-row CT is dealing with 'data explosion'. For our carotid/intracranial CT angiograms, we routinely have 375 images to review (300 mm coverage reconstructed every 0.8 mm); for aortic studies we have 450-500 images ( approximately 600 mm coverage reconstructed every 1.3 mm); and for a study of the lower extremity inflow and run-off, we may generate 900-1000 transverse reconstructions. While we could reconstruct fewer images for these data, experience with single-detector row CT scanners indicates that longitudinal resolution and disease detection is improved when at least 50% overlap of cross-sections is generated [Radiology 200 (1996) 312]. If we are to optimize our clinical protocols and take full advantage of these CT scanners, we will need to change the way that we interpret, transfer, and store CT data. Film is no longer a viable option. Workstation based review of transverse reconstructions for interpretation is a necessity, but the workstations must improve to provide efficient access to these data, and we must have a way of providing our clinicians with images that can be transported to clinics and the operating room. Alternative visualization and analysis using volumetric tools, including 3-D visualization must evolve from luxury to necessity. We cannot rest on historical precedent to interpret these near isotropically sampled volumetric data using transverse reconstructions alone [Radiology 173 (1989) 527]. Although the tools for volumetric analysis on 3-D workstations have evolved over recent years, they have probably not yet evolved to a level that routine interpretation can be performed as efficiently and accurately as transverse section review. Both hardware and software developments must occur. While current computer workstations and visualization software are certainly adequate for assessing these MDCT data volumetrically, the process is very time consuming. What follows are a description of current workstation capabilities and a brief discussion of where development needs to go to facilitate the complete integration of volumetric analysis into the interpretive process of CT data.

Humans↗
PubMed2001

Proteomics -- the protein expression technology to study connective tissue biology.

During the formation of peribronchial fibrosis in asthma, remodeling of connective tissue is due to an increase in deposition of extracellular matrix components like that of specific types of collagens and proteoglycans. By taking bronchial biopsies, we were able to isolate cell cultures derived from asthmatic patients and healthy volunteers, which provides a good model system to study differences regarding cell morphology and key connective tissue proteins in the remodeling process. Proteomics, utilizing two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. By using this powerful tool, it is possible to quantitatively study protein regulation and to obtain increased knowledge about the mechanism behind the inflammatory process and formation of peribronchial fibrosis. We have optimized a proteomic protocol enabling detailed investigation of the protein expression pattern in human lung cells. An increased expression pattern was obtained, whereby 20 protein spots could be detected by image analysis in the <45 kDa region. Out of these, specific regulations of four spots were found by quantitative image analysis and spots of interest were identified by MALDI TOF-MS. This protocol enables us to study 1000--2000 proteins simultaneously and the possibility to correlate protein expression to the physiological status of the cell culture investigated. We have found that two proteins, actin and tropomyosin, are increased in expression due to transforming growth factor-beta stimulation. These proteins are correlated to the transformation of normal fibroblasts to myofibroblasts which are involved in the remodeling processes observed in asthma.

Asthma↗
PubMed1997

Osmotherapy. Basic concepts and controversies.

Osmotherapy with compounds such as mannitol has become a mainstay of neurologic and neurosurgical intensive care. Elevated intracranial pressure is the most common indication. A substantive debate remains as to the appropriate timing of administration and the optimal fluid management protocol, and experts disagree about the clinically relevant mechanisms of action of osmotic diuretics. This article briefly summarizes the basic literature on the physical actions of mannitol, addresses commonly asked questions, and highlights some of the controversies that arise at the bedside.

Blood-Brain Barrier↗
PubMed1998

Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity.

In the absence of a universal specific molecular tracer of apoptosis, structural DNA alterations provide the basis of labeling systems: double-strand fragmentation for TUNEL (terminal transferase-mediated dUTP nick end-labeling), denaturation for poly (A) in situ hybridization, immunogenicity of single strand DNA, all methods which imply limited specificity due to the unavoidable presence of DNA breaks in virtually all cells. Thus, TUNEL application has been restrained to a narrow spectrum of sample conditions which has limited, in particular, retrospective surveys and apoptotic nuclei-protein double labelings. In the apoptotic nucleus two main obstacles intervene between TUNEL reagents and their targets: DNA hypercondensation and proteins around DNA. The former increases in the course of apoptosis and both are worsened by crosslinking and precipitating fixatives. This point out that TUNEL is an ambitious approach whose target, apoptotic DNA breaks, is less accessible than breaks occurring in non-apoptotic less compacted DNA. However, TUNEL has an advantage: the far greater degree of apoptotic DNA fragmentation. How to obtain a frank differential staining between apoptotic and non-apoptotic DNA? It appears that the answer relies on the pretreatment step and not in modifying the TUNEL staining protocol, which is optimal. Adapted pretreatments are able to circumvent accessibility obstacles and to extend TUNEL applicability to the most demanding conditions, those of archived tissue samples and of TUNEL--protein double labelings.

Apoptosis↗
PubMed1998

Three-dimensional CT angiography: renal applications.

CT angiography has become an effective noninvasive imaging modality for evaluating the renal vasculature. The current clinical applications for CT angiography and three-dimensional (3D) imaging related to the kidneys and renal vasculature include evaluation of renal artery stenosis, renal diseases related to aortic diseases, living-related renal donor candidates, and preoperative evaluation of renal masses for possible nephron sparing surgeries. Imaging parameters need to be individually prescribed for renal CT angiography depending on the diagnostic goal of the examination. Methods for optimizing spiral CT protocols including patient preparation, contrast administration, image acquisition parameters, and image reconstruction will be covered.

Angiography↗
PubMed2001

Nonlocalized lower gastrointestinal bleeding: provocative bleeding studies with intraarterial tPA, heparin, and tolazoline.

PURPOSE: The purpose of this study was to assess the efficacy and safety of provocative mesenteric angiography with tissue plasminogen activator (tPA), heparin, and tolazoline in patients with nonlocalized lower gastrointestinal (LGI) bleeding. Results were examined to assess the clinical impact of the study on patients who had positive or negative results from elective provocative bleeding studies. MATERIALS AND METHODS: Seventeen provocative bleeding studies for occult LGI bleeding were performed in 16 patients, nine of whom were women, aged 44-79 years. All patients had negative results from previous endoscopic and angiographic studies. Patients' requirements for blood transfusion ranged from 6 to 69 units. Studies were performed electively. Blood group matching and cross-matching were performed for all patients. To provoke bleeding, a combination of intravenous heparin, intraarterial tolazoline, and intraarterial tPA was used. Doses used included 3,000-10,000 U heparin, 25-100 mg intraarterial tolazoline, and 10-50 mg intraarterial tPA (mean, 20.3 mg). Duration of follow-up was 3-34 months. RESULTS: Seventeen elective provocative studies were performed in 16 patients with occult LGI bleeding, leading to provoked bleeding in six patients (37.5%). In addition, two previously undiagnosed vascular abnormalities were diagnosed, which did not bleed during provocation. Therefore, an abnormality was identified in eight of 16 patients (50%) overall. There were no procedural complications encountered during or after any of the 17 procedures. In six patients in whom bleeding was successfully provoked, four bleeding episodes occurred in the large bowel and two occurred in the small bowel. Five of the positively provoked patients had a previously positive tagged red cell scintigraphic study. Three patients had superselective embolization at the time of provoked bleeding. Two were treated with estrogen therapy, and one patient was treated palliatively. Five of these six patients required no further therapy for LGI bleeding. Ten patients (including two with vascular abnormalities) did not bleed during the provoked study with tPA. Follow-up of the group of eight patients with completely normal study results ranged from 3 to 34 months in duration, and during the follow-up period, five patients experienced repeated bleeding and one had no further bleeding. One patient was diagnosed with an ileal vascular lesion during subsequent intraoperative enteroscopy and underwent surgical resection. One patient was lost to follow-up. CONCLUSION: Intraarterial provocative mesenteric angiography with heparin, vasodilator, and tPA identified the site of bleeding in 37.5% of patients in our study group and contributed to treatment in 50%. This small study indicates that the procedure appears to be safe, with no complications encountered in this series. Larger prospective studies are needed to fully assess the safety and efficacy of the technique and to optimize the pharmacologic protocol and patient selection.

Adult↗
PubMed2002

MR imaging of the globe and optic nerve.

MR imaging is an important tool for the evaluation of the optic nerve and globe. A working knowledge of the clinical questions an ophthalmologist faces helps radiologists optimize MR imaging protocols and focus their attention on the important clinical issues.

Eye↗
PubMed2002

Comparison of accuracy and interreader agreement in side-by-side versus independent evaluations of MR imaging of the medial collateral ligament of the elbow.

RATIONALE AND OBJECTIVES: The authors compared independent and side-by-side evaluation of magnetic resonance (MR) images of the medial collateral ligament (MCL) of the elbow, with regard to sensitivity, specificity, and interreader agreement MATERIALS AND METHODS: Six MR imaging sequences were used to image the MCLs in 28 cadaveric specimens, eight with surgically created lesions. Two reading methods were used. For independent evaluation, the images were first evaluated independently and rated on a five-point scale by two musculoskeletal radiologists experienced in interpreting MR images and blinded to the MCL integrity. The images were then reevaluated on the same scale by both readers after at least 2 weeks, with images from all six sequences shown side by side. For each MR sequence and reading method, the sensitivity and specificity were estimated nonparametrically, and differences were tested with the McNemar test. Interreader agreement was assessed with a K statistic, and differences were tested with Z and chi2 tests after adjustment for the dependence structure between correlated K statistics. RESULTS: For all sequences, side-by-side evaluation generally yielded higher specificity than independent evaluation, as well as better agreement between readers. CONCLUSION: Observer performance is superior when multiple MR imaging pulse sequences are reviewed simultaneously rather than independently and separately. Side-by-side review of different MR pulse sequences enabled higher accuracy and lower interreader variability for evaluation of the elbow MCL. These findings have implications for the design of studies to optimize MR imaging protocols by using multiple pulse sequences and multiple readers.

Aged↗