Use of a simple light absorbance assay to measure lymphocyte proliferation

The proliferative response of human lymphocytes to stimuli such as foreign histocompatibility antigens or mitogens is generally assessed by measuring the amount of tritiated thymidine which the cells incorporated in culture. In this paper, the possibility of assessing lymphocyte proliferation and viability by an empirical assay, using measurement of light absorbance on a ELISA reader in the yellow wave length (450 nm/air-550 nm/air), has been studied. The correlation of these measurements with a colormetric viability assay using MTS/PMS, with tritiated thymidine incorporation and with trypan blue exclusion viability counting, was determined. The results showed that the light absorbance assay correlated well with cell proliferation during 48-120 hours culture period and with cell viability after a 72 hour period. The MTS/PMS colormetric assay as well as trypan blue exclusion cell counting confirmed that the light absorbance assay was not merely caused by dead cells. This data confirm that the light absorbance assay is sufficiently sensitive to low levels of proliferation to allow detection of such responses at least as effectively as thymidine incorporation. The light absorbance assay procedure avoids the expense, time and hazards associated with scintillation counting, and is simple to perform without the necessity for reagents and preparative steps required by other assays.     

Reduction of restenosis after angioplasty in an atheromatous rabbit model by suicide gene therapy

BACKGROUND: Gene delivery of the thymidine kinase (tk) gene combined with ganciclovir (GCV) limits intimal hyperplasia after abrasion of normal arteries. However, the low efficiency of adenoviral-mediated gene transfer to atherosclerotic arteries has raised concerns about the applicability of this strategy to the prevention of restenosis. METHODS AND RESULTS: A replication-defective adenoviral vector expressing tk (Ad-RSVtk) demonstrated selective toxicity toward GCV-treated arterial smooth muscle cells, with oligonucleolytic cleavage suggesting apoptosis. In vivo, after demonstration of tk expression after Ad-RSVtk delivery, the combination of Ad-RSVtk followed by GCV was tested in a rabbit model of angioplasty of atheromatous iliac arteries. Angioplasty (8 atm, 20 minutes) was performed by use of a hydrogel balloon coated with Ad-RSVtk (4x10(9) plaque forming units). GCV was infused (25 mg.kg(-1) I.V. BID) from days 2 through 7 after angioplasty in 8 of 12 rabbits. Four weeks later, morphometric analysis demonstrated a reduced intima-to-media ratio in the group receiving combination therapy compared with Ad-RSVtk alone (3.0+/-1.2 versus 5.2+/-0.5, P<.018). GCV per se had no effect on intimal hyperplasia after arterial injury. CONCLUSIONS: In vitro, Ad-RSVtk demonstrates selective toxicity toward GCV-treated arterial smooth muscle cells involving apoptosis. In vivo, GCV conditions reduction of neointimal formation after percutaneous delivery of Ad-RSVtk during angioplasty of atheromatous arteries.     

Studies of the cellular mechanisms underlying the vasorelaxant effects of rutaecarpine, a bioactive component extracted from an herbal drug

We conducted studies to investigate the nature and underlying mechanisms of the vascular effects of rutaecarpine (Rut), an alkaloid isolated from the Chinese herbal drug Evodia rutaecarpa. By using largely the effects on phenylephrine (PE)-induced contraction in the isolated rat aorta as the experimental index and by comparison with several known vascular muscle relaxants such as acetylcholine (ACh), histamine, and A23187, Rut relaxed PE-precontracted aorta in concentration-(10(-7)-10(-4) M) and endothelium-dependent manners. Studies with appropriate antagonists indicated that this was coupled to nitric oxide (NO) and guanylyl cyclase. Extracellular Ca2+ removal and treatment with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), suggested that influx of extracellular Ca2+ was the major factor contributing to the action of Rut. Pertussis toxin suppressed the relaxation potency of histamine but had no effects on the actions of Rut. NaF, the G proteins activator, attenuated the actions of ACh, but only minimally affected Na-NP, A23187, and Rut. 1-[6-{[17 beta-3-methoxyestra-1,2,3(10)-trien-17-yl]amino} hexyl]-1H-pyrrole-2,5-dione (U73122), the phospholipase C inhibitor, again suppressed the actions of ACh but had few effects on A23187 and Rut. Taken together, these results suggest that these vasorelaxants had different cellular mechanisms and that neither pertussis toxin-sensitive Gi protein, other G proteins, nor phospholipase C activation was involved in the cellular response to rutaecarpine.     

Three-dimensional contrast-enhanced magnetic resonance angiography of the thoracic vasculature

Magnetic resonance angiography (MRA) has become a useful non-invasive imaging technique for the assessment of vascular disease. Due to limitations such as respiratory motion artefacts, saturations problems, and long acquisition times, applications of MRA in the thorax have largely been restricted to imaging of the aorta. The recent introduction of breath-hold three-dimensional (3D) contrast-enhanced MRA promises not only to enhance conventional MR protocols for aortic imaging, but to extend the clinical indications of MRI to diseases affecting other vascular structures of the thorax, most notably the pulmonary arteries. This article describes the technical aspects of contrast-enhanced 3D MRA and reviews existing and potential future clinical applications.     

Molecular genetics and clinical-pathology features of hereditary nonpolyposis colorectal carcinoma (Lynch syndrome): historical journey from pedigree anecdote to molecular genetic confirmation

The myocardium of the left ventricle of the heart is a fibrous structure, the fibres being wound helically anticlockwise on the epicardium and changing progressively through the wall thickness to being wound helically clockwise on the endocardium. At any point, therefore, the material can be considered as transversely isotropic. For mechanically modelling the ventricle, two independent material properties, the along-fibre and across-fibre moduli of elasticity are required. This paper describes a computer method for determining these by using a finite element model and matching cavity volume and ventricle length against values derived from cineangiographic and pressure data. The method is applied to a group of patients with various cardiac diagnoses and the modular ratios for their myocardia are derived.     

Changes in specific markers of haemostasis during reduction mammoplasty

We herein report a case of pure clear cell papillary thyroid carcinoma, which is the first reported case in Japan. The tumors measured 1.0 x 0.9 and 0.7 x 0.4 cm in size. An ultrasonographical examination revealed hypoechoic irregular-shaped lesions with fine internal calcifications. No lymph node metastasis was observed in any of the surgical specimens. Distant metastasis had not been observed as of 6 years after surgical treatment. The number of cases of pure clear cell papillary carcinoma reported so far are too few to clearly elucidate its characteristics; however, the ultrasonographical findings and biological behavior of this case were compatible with those of non-clear cell papillary thyroid carcinoma.     

Involvement of dsRNA virus in the protein composition and growth kinetics of host Trichomonas vaginalis

Trichomonas vaginalis harbors a double-stranded (ds)-RNA virus, and the presence of virus is related to upregulated expression and phenotypic variation of a prominent immunogen (Khoshnan A, Alderete JF (1994) J Virol 68: 4035-4038). To further test the influence of virus on T. vaginalis, virus-infected (V+) isolates were compared to virus-free (V-), agar-cloned progeny trichomonads derived from the parental isolates for accumulation of total proteins and cysteine proteinases. Comparative high resolution two dimensional (2D)-SDS-PAGE was performed of trichomonads grown in a chemostat under identical conditions. At least 47 proteins were identified as specifically expressed by representative V+ isolate 347, and approximately 41 spots were specific to the corresponding V- progeny, showing an association between virus and the presence and absence of parasite proteins. Qualitatively and quantitatively dissimilar cysteine proteinase patterns were detected from numerous V+ isolates and the V- progeny. A 2D analysis for isolate 347 showed the appearance of unique proteinase activities for parental parasites and presence of at least one proteinase in the V- progeny. Finally, the V+ T. vaginalis isolate 347, but not the V- isolate 347 progeny nor other V+ isolates, underwent fluctuations in density during chemostat growth allowing for purification of virus particles from the V+ isolate 347 supernatants during decreased parasite density.     

Late-life depression and functional disability: the role of goal-focused group psychotherapy

This study compares the efficacy of two time-limited group psychotherapies for depression and functional disability in late life. Goal-focused group psychotherapy (GFGP) utilized focused psychoeducation and skills training to assist each patient in the achievement of individualized goals. Reminiscence therapy (RT) emphasized individual life review to facilitate discussion. Subjects (N = 13), ages 55 and above, with major depression were randomly assigned to one of the two groups. Most were receiving antidepressant treatment. All had failed to achieve full remission. Whereas both treatment groups improved in depressed mood and disability, GFGP subjects had a far greater change in depressive symptomatology and also improved in the areas of hope, hopelessness, anxiety and social functioning.     

Production of anti-Ro/SS-A and anti-La/SS-B autoantibodies is closely coordinated in systemic lupus erythematosus and independent of anti-dsDNA production

Capillary zone electrophoresis (CZE) was utilized to identify a synaptobrevin-thioredoxin fusion protein (TSB-51). TSB-51 is a substrate for cleavage by botulinum toxin B at the Q(76)-F(77) site. TSB-51 was derivatized with a fluorophore, CBQCA [3-(4-carboxy-benzoyl)-2-quinoline-carboxaldehyde], for 4 h at room temperature. Optimal conditions for CZE separation of the TSB-51-CBQCA complex were determined: buffer (sodium borate), pH (9.0), applied voltage (25 kV), temperature (25 degrees C) and forward polarity. SDS-PAGE showed that TSB-51 had a molecular mass of approximately 19 kDa. The protein was transferred to PVDF membrane and sequenced by the Edman degradation method verifying the first twelve amino acids as SDKIIHLTDDSF. TSB-51 was also collected during CZE separation and subsequently sequenced yielding the first three amino acids as SDK. This CZE-LIF method coupled with the CBQCA derivatization, fraction collection and Edman sequencing allowed for identification of the recombinant protein, a fast separation run time and utilization of small volumes of peptide (1.5 ng protein/23.6 nl injection). This method will be used for monitoring the endopeptidase activity of botulinum toxin B on TSB-51.     

Early diagnosis of lentivirus-induced infiltrative lung disease in sheep by high resolution computed tomography

The visna-maedi lentivirus can induce an interstitial pneumonitis in sheep, and provides a convenient example to study natural or experimental lentiviral pathology. We wanted to determine whether high resolution computed tomography (HRCT) is able to detect early morphological changes following lentiviral infection in the lungs. Spontaneously infected adult sheep (n=3) and experimentally infected lambs (n=5) were compared to uninfected controls (n=4). The HRCT scans generally showed abnormal features in infected animals, including: increased parenchymal density; alveolar oedema; thickened interlobular septa; and increased density in peribronchiolar areas. HRCT was more sensitive than chest radiography for the early diagnosis of interstitial pneumonitis, although one sheep with advanced disease and radiographic and histopathological abnormalities had a paradoxically normal scan. One control animal showed minor abnormalities on scanning, which were probably due to the procedure used for anaesthesia. The HRCT observations were confirmed by postmortem histological examination of the lungs. In conclusion, high resolution computed tomography provides a noninvasive means of following the development of lung pathology in a natural ovine model of lentiviral disease.