Identifying and quantifying apoptosis: navigating technical pitfalls.

Apoptosis or programmed cell death is often altered in malignancies and is frequently determined by the terminal transferase-mediated nick end labeling technique (TUNEL). However, commercially available protocols can produce high background and false-positive staining, which renders the distinction between apoptosis and necrosis difficult. In an attempt to develop a rapid and reproducible method for detecting and quantifying apoptosis, we coupled optimization of the Apoptag Plus Peroxidase In Situ Apoptosis Detection kit with quantitative histomorphometric computer imaging software using the Bacus Laboratories Incorporated Slide Scanner (BLISS). Multiple (200-350) unique 40x images were scanned using the BLISS system and downloaded into the WebSlide Browser program, creating a permanent, scanned record of the area assessed. The stored images were counted, with the final analysis simultaneously taking into account cells that were immunohistochemically positive and the histology of the surrounding cells to reduce the possibility of false positive and negative staining. In addition, cells with equivocal staining can be simultaneously reviewed by other technologists with networked WebSlide Browser access to the same images. Our data show that the advantages offered by the BLISS imaging software greatly reduce the potential drawbacks of using the TUNEL method as a sole means of quantification.     

Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting.

We have developed a simplified procedure for the ligation-mediated polymerase chain reaction (LMPCR) using Thermococcus litoralis DNA polymerase (Vent DNA polymerase). We show that Vent DNA polymerase produces correct, blunt-ended primer extension products with substantially higher efficiency than Thermus aquaticus (Taq) DNA polymerase or modified T7 DNA polymerase (Sequenase). This difference leads to significantly improved genomic sequencing, methylation analysis, and in vivo footprinting with LMPCR. These improvements include representation of all bands with more uniform intensity, clear visualization of previously difficult regions of sequence, and reduction in the occurrence of spurious bands. It also simplifies the use of DNase I cut DNA for LMPCR footprinting.     

Nosocomial Legionnaires' disease. Epidemiologic demonstration of cooling towers as a source

Investigation of a recent outbreak of nosocomial legionnaires' disease--initially thought to be due to the documented presence of Legionella pneumophila in the hospital potable water--showed that aerosols from one or more cooling towers were the actual source of infection. From June 27 to Aug 25, 1983, nosocomial legionnaires' disease developed in 15 persons at a hospital in Rhode Island. Twelve (80%) of 15 case-patients occupied rooms in building 1, unit B, compared with eight (28%) of 29 control patients (odds ratio = 10.8; 95% confidence interval = 1.4 to 85.6). Subsequent investigation demonstrated that water in a cooling tower located 100 ft upwind of unit B was heavily contaminated with L pneumophila, serogroup 1, subgroup 1, 2, 4, 5. The same strain was isolated from nine of the patients and from the make-up water for the tower. Active surveillance during the ten months following decontamination of the cooling tower identified no additional cases of nosocomial legionnaires' disease, although the hospital potable water had not been treated. While recommendations have been made for controlling nosocomial legionnaires' disease by heating or hyperchlorination of hospital potable water, this outbreak demonstrates the importance of an adequate epidemiologic-environmental investigation in choosing the appropriate control strategy.     

Torsional diplopia after transantral orbital decompression and extraocular muscle surgery associated with Graves' orbitopathy.

Graves' orbitopathy can be associated with horizontal, vertical, and torsional diplopia. Of 428 patients treated with transantral orbital decompression, 21 had incycloduction (mean, 12.8 degrees; range, 5 to 20 degrees) and five had excycloduction (mean, 12 degrees; range, 5 to 20 degrees). All 26 patients had had recessions of the medial or inferior rectus muscle (or both) before onset of torsional diplopia. Mean recession was 5.5 mm (range, 4 to 10 mm) and 5.3 mm (range, 2 to 10 mm) of medial rectus muscle and inferior rectus muscle, respectively. An A pattern was often associated with the condition. Superior oblique tenectomy and inferior oblique myectomy were performed most frequently for incycloduction and excycloduction, respectively. Superior oblique tenectomy induced a mean incycloduction decrease of 7.1 degrees (range, 0 to 12 degrees). Exotropia in downgaze was decreased, and a small ipsilateral hyperdeviation was induced. Bilateral inferior oblique myectomy in one patient decreased excycloduction 10 degrees without inducing new deviation. At follow-up (mean, 63.7 months) after last strabismus operation, 15 patients with incycloduction and two with excycloduction had no diplopia.     

Characterization of technetium-99m-L,L-ECD for brain perfusion imaging, Part 1: Pharmacology of technetium-99m ECD in nonhuman primates

Technetium-99m ethyl cysteinate dimer ([99mTc]ECD) is a neutral, lipophilic complex which rapidly crosses the blood-brain barrier. Brain retention and tissue metabolism of [99mTc]ECD is dependent upon the stereochemical configuration of the complex. While both L,L and D,D enantiomers are extracted by the brain, only the L,L but not the D,D form, is metabolized and retained in the monkey brain (4.7% injected dose initially, T 1/2 greater than 24 hr). Dynamic single photon emission computed tomography imaging studies in one monkey indicates 99mTc-L,L-ECD to be distributed in a pattern consistent with regional cerebral blood flow for up to 16 hr postinjection. Dual-labeled 99mTc-L,L-ECD and [14C]iodoantipyrine autoradiography studies performed 1 hr after administration show cortical gray to white matter ratios of both isotopes to be equivalent (approximately 4-5:1). These data suggest that 99mTc-L,L-ECD will be useful for the scintigraphic assessment of cerebral perfusion in humans.     

Isolated congenital tricuspid valve dysplasia: A rare condition mimicking persistent pulmonary hypertension of the newborn

ABSTRACT Isolated congenital tricuspid valve dysplasia is a rare and potentially lethal congenital heart disease that can be easily confused with persistent pulmonary hypertension of the newborn. We describe a neonate with isolated congenital tricuspid valve dysplasia who did not respond to mechanical ventilation but improved by tolazoline. Clinicians should be aware that the initial fulminant course of this condition may be reversed by reducing the pulmonary vascular resistance, thereby allowing time for spontaneous recovery.     

Digital subtraction angiography of the thoracic aorta

Forty-three patients with acquired and congenital abnormalities of the thoracic aorta were studied using digital subtraction angiography (DSA) after an intravenous bolus injection of 40 ml of contrast material. Abnormalities studied included coarctation, pseudocoarctation, Marfan syndrome, cervical aorta, double aortic arch, aneurysm, dissection, and tumor. Twenty-four patients also had conventional angiography. DSA was accurate in 95% of cases; in the other 5%, involving patients with acute type I dissection, the coronary arteries could not be seen. The authors concluded that in 92% of their patients, DSA could have replaced the standard aortogram.     

Personality factors in resistance to illness after recent life changes

Three personality dimensions, derived by factor analysis from the Omnibus Personality Inventory, are examined as possible intervening variables between recent life experience and subsequent health change in a college population. All three personality measures, social conformity, liberal intellectualism and emotional sensitivity, are found to be independently and significantly predictive of health change; these relationships hold even when recent life experience is introduced as a significant predictor of health change. The addition of personality measures to information about recent life experience sinificantly improves the predicability of deleterious health change. The results are discussed in the context of factors which promote resistance to health breakdown after life changes.