The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.     

A screening strategy for population studies in systemic lupus erythematosus

A survey design which uses two tests in series greatly increases the efficiency of screening. We conducted a study to ascertain the sensitivity, specificity, and predictive value of a screening questionnaire for systemic lupus erythematosus (SLE) patients. A series design in which this questionnaire is used, followed by an antinuclear antibody test, increased the predictive value of a positive screening result by 5 to 10-fold, depending on estimates of disease prevalence. An empirical study of the series design identified two new cases of SLE and indicated that the use of a questionnaire based on accepted criteria is theoretically sound and practical. This approach should prove useful for population surverys of SLE to avoid the sampling and detection biases of previous epidemiologic studies.     

Unresolved questions regarding human hereditary deafness

Human hearing loss is a common neurosensory disorder about which many basic research and clinically relevant questions are unresolved. This review on hereditary deafness focuses on three examples considered at first glance to be uncomplicated, however, upon inspection, are enigmatic and ripe for future research efforts. The three examples of clinical and genetic complexities are drawn from studies of (i) Pendred syndrome/DFNB4 (PDS, OMIM 274600), (ii) Perrault syndrome (deafness and infertility) due to mutations of CLPP (PRTLS3, OMIM 614129), and (iii) the unexplained extensive clinical variability associated with TBC1D24 mutations. At present, it is unknown how different mutations of TBC1D24 cause non‐syndromic deafness (DFNB86, OMIM 614617), epilepsy (OMIM 605021), epilepsy with deafness, or DOORS syndrome (OMIM 220500) that is characterized by d eafness, o nychodystrophy (alteration of toenail or fingernail morphology), o steodystrophy (defective development of bone), mental r etardation, and s eizures. A comprehensive understanding of the multifaceted roles of each gene associated with human deafness is expected to provide future opportunities for restoration as well as preservation of normal hearing.     

Attitudes about genetic risk of couples undergoing in-vitro fertilization

Many couples undergoing in-vitro fertilization (IVF) are at a higher risk of having a child with a genetic abnormality. In a sample of 55 consecutive couples starting IVF, only 33% had no genetic risk factor. The most common genetic risks were advanced maternal age and possible abnormalities associated with severe male infertility. Despite education on these risks, 71% of couples had no interest in receiving formal genetic counselling. Only 14% of couples at risk would consider using a gamete donor to avoid transmitting a genetic disorder to a child. The triple test to screen for fetal abnormalities was acceptable to 82% of couples, but only 47% planned to have amniocentesis or chorionic villi sampling. Couples were significantly more likely to opt for prenatal testing if they would consider terminating a pregnancy should the fetus have a severe genetic abnormality (P < 0.01). Roman Catholic couples tended to have more conservative attitudes about pregnancy termination. Socio-economic status and whether the infertility factor was male or female were not predictors of a couple's attitudes.      

Evaluation of [18F]-JNJ-64326067-AAA tau PET tracer in humans

The [¹⁸F]-JNJ-64326067-AAA ([¹⁸F]-JNJ-067) tau tracer was evaluated in healthy older controls (HCs), mild cognitive impairment (MCI), Alzheimer’s disease (AD), and progressive supranuclear palsy (PSP) participants. Seventeen subjects (4 HCs, 5 MCIs, 5 ADs, and 3 PSPs) received a [¹¹C]-PIB amyloid PET scan, and a tau [¹⁸F]-JNJ-067 PET scan 0-90 minutes post-injection. Only MCIs and ADs were amyloid positive. The simplified reference tissue model, Logan graphical analysis distribution volume ratio, and SUVR were evaluated for quantification. The [¹⁸F]-JNJ-067 tau signal relative to the reference region continued to increase to 90 min, indicating the tracer had not reached steady state. There was no significant difference in any bilateral ROIs for MCIs or PSPs relative to HCs; AD participants showed elevated tracer relative to controls in most cortical ROIs (P < 0.05). Only AD participants showed elevated retention in the entorhinal cortex. There was off-target signal in the putamen, pallidum, thalamus, midbrain, superior cerebellar gray, and white matter. [¹⁸F]-JNJ-067 significantly correlated (p < 0.05) with Mini-Mental State Exam in entorhinal cortex and temporal meta regions. There is clear binding of [¹⁸F]-JNJ-067 in AD participants. Lack of binding in HCs, MCIs and PSPs suggests [¹⁸F]-JNJ-067 may not bind to low levels of AD-related tau or 4 R tau.     

Radiation-induced red cell damage: role of reactive oxygen species

BACKGROUND: Cellular blood components are irradiated to prevent graft- versus-host disease in transfusion recipients at risk for this syndrome. Because gamma radiation can result in the production of reactive oxygen species, the role of reactive oxygen species was investigated in radiation-induced red cell damage. STUDY DESIGN AND METHODS: Whole blood from normal donors was exposed to various doses of t-butyl hydroperoxide (0-1 mM) and/or to gamma-radiation (0-50 Gy). Oxidative damage was assessed by the extent of lipid peroxidation (measured by thiobarbituric acid-reactive substances [TBARS]) and hemoglobin oxidation. Fresh blood was divided into three parts-one initially irradiated and stored, another stored with portions irradiated weekly, and a third stored without irradiation. TBARS and hemoglobin oxidation were measured weekly. RESULTS: As expected, t- butyl hydroperoxide induced TBARS formation and hemoglobin oxidation in a dose-dependent fashion. The gamma-radiation not only increased hemoglobin oxidation and TBARS formation, but also enhanced the t-butyl hydroperoxide effect on red cells. Red cell storage increased TBARS generation and hemoglobin oxidation in a time-dependent fashion. When radiation was administered either initially or after weekly storage, TBARS production and hemoglobin oxidation were increased over that measured in unirradiated paired controls. CONCLUSION: Gamma radiation at clinically used doses increases lipid peroxidation and hemoglobin oxidation in human red cells. The effect of gamma-radiation is accentuated by blood storage and induces damage independent of time of storage.     

A neural hypothesis for stress-induced headache

The mechanisms by which stress contributes to CTH are not clearly understood. The commonly accepted notion of muscle hyper-reactivity to stress in CTH sufferers is not supported in the research data. We propose a neural model whereby stress acts supra-spinally to aggravate already increased pain sensitivity in CTH sufferers. Indirect support for the model comes from emerging research elucidating complex supra-spinal networks through which psychological stress may contribute to and even cause pain. Similarly, emerging research demonstrates supra-spinal pain processing abnormalities in CTH sufferers. While research with CTH sufferers offering direct support for the model is lacking at present, initial work by our group is consistent with the models predictions, particularly, that stress aggravates already increased pain sensitivity in CTH sufferers.     

Man’s best friend? Infective endocarditis due to Capnocytophaga canimorsus

Infective endocarditis caused by zoonotic microorganisms is an uncommon clinical entity. A 55-year-old man was diagnosed with endocarditis due to Capnocytophaga canimorsus, a commensal bacterium contained in the saliva of dogs, that involved the aortic and tricuspid valves and was complicated by a para-aortic valve abscess and aorta-to-right atrial fistula. The patient was successfully treated with antibiotic therapy and surgical intervention. C canimorsus endocarditis should be considered in patients with culture-negative endocarditis, particularly in immunosuppressed, asplenic or alcoholic individuals who have recently suffered a dog bite or have had close contact with dogs.