Test-retest variability of resting-state networks in healthy aging and prodromal Alzheimer's disease.

In recent years, changes in resting-state networks (RSN), identified by functional magnetic resonance imaging (fMRI), have gained increasing attention as potential biomarkers and trackers of neurological disorders such as Alzheimer's disease (AD). Intersession reliability of RSN is fundamental to this approach. In this study, we investigated the test-retest reliability of three memory related RSN (i.e., the default mode, salience, and executive control network) in 15 young, 15 healthy seniors (HS), and 15 subjects affected by mild cognitive impairment (MCI) with positive biomarkers suggestive of incipient AD (6 females each). FMRI was conducted on three separate occasions. Independent Component Analysis decomposed the resting-state data into RSNs. Comparisons of variation in functional connectivity between groups were made applying different thresholds in an explorative approach. Intersession test-retest reliability was evaluated by intraclass correlation coefficient (ICC) comparisons. To assess the effect of gray matter volume loss, motion, cerebrospinal fluid based biomarkers and the time gap between sessions on intersession variation, the former four were correlated separately with the latter. Data showed that i) young subjects ICCs (relative to HS/MCI-subjects) had higher intersession reliability, ii) stringent statistical thresholds need to be applied to prevent false-positives, iii) both HS and MCI-subjects (relative to young) showed significantly more clusters of intersession variation in all three RSN, iv) while intersession variation was highly correlated with head motion, it was also correlated with biomarkers (especially phospho-tau), the time gap between sessions and local GMV. Results indicate that time gaps between sessions should be kept constant and that head motion must be taken into account when using RSN to assess aging and neurodegeneration. In patients with prodromal AD, re-test reliability may be increased by accouting for overall disease burden by including biomarkers of neuronal injury (especially phospho-tau) in statistical analyses. Local atrophy however, does not seem to play a major role in regards to reliability, but should be used as covariate depending on the research question.

The morphological and spectral phenotype of apoptosis in HeLa cells varies following exposure to UV-C and the addition of inhibitors of ICE and CPP32.

Numerous extra- and intracellular factors, including UV radiation, can initiate a programme of cell death by apoptosis. While apoptosis is commonly defined morphologically, the relationships between morphology and molecular events are not well established. To investigate these relationships in HeLa cells, eight morphometric criteria for cell proliferation and damage and 10 criteria for apoptotic phenotype were examined using light microscopy, and corroborated by ultrastructure and spectral imaging. They were identified (1) during a time course after irradiation with 0, 10 or 30 J/m2 UV-C; (2) after separation of apoptotic from normal cells on a Percoll gradient; and (3) after irradiation with UV-C plus perturbation of the apoptotic pathway by treatment with inhibitors of two caspases, ICE and CPP32. The number of cells in apoptosis increased in a dose-dependent manner after UV-C treatment. Centrifugation of irradiated cells on a Percoll gradient increased the collection of apoptotic cells tenfold. The stereotypical apoptotic phenotype, in which cells have deep cytoplasmic blebbing and highly condensed DNA, comprised only a few percent of all apoptosis, and was rarely seen in groups receiving caspase inhibitors. The most common apoptotic phenotype was a rounded cell with large spherical nucleolus and associated DNA. After treatment with UV-C plus inhibitors the apoptotic index was decreased by about 30% compared to UV-C radiation alone. These apoptotic cells had dark spherical cytoplasm with small blebs, greatly increased numbers of cytoplasmic ribosomes, abundant nucleolar material with a large separate granular component, and chromatin condensed at the nuclear membrane. Using the technique of spectral imaging, it was found that the spectrum obtained from the granular component of the nucleolus, which was elevated in apoptotic cells treated with UV-C plus inhibitors, was similar to the dense accumulation of ribosomes in the apoptotic cytoplasm. The data indicate that spectral imaging may be a useful tool for identifying and characterizing variations in the apoptotic process, and that the caspase inhibitors used here do not completely abolish UV-C induced apoptosis, but rather alter its incidence and progression.

Saccomanno smear slides and Megafunnel slides for sputum specimens. A comparison.

OBJECTIVE: To compare Megafunnel slides to standard Saccomanno smear slides of sputum specimens and evaluate the use of Megafunnel slides for retrospective studies. STUDY DESIGN: Papanicolaou-stained Saccomanno smear and Megafunnel slides (Shandon Lipshaw, Inc., Shandon Inc., Pittsburgh, Pennsylvania, U.S.A.) of 65 clinical sputum specimens from 51 patients were compared for cellular morphology, staining, background and cytologic diagnosis. Recovery of diagnostic cells was quantitated using 10 of these specimens. Megafunnel slides prepared from the clinical sputum samples were immunocytochemically stained. Diagnostic cells were quantitated both before removal from 64 archived Saccomanno smear slides and after placement of these cells onto 238 Megafunnel slides. RESULTS: Saccomanno smear slides and Megafunnel slides of clinical specimens were similar in morphology, background, staining, diagnosis and cell recovery. Megafunnel slides were superior for multiple immunocytochemical stains. The production of multiple Megafunnel slides from archival smear slides provided a method of performing numerous retrospective studies. CONCLUSION: Megafunnel slides compared favorably to Saccomanno smear slides in the quality of specimens but are more expensive and labor intensive to prepare. However, the reduction in screening time by cytotechnologists may be advantageous. Additionally, their potential use for immunocytochemistry, fluorescence in situ hybridization, or other special clinical and research analyses is very promising.

Examination of p53 alterations and cytokeratin expression in sputa collected from patients prior to histological diagnosis of squamous cell carcinoma.

Mutations in the p53 gene are detected in greater than 50% of squamous cell carcinomas of the lung and to a lesser extent in adenocarcinomas. The p53 protein is also overexpressed in a relatively high percentage of preinvasive lesions of the bronchial epithelium. However, unlike tumor tissue, immunoreactivity does not necessarily imply that cells in preinvasive lesions carry a mutant p53 allele. In some cases, overexpression may result from a cellular checkpoint reaction to a toxic or mutagenic substance such as exposure to tobacco smoke. In any case, p53 overexpression in preinvasive lesions may serve as a biomarker for high risk assessment of lung cancer and other tumors in the aerodigestive tract. A study was designed to retrospectively analyze p53 overexpression in cells from sputum samples collected prior to histological tumor diagnosis. The rationale was based on the observation that both preinvasive and tumor cells from the bronchial epithelium are exfoliated into the airways and can be detected based on morphology in sputa. Two sets of cases were chosen: 1) patients whose first primary tumor was a squamous cell carcinoma containing a mutant p53 allele with overexpression observed in most of the tumor cells; and 2) patients whose squamous cell tumor did not contain a mutant p53 allele. Cells which stained positive for p53 expression were observed in sputum samples collected from all six patients whose tumors were positive for a mutant p53 allele. Also p53 positive cells were detected on sputum slides for two of the five cases where the tumor DNA did not contain a mutation and/or tumor cells which overexpress p53 were not detected in tissue sections. Although cells which stained positive for p53 were present in sputum from patients whose tumors contained a missense mutation, the presence of p53 overexpression was not specific for tumors which contain an altered p53 allele since overexpression was detected in sputum cells from patients whose tumor DNA did not contain a p53 mutation and/or tumor cells which stained positive for p53 were not observed in tissue sections. However, the p53 positive cells in sputa collected from the latter group of patients could have been exfoliated from other lesions which contained a mutant p53 allele. The accumulation of p53 in some sputum cells was concomitant with expression of simple epithelial type cytokeratins (CK) 8 and 18 or at least one of the other cytokeratins detected by a broad spectrum (PAN) CK antibody mixture. These data imply that most of the sputum cells which overexpress p53 are epithelial cells. Moreover, our results are consistent, at least in part, with other observations that cells which overexpress p53 in dyplasias and hyperplasias express CK 8, 18. We will continue to explore the possibility that expression of cytokeratins 8, 18 and/or other cytokeratins in conjunction with p53 overexpression and/or morphological criteria could define a new class of atypical cells which are predisposed to cancer development.