Transcontinental communication and quantitative digital histopathology via the Internet; with special reference to prostate neoplasia

OBJECTIVE: To describe practical experiences in the sharing of very large digital data bases of histopathological imagery via the Internet, by investigators working in Europe, North America, and South America. MATERIALS: Experiences derived from medium power (sampling density 2.4 pixels/microm) and high power (6 pixels/microm) imagery of prostatic tissues, skin shave biopsies, breast lesions, endometrial sections, and colonic lesions. Most of the data included in this paper were from prostate. In particular, 1168 histological images of normal prostate, high grade prostatic intraepithelial neoplasia (PIN), and prostate cancer (PCa) were recorded, archived in an image format developed at the Optical Sciences Center (OSC), University of Arizona, and transmitted to Ancona, Italy, as JPEG (joint photographic experts group) files. Images were downloaded for review using the Internet application FTP (file transfer protocol). The images were then sent from Ancona to other laboratories for additional histopathological review and quantitative analyses. They were viewed using Adobe Photoshop, Paint Shop Pro, and Imaging for Windows. For karyometric analysis full resolution imagery was used, whereas histometric analyses were carried out on JPEG imagery also. RESULTS: The three applications of the telecommunication system were remote histopathological assessment, remote data acquisition, and selection of material. Typical data volumes for each project ranged from 120 megabytes to one gigabyte, and transmission times were usually less than one hour. There were only negligible transmission errors, and no problem in efficient communication, although real time communication was an exception, because of the time zone differences. As far as the remote histopathological assessment of the prostate was concerned, agreement between the pathologist's electronic diagnosis and the diagnostic label applied to the images by the recording scientist was present in 96.6% of instances. When these images were forwarded to two pathologists, the level of concordance with the reviewing pathologist who originally downloaded the files from Tucson was as high as 97.2% and 98.0%. Initial results of studies made by researchers belonging to our group but located in others laboratories showed the feasibility of making quantitative analysis on the same images. CONCLUSIONS: These experiences show that diagnostic teleconsultation and quantitative image analyses via the Internet are not only feasible, but practical, and allow a close collaboration between researchers widely separated by geographical distance and analytical resources.     

Correlation between extent of osteolytic damage and metastatic burden of human breast cancer metastasis in nude mice: real-time PCR quantitation

Orthotopic or intracardiac injection of human breast cancer cell lines into immunocompromised mice allows study of the molecular basis of breast cancer metastasis. We have established a quantitative real-time PCR approach to analyze metastatic spread of human breast cancer cells inoculated into nude mice via these routes. We employed MDA-MB-231 human breast cancer cells genetically tagged with a bacterial beta-galactosidase (Lac-Z) retroviral vector, enabling their detection by TaqMan real-time PCR. PCR detection was linear, specific, more sensitive than conventional PCR, and could be used to directly quantitative metastatic burden in bone and soft organs. Attesting to the sensitivity and specificity of the PCR detection strategy, as few as several hundred metastatic MDA-MB-231 cells were detectable in 100 microns segments of paraffin-embedded lung tissue, and only in samples adjacent to sections that scored positive by histological detection. Moreover, the measured real-time PCR metastatic burden in the bone environment (mouse hind-limbs, n = 48) displayed a high correlation to the degree of osteolytic damage observed by high resolution X-ray analysis (r2 = 0.972). Such a direct linear relationship to tumor burden and bone damage substantiates the so-called 'vicious cycle' hypothesis in which metastatic tumor cells promote the release of factors from the bone which continue to stimulate the tumor cells. The technique provides a useful tool for molecular and cellular analysis of human breast cancer metastasis to bone and soft organs, can easily be extended to other cell/marker/organ systems, and should also find application in preclinical assessment of anti-metastatic modalities.     

Surface antigen profiles of leukocytes and melanoma cells in lymph node metastases are associated with survival in AJCC stage III melanoma patients

There is an urgent need to identify more accurate prognostic biomarkers in melanoma patients, particularly in those with metastatic disease. This study aimed to identify melanoma and leukocyte surface antigens predictive of survival in a prospective series of AJCC stage IIIb/c melanoma patients (n = 29). Live cell suspensions were prepared from melanoma metastases within lymph nodes (LN). The suspensions were immuno-magnetically separated into CD45⁺ (leukocyte) and CD45^? (non-hematopoietic, enriched melanoma cell) fractions. Surface antigens on CD45^? and CD45⁺ cell populations were profiled using DotScan? microarrays (Medsaic Pty. Ltd.) and showed differential abundance levels for 52 and 78 antigens respectively. Associations of the surface profiles with clinicopathologic and outcome data (median follow-up 35.4 months post LN resection) were sought using univariate (log-rank test) and multivariate (Wald’s test; modelled with patient’s age, gender and AJCC staging at LN recurrence) survival models. CD9 (p = 0.036), CD39 (p = 0.004) and CD55 (p = 0.005) on CD45⁺ leukocytes were independently associated with distant metastasis-free survival using multivariate analysis. Leukocytes with high CD39 levels were also significantly associated with increased overall survival (OS) in multivariate analysis (p = 0.016). LNs containing leukocytes expressing CD11b (p = 0.025), CD49d (p = 0.043) and CD79b (p = 0.044) were associated with reduced OS on univariate analysis. For enriched melanoma cells (CD45^? cell populations), 11 surface antigens were significantly correlated with the disease-free interval (DFI) between diagnosis of culprit primary melanoma and LN metastasis resection. Nine antigens on CD45⁺ leukocytes also correlated with DFI. Following validation in independent datasets, surface markers identified here should enable more accurate determination of prognosis in stage III melanoma patients and provide better risk stratification of patients entering clinical trials.     

Time‐dependent effects of isoflurane and dexmedetomidine on functional connectivity,spectral characteristics,and spatial distribution of spontaneous BOLD fluctuations

Anesthesia is often necessary to perform fMRI experiments in the rodent model; however, commonly used anesthetic protocols may manifest changing brain conditions over the duration of the study. This possibility was explored in the current work. Eleven rats were anesthetized with 2% isoflurane anesthesia; four rats were anesthetized for a short period (30 min, simulating induction and fMRI setup) and seven rats were anesthetized for a long period (3 h, simulating surgical preparation). Following the initial anesthetic period, isoflurane was discontinued, and a dexmedetomidine bolus (0.025 mg/kg) and continuous subcutaneous infusion (0.05 mg/kg/h) were administered. Blood‐oxygen‐level dependent resting state imaging was performed every 30 min from 0.75 h post dexmedetomidine bolus until 5.75 h post‐bolus. Evaluation of power spectra obtained from time courses in the primary somatosensory cortex revealed, in general, a monotonic increase in low‐frequency power (0.05–0.3 Hz) in both groups over the duration of resting state imaging. Greater low‐band spectral power (0.05–0.15 Hz) is present in the short isoflurane group for the first 2.75 h, but the spectra become highly uniform at 3.25 h. The emergence of a ~0.18 Hz peak, beginning at the 3.75 h time point, exists in both groups and evolves similarly, increasing in strength as the duration of dexmedetomidine sedation (and time since isoflurane cessation) extends. In the long isoflurane group only, bilateral functional connectivity strengthens with anesthetic duration, and correlation is linearly linked to low‐band spectral power. Convergence of connectivity and spectral metrics between the short and long isoflurane groups occurs at ~3.25 h, suggesting the effects of isoflurane have subsided. Researchers using dexmedetomidine following isoflurane for functional studies should be aware of the duration specific effects of the pre‐scan isoflurane durations as well as the continuing influences of long‐term imaging under dexmedetomidine. Copyright © 2014 John Wiley & Sons, Ltd.     

Immunoproteasome in animal models of Duchenne muscular dystrophy

Increased proteasome activity has been implicated in the atrophy and deterioration associated with dystrophic muscles of Duchenne muscular dystrophy (DMD). While proteasome inhibitors show promise in the attenuation of muscle degeneration, proteasome inhibition-induced toxicity was a major drawback of this therapeutic strategy. Inhibitors that selectively target the proteasome subtype that is responsible for the loss in muscle mass and quality would reduce side effects and be less toxic. This study examined proteasome activity and subtype populations, along with muscle function, morphology and damage in wild-type (WT) mice and two murine models of DMD, dystrophin-deficient (MDX) and dystrophin- and utrophin-double-knockout (DKO) mice. We found that immunoproteasome content was increased in dystrophic muscles while the total proteasome content was unchanged among the three genotypes of mice. Proteasome proteolytic activity was elevated in dystrophic muscles, especially in DKO mice. These mice also exhibited more severe muscle atrophy than either WT or MDX mice. Muscle damage and regeneration, characterized by the activity of muscle creatine kinase in the blood and the percentage of central nuclei were equally increased in dystrophic mice. Accordingly, the overall muscle function was similarly reduced in both dystrophic mice compared with WT. These data demonstrated that there was transformation of standard proteasomes to immunoproteasomes in dystrophic muscles. In addition, DKO that showed greatest increase in proteasome activities also demonstrated more severe atrophy compared with MDX and WT. These results suggest a putative role for the immunoproteasome in muscle deterioration associated with DMD and provide a potential target for therapeutic intervention.     

Attachment security and parenting quality predict children's problem-solving,attributions, and loneliness with peers

The influence of early relational experience on later social understanding has evoked rich theoretical discussion but relatively little empirical inquiry. Enlisting data from the NICHD Study of Early Child Care and Youth Development, measures of the security of attachment in infancy, toddlerhood, and early childhood, together with measures of parenting quality (maternal sensitivity and depressive symptoms) gathered longitudinally throughout infancy and early childhood, were used to predict differences in children's thoughts and feelings about peers (i.e., social problem solving, negative attributional biases, aggressive solutions to ambiguous social situations, and self-reported loneliness) when children were 54 months and in first grade. Relational experiences, especially before 36 months, were significantly predictive of later peer-related representations. Attachment security at 24 and 36 months was associated with enhanced social problem-solving skills and less loneliness, but security of attachment at 15 months was nonpredictive. Early maternal sensitivity was positively associated with later social problem-solving and negatively with aggressive responses, and early maternal depressive symptoms were positively associated with children's negative attributions. Concurrent parenting quality was also associated with children's thoughts and feelings about peers, but less consistently. These findings shed new light on how early relational experiences may contribute to social information processing with peers at the end of the preschool years, and that the timing of relational influences may be crucial.     

Activated γδ T cells inhibit osteoclast differentiation and resorptive activity in vitro

Extensive evidence suggests that the immune system exerts powerful effects on bone cells, particularly in chronic disease pathologies such as rheumatoid arthritis (RA). The chronic inflammatory state in RA, particularly the excessive production of T cell‐derived proinflammatory cytokines such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐17, triggers bone erosions through the increased stimulation of osteoclast formation and activity. While evidence supports a role for IL‐17 and TNF‐α secreted by conventional CD4⁺ T cells in RA, recent evidence in animal models of RA have implicated γδ T cells as a major producer of pathogenic IL‐17. However, the capacity of γδ T cells to influence osteoclast formation and activity in humans has not yet been investigated widely. To address this issue we investigated the effects of γδ T cells on osteoclast differentiation and resorptive activity. We have demonstrated that anti‐CD3/CD28‐stimulated γδ T cells or CD4⁺ T cells inhibit human osteoclast formation and resorptive activity in vitro. Furthermore, we assessed cytokine production by CD3/CD28‐stimulated γδ T cells and observed a lack of IL‐17 production, with activated γδ T cells producing abundant interferon (IFN)‐γ. The neutralization of IFN‐γ markedly restored the formation of osteoclasts from precursor cells and the resorptive activity of mature osteoclasts, suggesting that IFN‐γ is the major factor responsible for the inhibitory role of activated γδ T cells on osteoclastogenesis and resorptive activity of mature osteoclasts. Our work therefore provides new insights on the interactions between γδ T cells and osteoclasts in humans.