Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.

AIMS/BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant tumours, but is inactive in normal somatic cells except for male germ cells and proliferating stem cells. Thus, the measurement of telomerase activity in tissue samples may provide useful diagnostic and prognostic information. The aim of this study was to determine whether telomerase expression is useful for the detection of occult malignant cells in lymph nodes. METHODS: Telomerase activity was compared with histological findings in 123 surgically removed lymph nodes submitted for routine or frozen section diagnosis. Telomerase activity was measured using a modified, semi-quantitative PCR-based telomeric repeat amplification protocol (TRAP). The assay was adapted for single 5 microns OCT embedded cryostat sections. In either fresh tissues or cryostat sections, normalised activity was linear when compared with protein concentration. Furthermore, using an in situ hybridisation method, the human telomerase RNA (hTR) component was measured in a subset of negative and positive nodes. RESULTS: Most (96%) of the 97 histologically negative nodes expressed low levels of activity (mean value of positive samples = 3.0 units/microgram protein) which may be derived from activated lymphocytes that express telomerase activity. All 26 malignant nodes (17 metastases, nine lymphomas) expressed telomerase (mean value = 17.8 units/microgram protein). The rank order levels between the two groups differed significantly (p = 0.0002). In situ results showed clearly that the hTR was expressed relatively highly in metastatic cancer cells and at lower levels in germinal centres of secondary follicles. CONCLUSIONS: Although expression of telomerase by activated lymphocytes may limit its usefulness, measurement of enzyme activity combined with detection of hTR using in situ hybridisation may assist in the histopathological diagnosis of lymph nodes.     

Multivariate analysis of the factors associated with live births during in vitro fertilisation in Southeast Asia: a cross-sectional study of 104,015 in vitro fertilisation records in Taiwan

Purpose

To investigate the factors associated with live births and the interaction between age and the number of embryos transferred after in vitro fertilisation (IVF) treatment.

Methods

This study analyses data from a population-based-assisted reproductive database of all registered artificial reproduction institutions (n = 80) from 2010 to 2016 in Taiwan. The probability of a live birth in correlation with the treatment parameters was measured with multivariate logistic regression analyses using the generalised additive model (GAM) and Pearson’s chi-square exact test.

Results

A total of 104,015 IVF treatments performed between 2010 and 2016 were included in our analysis. From these treatments, 31,467 (30.3%) were successfully delivered, and 40,565 test-tube babies were born. Pearson’s chi-square exact test indicated that parents’ age, cause of infertility, type of ovarian stimulation, additional assisted reproductive technology techniques, donated egg or sperm, fresh or frozen embryo, presence or absence of ovarian hyperstimulation syndrome, and day of embryo transfer were significantly associated with live births after an IVF cycle (p < 0.05). Multiple logistic regression analysis with the GAM revealed that the odds of a live birth with IVF treatment in patients < 34 years of age were 2.55 times higher than that in patients ≥ 45 years of age (odds ratio = 2.55, 95% confidence interval = 1.69–2.90) for patients who underwent a single-embryo transfer (SET); a similar pattern was observed when two or more embryos were transferred. Egg donation, the assisted hatching technique, oral ovarian stimulation agents, and implantation of frozen embryos during SET were shown to improve the chance of a live birth by 29–90%. Implantation of the embryo after the 5th day of culture yielded the highest odds of a live birth. The interaction plot revealed that maternal age, especially < 40 years, was associated with the probability of a live birth. SET and double-embryo transfer showed similar associations with the probability of a live birth across age groups. Transferring more than two embryos might reduce the probability of a live birth during IVF treatment for women ≥ 40 years of age.

Conclusions

Implanting a greater number of embryos did not improve the age-related decrease in fertility for patients undergoing IVF. Therefore, we suggested that ≤ 2 blastocysts could be transferred during IVF treatments for women ≥ 40 years. Transferring a blastocyst on day 5 of culture was associated with a significant increase in the odds of a live birth resulting from IVF.

     

Napsin A (TA02) is a useful alternative to thyroid transcription factor-1 (TTF-1) for the identification of pulmonary adenocarcinoma cells in pleural effusions

The purpose of this study was to test napsin A as a diagnostic marker of metastatic lung adenocarcinoma in pleural effusions, and to compare its performance with TTF-1. Napsin A and TTF-1 reactivities were determined immunohistochemically on formalin-fixed paraffin embedded cell blocks from 50 pleural effusion (5 mesotheliomas, 10 mesothelial proliferations, 12 pulmonary, and 23 nonpulmonary metastases). The results were evaluated separately, and correlated to the final diagnoses. Concordant results were obtained in 48/50 cases. TTF-1 and Napsin A were positive in 8/12 and 10/12 pulmonary adenocarcinomas, respectively. Both markers were negative in 42 cases, including two lung carcinomas. Napsin reactivity was found in more than 75% of the tumor cells in 9/10 positive cases, whereas TTF-1 reactivity was seen in more than 75% of the tumor cells in 2/8 positive cases only (P < 0.05). This makes napsin A an alternative to TTF-1 in cytological diagnosis of effusions in which tumor cells may be scanty.     

Occult hepatitis B virus infection in HBs antigen-negative hepatocellular carcinoma in a Japanese population: involvement of HBx and p53

Hepatitis B virus (HBV) genome was reported to be detected in serum or liver tissues in hepatocellular carcinoma (HCC) patients negative for hepatitis B surface antigen (HBsAg). Hepatitis B x (HBx) and p53 protein were reported to play an important role in HBV-related hepatocarcinogenesis. To clarify latent HBV infection in HBsAg- and anti-hepatitis C virus (anti-HCV)-negative HCC in a Japanese population and involvement of HBx and p53 protein in these patients, we performed the sensitive and specific nested polymerase chain reaction (PCR) and immunohistochemical analysis. Of 1,024 HCC patients we saw between 1974 and 1998, 66 (6.4%) were negative for HBsAg and anti-HCV. Serum DNA was amplified by nested PCR by using specific primers of surface (S), core (C) and X regions in 26 patients negative for HBsAg and anti-HCV. Eighteen (69%) patients were positive for either S, C, or X region and the results of PCR were confirmed by Southern blotting. Of 18 PCR-positive patients, 3 were positive for anti-HBs and 9 were positive for anti-HBc, however, one was negative for any HBV markers. In HBsAg-negative and PCR-positive patients, the positive rates of expression of HBx and p53 were 8/13 (62%) and 7/13 (54%), being comparable to those in HBsAg-positive HCC patients. The results of the present study suggest that high prevalence of HBV infection is observed in HBsAg-negative HCC in a Japanese population and expression of HBx and p53 is consistent with a role, in these patients, for the transforming ability of these proteins.     

New CR system with pixel size of 50 microm for digital mammography: physical imaging properties and detection of subtle microcalcifications

PURPOSE: To investigate the physical imaging properties and detection of simulated microcalcifications of a new computed radiography (CR) system with a pixel size of 50 microm for digital mammography. MATERIALS AND METHODS: New and conventional CR were employed in this study. The new CR system included a high-resolution imaging plate coupled with the FCR5000MA (50 microm pixel pitch) including transparent support and a dual-sided reader. The conventional CR system was coupled with the FCR9000 (100 microm pixel pitch). Modulation transfer functions (MTFs) and Wiener spectra (WS) of the new and conventional CR systems were measured. Observer performance tests were conducted to compare the effects of pixel size (50 microm vs. 100 microm) on the diagnostic accuracy of CR systems in the detection of simulated microcalcifications. RESULTS: The presampling MTF of the new CR system was higher at high frequencies than the conventional CR system. The WS of the new CR system was comparable to that of the conventional CR system at all frequencies. The area under the receiver operating characteristic (ROC) curve (Az) obtained with the new CR and the conventional CR systems were 0.84 and 0.79, respectively. Results showed that the detection of simulated clustered microcalcifications was significantly improved by use of the new CR system compared with the conventional CR system (p<0.05). CONCLUSION: The new CR mammography system improved physical imaging properties and detection of simulated microcalcifications over conventional CR mammography.     

Regeneration of hair and other skin appendages: A microenvironment‐centric view

Advances in skin regeneration have resulted in techniques and products that have allowed regeneration of both the dermis and epidermis. Yet complete skin regeneration requires the adnexal skin structures. Thus it is crucial to understand the regenerative potential of hair follicles where genetic, nutritional, and hormonal influences have important effects and are critical for skin regeneration. The follicular stem cell niche serves as an anatomical compartment, a structural unit, a functional integrator, and a dynamic regulator necessary to sustain internal homeostasis and respond to outside stimuli. In particular, mechanics such as pressure, compression, friction, traction, stretch, shear, and mechanical wounding can influence hair loss or growth. Relevant niche signaling pathways such as Wnt, bone morphogenetic protein, fibroblast growth factor, Shh, and Notch may yield potential targets for therapeutic interventions.     

Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing platelet-derived growth factor receptor in the muscle, but it does not affect blood glucose levels

Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached approximately 60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.     

Suppressed complement activation in human decay accelerating factor transgenic porcine liver cross-circulated with nonhuman primates

BACKGROUND: We developed an extracorporeal liver perfusion (ECLP) system as a liver-assist device. In this study, we evaluated the safety of the ECLP using human decay accelerating factor (hDAF) transgenic porcine livers in healthy baboons. METHODS: Livers were isolated from five hDAF transgenic pigs and five nontransgenic pigs for the ECLP. Ten cross-circulations between the ECLP and healthy baboons were performed without immunosuppressive agents. Cross-circulation was discontinued in any of the following circumstances: elevated hepatic arterial (>200 mm Hg) or portal (>60 mm Hg) perfusion pressure, massive exudate from the graft liver, mild macroscopic hemolysis, thrombocytopenia, or 24-hr well-conditioned cross-circulation. RESULTS: The cross-circulations with nontransgenic porcine livers were discontinued at 4.4+/-1.2 hr (mean+/-standard deviation) because of high perfusion pressure (n=2) or hemolysis (n=3). Three cross-circulations with hDAF transgenic porcine livers were performed for 24 hr; the other two cross-circulations were discontinued at 13 and 17 hr because of massive exudate and thrombocytopenia, respectively. The duration was 20.4+/-5.1 hr. Deposition of membrane attack complex in the hDAF transgenic porcine liver was less than that in the nontransgenic liver, although immunoglobulin-M deposition was comparable. The porcine livers showed no apparent interlobular bleeding or lobular necrosis. All porcine livers maintained bile production during the cross-circulation. No baboons showed any serious complications after the cross-circulation. CONCLUSION: The hDAF transgenic porcine liver reduced complement activation in xenoperfusion with healthy nonhuman primate blood and led to extended duration of cross-circulation.     

Serum levels of IgE anti-BP180 and anti-BP230 autoantibodies in patients with bullous pemphigoid

BACKGROUND: Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by autoantibodies against the hemidesmosomal proteins, BP180 and BP230. NC16A, a non-collagenous stretch of the BP180 ectodomain is the primary target of pathogenic IgG antibodies. Whereas IgG anti-BP180 autoantibodies play a primary role in the pathogenesis, there is a growing number of data regarding the potential pathogenic roles of IgE class autoantibodies in BP. OBJECTIVES: To examine the levels of IgG and IgE autoantibodies against BP180 and BP230, and to investigate mutual association and clinical relevance. METHODS: Sera obtained from 67BP patients and 36 healthy donors were subjected to ELISA assays to measure serum IgG and IgE levels of anti-BP180 and anti-BP230 antibodies. RESULTS: IgG anti-BP180 antibodies were positive in 63 (94%) of 67BP patients. IgG anti-BP230, IgE anti-BP180, and IgE anti-BP230 antibodies were found in 48 (72%), 20 (30%) and 45 (67%), respectively. IgG anti-BP180 levels were correlated with the affected areas. IgG anti-BP230 antibodies tended to increase in proportion to elongation of disease duration. IgE anti-BP230 levels showed a strong association with local eosinophil accumulation, while the levels were reversely related with the affected areas in BP. CONCLUSIONS: IgE autoantibodies to BP180 and BP230 are detected at high frequencies in BP. IgE anti-BP230 antibodies may have a role in attracting eosinophils to the skin lesions.     

Anti‐ACSA‐2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes

Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti‐astrocyte cell surface antigen‐2 (Anti‐ACSA‐2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti‐ACSA‐2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non‐astroglial cells such as neurons, oligodendrocytes, NG2⁺ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co‐labeling studies of GLAST and ACSA‐2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single‐positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA‐2 was unlike GLAST not sensitive to papain‐based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti‐ACSA‐2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA‐2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages.