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Length-dependent activation (LDA) of contraction is an important mechanism of proper myocardial function that is often blunted in diseases accompanied by deficient contractility and impaired calcium homeostasis. We evaluated how the extent of LDA is related to the decreased force in healthy rat myocardium under negative inotropic conditions that affect the calcium cycle. The length-dependent effects on auxotonic twitch and Ca-transient were compared in isolated rat ventricular cardiomyocytes at room temperature (“25C”) and near-physiological temperature (“35C”) in normal Tyrode and at 25°C with thapsigargin-depleted sarcoplasmic reticulum (“25C + Thap”). At the slack length, a similar negative inotropy in “35C” and “25C + Thap” was accompanied by totally different changes in Ca-transient amplitude, time-to-peak, and time-to-decline from peak to 50% amplitude. End-systolic/end-diastolic tension-sarcomere length relationships were obtained for each individual cell, and the ratio of their slopes, the dimensionless Frank-Starling Gain index, was 2.32 ± 0.16, 1.78 ± 0.09, and 1.37 ± 0.06 in “25C,” “35C” and “25C + Thap,” respectively (mean ± S.E.M.). Ca-transient diastolic level and amplitude did not differ between “25C” and “35C” at any SL, but in “35C” it developed and declined significantly faster. In contrast, thapsigargin-induced depletion of SERCA2a significantly attenuated and retarded Ca-transient. The relative amount of Ca2+ utilized by troponin C, evaluated by the integral magnitude of a short-lived component of Ca-transient decline (“bump”), increased by ~25% per each 0.05 μm increase in SL in all groups. The kinetics of the Ca-TnC dissociation, evaluated by the bump time-to-peak, was significantly faster in “35C” and slower in “25C + Thap” vs. “25C” (respectively, 63.7 ± 5.3 and 253.6 ± 8.3% of the value in “25C,” mean ± S.E.M.). In conclusion, a similar inotropic effect can be observed in rat ventricular myocardium under totally different kinetics of free cy

Macrophage behavior is of great interest in response to tissue injury and promotion of regeneration. With increasing numbers of zebrafish reporter-based assays, new capabilities now exist to characterize macrophage migration, and their responses to biochemical cues, such as reactive oxygen species. Real time detection of macrophage behavior in response to oxidative stress using quantitative measures is currently beyond the scope of commercially available software solutions, presenting a gap in understanding macrophage behavior. To address this gap, we developed an image analysis pipeline solution to provide real time quantitative measures of cellular kinetics and reactive oxygen species content in vivo after tissue injury. This approach, termed Zirmi, differs from current software solutions that may only provide qualitative, single image analysis, or cell tracking solutions. Zirmi is equipped with user-defined algorithm parameters to customize quantitative data measures with visualization checks for an analysis pipeline of time-based changes. Moreover, this pipeline leverages open-source PhagoSight, as an automated keyhole cell tracking solution, to avoid parallel developments and build upon readily available tools. This approach demonstrated standardized space- and time-based quantitative measures of (1) fluorescent probe based oxidative stress and (2) macrophage recruitment kinetic based changes after tissue injury. Zirmi image analysis pipeline performed at execution speeds up to 10-times faster than manual image-based approaches. Automated segmentation methods were comparable to manual methods with a DICE Similarity coefficient > 0.70. Zirmi provides an open-source, quantitative, and non-generic image analysis pipeline. This strategy complements current wide-spread zebrafish strategies, for automated standardizations of analysis and data measures.

Macrophage behavior is of great interest in response to tissue injury and promotion of regeneration. With increasing numbers of zebrafish reporter-based assays, new capabilities now exist to characterize macrophage migration, and their responses to biochemical cues, such as reactive oxygen species. Real time detection of macrophage behavior in response to oxidative stress using quantitative measures is currently beyond the scope of commercially available software solutions, presenting a gap in understanding macrophage behavior. To address this gap, we developed an image analysis pipeline solution to provide real time quantitative measures of cellular kinetics and reactive oxygen species content in vivo after tissue injury. This approach, termed Zirmi, differs from current software solutions that may only provide qualitative, single image analysis, or cell tracking solutions. Zirmi is equipped with user-defined algorithm parameters to customize quantitative data measures with visualization checks for an analysis pipeline of time-based changes. Moreover, this pipeline leverages open-source PhagoSight, as an automated keyhole cell tracking solution, to avoid parallel developments and build upon readily available tools. This approach demonstrated standardized space- and time-based quantitative measures of (1) fluorescent probe based oxidative stress and (2) macrophage recruitment kinetic based changes after tissue injury. Zirmi image analysis pipeline performed at execution speeds up to 10-times faster than manual image-based approaches. Automated segmentation methods were comparable to manual methods with a DICE Similarity coefficient > 0.70. Zirmi provides an open-source, quantitative, and non-generic image analysis pipeline. This strategy complements current wide-spread zebrafish strategies, for automated standardizations of analysis and data measures.

type:Original Article, identifier:http://igakkai.kms-igakkai.com/wp/wp-content/uploads/1985en/11(3)135-141.1985.pdf

Asplenic patients have a lifelong risk of overwhelming post-splenectomy infection and have been reported to have low numbers of peripheral blood IgM memory B cells. The clinical value of quantitation of memory B cells as an indicator of splenic abnormality or risk of infection has been unclear. To assess changes in B cell sub-populations after splenectomy we studied patients recruited to a spleen registry (n = 591). A subset of 209 adult asplenic or hyposplenic subjects, and normal controls (n = 140) were tested for IgM memory B cells. We also determined a) changes in IgM memory B cells with time after splenectomy using the cross-sectional data from patients on the registry and b) the kinetics of changes in haematological markers associated with splenectomy(n = 45). Total B cells in splenectomy patients did not differ from controls, but memory B cells, IgM memory B cells and switched B cells were significantly (p<0.001) reduced. The reduction was similar for different indications for splenectomy. Changes of asplenia in routine blood films including presence of Howell-Jolly bodies (HJB), occurred early (median 25 days) and splenectomy associated thrombocytosis and lymphocytosis peaked by 50 days. There was a more gradual decrease in IgM memory B cells reaching a stable level within 6 months after splenectomy. IgM memory B cells as proportion of B cells was the best discriminator between splenectomized patients and normal controls and at the optimal cut-off of 4.53, showed a true positive rate of 95% and false positive rate of 20%. In a survey of 152 registry patients stratified by IgM memory B cells around this cut-off there was no association with minor infections and no registry patients experienced OPSI during the study. Despite significant changes after splenectomy, conventional measures of IgM memory cells have limited clinical utility in this population. © 2011 Cameron et al.

Asplenic patients have a lifelong risk of overwhelming post-splenectomy infection and have been reported to have low numbers of peripheral blood IgM memory B cells. The clinical value of quantitation of memory B cells as an indicator of splenic abnormality or risk of infection has been unclear. To assess changes in B cell sub-populations after splenectomy we studied patients recruited to a spleen registry (n = 591). A subset of 209 adult asplenic or hyposplenic subjects, and normal controls (n = 140) were tested for IgM memory B cells. We also determined a) changes in IgM memory B cells with time after splenectomy using the cross-sectional data from patients on the registry and b) the kinetics of changes in haematological markers associated with splenectomy(n = 45). Total B cells in splenectomy patients did not differ from controls, but memory B cells, IgM memory B cells and switched B cells were significantly (p<0.001) reduced. The reduction was similar for different indications for splenectomy. Changes of asplenia in routine blood films including presence of Howell-Jolly bodies (HJB), occurred early (median 25 days) and splenectomy associated thrombocytosis and lymphocytosis peaked by 50 days. There was a more gradual decrease in IgM memory B cells reaching a stable level within 6 months after splenectomy. IgM memory B cells as proportion of B cells was the best discriminator between splenectomized patients and normal controls and at the optimal cut-off of 4.53, showed a true positive rate of 95% and false positive rate of 20%. In a survey of 152 registry patients stratified by IgM memory B cells around this cut-off there was no association with minor infections and no registry patients experienced OPSI during the study. Despite significant changes after splenectomy, conventional measures of IgM memory cells have limited clinical utility in this population. © 2011 Cameron et al.

Letter, Research Support, Non-U.S. Gov't, FLWIN, SCOPUS: le.j, info:eu-repo/semantics/published

Letter, Research Support, Non-U.S. Gov't, FLWIN, SCOPUS: le.j, info:eu-repo/semantics/published

In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated calcein transport in a cellular model. Several of the methoxylated flavonoids are among the best MRP1 inhibitors (IC50 values, ranging between 2.7 and 14.3 μM) followed by robinetin, myricetin and quercetin (IC50 values ranging between 13.6 and 21.8 μM). Regarding inhibition of MRP2 activity especially robinetin and myricetin appeared to be good inhibitors (IC 50 values of 15.0 and 22.2 μM, respectively). Kinetic characterization revealed that the two transporters differ marginally in the apparent Km for the substrate calcein. For one flavonoid, robinetin, the kinetics of inhibition were studied in more detail and revealed competitive inhibition with respect to calcein, with apparent inhibition constants of 5.0 μM for MRP1 and 8.5 μM for MRP2. For inhibition of MRP1, a quantitative structure activity relationship (QSAR) was obtained that indicates three structural characteristics to be of major importance for MRP1 inhibition by flavonoids: the total number of methoxylated moieties, the total number of hydroxyl groups and the dihedral angle between the B- and C-ring. Regarding MRP2 mediated calcein efflux inhibition, only the presence of a flavonol B-ring pyrogallol group seems to be an important structural characteristic. Overall, this study provides insight in the structural characteristics involved in MRP inhibition and explores the differences between inhibitors of these two transporters, MRP1 and MRP2. Ultimately, MRP2 displays higher selectivity for flavonoid type inhibition than MRP1. © 2004 Elsevier Inc. All rights reserved.

type:text, The present study aimed at clarifying the cell kinetics of the mouse ureteral epithelium by focusing on vesicle maturation in the cells and labeling with bromodeoxyuridine (BrdU). Electron microscopically, superficial cells were characterized by concave plaques in the apical plasma membrane and numerous fusiform vesicles in the cytoplasm. Intermediate cells were laden with ellipsoid vesicles, and basal cells had a few or no round vesicles. From the difference in number and form of vesicles among the three types of cells, it can be inferred that intermediate cells are immature in comparison with superficial cells, and likewise basal cells in comparison with intermediate cells. When BrdU was injected intraperitoneally once a day for seven days, most BrdU-labeled cells were located in the basal layer. Twelve days after the last injection, BrdU was detected in the intermediate or superficial layer in addition to the basal layer. These findings suggest that the basal cell is a progenitor cell giving rise to daughter cells that migrate upward to replace intermediate and superficial cells., This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Health Science Research Grants for Research on Environmental Health from the Ministry of Health, Labour and Welfare of Japan.

The present study aimed at clarifying the cell kinetics of the mouse ureteral epithelium by focusing on vesicle maturation in the cells and labeling with bromodeoxyuridine (BrdU). Electron microscopically, superficial cells were characterized by concave plaques in the apical plasma membrane and numerous fusiform vesicles in the cytoplasm. Intermediate cells were laden with ellipsoid vesicles, and basal cells had a few or no round vesicles. From the difference in number and form of vesicles among the three types of cells, it can be inferred that intermediate cells are immature in comparison with superficial cells, and likewise basal cells in comparison with intermediate cells. When BrdU was injected intraperitoneally once a day for seven days, most BrdU-labeled cells were located in the basal layer. Twelve days after the last injection, BrdU was detected in the intermediate or superficial layer in addition to the basal layer. These findings suggest that the basal cell is a progenitor cell giving rise to daughter cells that migrate upward to replace intermediate and superficial cells., This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Health Science Research Grants for Research on Environmental Health from the Ministry of Health, Labour and Welfare of Japan.

The present study aimed at clarifying the cell kinetics of the mouse ureteral epithelium by focusing on vesicle maturation in the cells and labeling with bromodeoxyuridine (BrdU). Electron microscopically, superficial cells were characterized by concave plaques in the apical plasma membrane and numerous fusiform vesicles in the cytoplasm. Intermediate cells were laden with ellipsoid vesicles, and basal cells had a few or no round vesicles. From the difference in number and form of vesicles among the three types of cells, it can be inferred that intermediate cells are immature in comparison with superficial cells, and likewise basal cells in comparison with intermediate cells. When BrdU was injected intraperitoneally once a day for seven days, most BrdU-labeled cells were located in the basal layer. Twelve days after the last injection, BrdU was detected in the intermediate or superficial layer in addition to the basal layer. These findings suggest that the basal cell is a progenitor cell giving rise to daughter cells that migrate upward to replace intermediate and superficial cells., This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Health Science Research Grants for Research on Environmental Health from the Ministry of Health, Labour and Welfare of Japan.

Background: Epidemiological studies have established that heavy alcohol consumption in persons with chronic hepatitis C infection is associated with advanced liver disease, including cirrhosis. The cellular mechanisms underlying this process, which appear to occur over decades, are unknown. Increased hepatocyte apoptosis has been observed in association with hepatitis C infection. The aim of this study was to evaluate the relationship between alcohol consumption and hepatocyte apoptosis in hepatitis C-infected patients, Methods: Liver tissue from 20 hepatitis C-infected patients with variable alcohol consumption, and 10 normal control subjects was examined for hepatocyte apoptosis, proliferation and bcl-2 expression. Result(s): Hepatocyte apoptosis was significantly greater in hepatitis C-infected patients than in controls. In hepatitis C-infected patients, significantly more hepatocyte apoptosis was seen in those consuming at least 30g per day of alcohol compared with those drinking less than 10g daily. Bcl-2, an inhibitor of apoptosis, was not detected in liver tissue from patients with the highest ethanol intake and rate of hepatocyte apoptosis. In contrast, patients drinking lesser amounts of ethanol had lower rates of hepatocyte apoptosis and more frequent bcl-2 expression. Conclusion(s): This study confirms that both hepatitis C infection and ethanol consumption induce hepatocyte apoptosis in humans. Ethanol-induced hepatocyte apoptosis has previously been shown only in animal models of alcohol-related liver injury. The precise role of apoptosis in the pathogenesis of hepatitis C-related liver injury remains unclear, but its induction may be related to downregulation of bcl-2 expression associated with ethanol consumption. (C) 2000 Blackwell Science Asia Pty Ltd.

Background: Epidemiological studies have established that heavy alcohol consumption in persons with chronic hepatitis C infection is associated with advanced liver disease, including cirrhosis. The cellular mechanisms underlying this process, which appear to occur over decades, are unknown. Increased hepatocyte apoptosis has been observed in association with hepatitis C infection. The aim of this study was to evaluate the relationship between alcohol consumption and hepatocyte apoptosis in hepatitis C-infected patients, Methods: Liver tissue from 20 hepatitis C-infected patients with variable alcohol consumption, and 10 normal control subjects was examined for hepatocyte apoptosis, proliferation and bcl-2 expression. Result(s): Hepatocyte apoptosis was significantly greater in hepatitis C-infected patients than in controls. In hepatitis C-infected patients, significantly more hepatocyte apoptosis was seen in those consuming at least 30g per day of alcohol compared with those drinking less than 10g daily. Bcl-2, an inhibitor of apoptosis, was not detected in liver tissue from patients with the highest ethanol intake and rate of hepatocyte apoptosis. In contrast, patients drinking lesser amounts of ethanol had lower rates of hepatocyte apoptosis and more frequent bcl-2 expression. Conclusion(s): This study confirms that both hepatitis C infection and ethanol consumption induce hepatocyte apoptosis in humans. Ethanol-induced hepatocyte apoptosis has previously been shown only in animal models of alcohol-related liver injury. The precise role of apoptosis in the pathogenesis of hepatitis C-related liver injury remains unclear, but its induction may be related to downregulation of bcl-2 expression associated with ethanol consumption. (C) 2000 Blackwell Science Asia Pty Ltd.

Gastrin can induce mitotic stimulation in the oxyntic mucosa of the stomach, sometimes leading to abnormal growth. We examined whether gastrin was able to influence cell proliferation in the oesophageal epithelium. Rats were treated with gastrin, omeprazole or saline for 3 days, or were subjected to fundectomy or sham operation. Bromodeoxyuridine labelling (LI) and mitotic (MI) indices were counted in the proliferative zone of the squamous epithelium. Infusion of exogenous gastrin, treatment with omeprazole or fundectomy raised the LI and the MI values in the oesophageal epithelium, indicating that gastrin stimulates cell proliferation in the oesophageal mucosa in the rat., SCOPUS: ar.j, info:eu-repo/semantics/published

Gastrin can induce mitotic stimulation in the oxyntic mucosa of the stomach, sometimes leading to abnormal growth. We examined whether gastrin was able to influence cell proliferation in the oesophageal epithelium. Rats were treated with gastrin, omeprazole or saline for 3 days, or were subjected to fundectomy or sham operation. Bromodeoxyuridine labelling (LI) and mitotic (MI) indices were counted in the proliferative zone of the squamous epithelium. Infusion of exogenous gastrin, treatment with omeprazole or fundectomy raised the LI and the MI values in the oesophageal epithelium, indicating that gastrin stimulates cell proliferation in the oesophageal mucosa in the rat., SCOPUS: ar.j, info:eu-repo/semantics/published

SCOPUS: ar.j, info:eu-repo/semantics/published

SCOPUS: ar.j, info:eu-repo/semantics/published

Large gemistocytic cells are well-known elements of glial tumors. Recently, miniature gemistocytic cells and neoplastic glial fibrillary acidic protein (GFAP)-positive oligodendroglial cells, which are regularly seen in oligodendrogliomas, have been termed "transitional cells". The proliferative activity of the gemistocytic cell types and the GFAP-positive (gliofibrillary) oligodendrocytes was determined in eight astrocytomas, seven gemistocytic astrocytomas, eight glioblastomas, two monstrocellular glioblastomas, seven oligodendrogliomas and three mixed oligo-astrocytomas by immunohistochemical staining of the proliferation marker MIB-1 in combination with immunostaining for GFAP. Both large gemistocytic cells and the transitional cells showed cytoplasmic GFAP-positive staining. Neither in the classic gemistocytes nor in the minigemistocytes nuclear immunostaining for the MIB-1 antibody was observed. In contrast, MIB-1 staining was seen in the gliofibrillary oligodendrocytes. It is concluded that both large and miniature gemistocytic cell types contrast with gliofibrillary oligodendrocytes by their inability to proliferate.

Accurate descriptions of the kinetics of cell growth in semi-solid agar clonogenic systems have been difficult because the number of cells in colonies of different sizes is largely unknown. We stained and removed tumor cell colonies from agar, directly counted their cells, and established equations to quantitate the number of cells within colonies of different sizes. We used these equations to quantitate, in terms of cell number and volume, the total amount and kinetics of clonogenic cell proliferation from biopsies of human melanoma and cell lines of several different tumor types. Daily observations of cells in agar and serial photography indicated a 0- to 4-day delay in the onset of proliferation in agar followed by rapid growth and then abrupt cessation of proliferation. We quantified the extent of proliferation of cells from melanoma biopsies of seven patients and 11 cell lines after they were allowed to proliferate in agar until they stopped. Approximately 10% of cells divided one to five times while only 0.01% divided six to nine times. The total number of cells within the colonies at the end of growth was different while the total volume of cells within the colonies per plate was similar; approximately 10(9) microns 3 cellular volume per plate represents an upper limit for proliferation within the closed, nonrefed bilayer agar system. Previous replating studies using the same biopsy cells have shown that clonogenic melanoma cells can self-renew and have more proliferative capacity than that expressed during primary colony formation. Thus, the clonogenic assay only measured initial proliferative capacities. Furthermore, variable delays in the onset of proliferation may contribute to the heterogeneity of colony size within clonogenic assays.

Accurate descriptions of the kinetics of cell growth in semi-solid agar clonogenic systems have been difficult because the number of cells in colonies of different sizes is largely unknown. We stained and removed tumor cell colonies from agar, directly counted their cells, and established equations to quantitate the number of cells within colonies of different sizes. We used these equations to quantitate, in terms of cell number and volume, the total amount and kinetics of clonogenic cell proliferation from biopsies of human melanoma and cell lines of several different tumor types. Daily observations of cells in agar and serial photography indicated a 0- to 4-day delay in the onset of proliferation in agar followed by rapid growth and then abrupt cessation of proliferation. We quantified the extent of proliferation of cells from melanoma biopsies of seven patients and 11 cell lines after they were allowed to proliferate in agar until they stopped. Approximately 10% of cells divided one to five times while only 0.01% divided six to nine times. The total number of cells within the colonies at the end of growth was different while the total volume of cells within the colonies per plate was similar; approximately 10(9) microns 3 cellular volume per plate represents an upper limit for proliferation within the closed, nonrefed bilayer agar system. Previous replating studies using the same biopsy cells have shown that clonogenic melanoma cells can self-renew and have more proliferative capacity than that expressed during primary colony formation. Thus, the clonogenic assay only measured initial proliferative capacities. Furthermore, variable delays in the onset of proliferation may contribute to the heterogeneity of colony size within clonogenic assays.

Accurate descriptions of the kinetics of cell growth in semi-solid agar clonogenic systems have been difficult because the number of cells in colonies of different sizes is largely unknown. We stained and removed tumor cell colonies from agar, directly counted their cells, and established equations to quantitate the number of cells within colonies of different sizes. We used these equations to quantitate, in terms of cell number and volume, the total amount and kinetics of clonogenic cell proliferation from biopsies of human melanoma and cell lines of several different tumor types. Daily observations of cells in agar and serial photography indicated a 0- to 4-day delay in the onset of proliferation in agar followed by rapid growth and then abrupt cessation of proliferation. We quantified the extent of proliferation of cells from melanoma biopsies of seven patients and 11 cell lines after they were allowed to proliferate in agar until they stopped. Approximately 10% of cells divided one to five times while only 0.01% divided six to nine times. The total number of cells within the colonies at the end of growth was different while the total volume of cells within the colonies per plate was similar; approximately 10(9) microns 3 cellular volume per plate represents an upper limit for proliferation within the closed, nonrefed bilayer agar system. Previous replating studies using the same biopsy cells have shown that clonogenic melanoma cells can self-renew and have more proliferative capacity than that expressed during primary colony formation. Thus, the clonogenic assay only measured initial proliferative capacities. Furthermore, variable delays in the onset of proliferation may contribute to the heterogeneity of colony size within clonogenic assays.

The present study aimed at clarifying the cell kinetics of the mouse ureteral epithelium by focusing on vesicle maturation in the cells and labeling with bromodeoxyuridine (BrdU). Electron microscopically, superficial cells were characterized by concave plaques in the apical plasma membrane and numerous fusiform vesicles in the cytoplasm. Intermediate cells were laden with ellipsoid vesicles, and basal cells had a few or no round vesicles. From the difference in number and form of vesicles among the three types of cells, it can be inferred that intermediate cells are immature in comparison with superficial cells, and likewise basal cells in comparison with intermediate cells. When BrdU was injected intraperitoneally once a day for seven days, most BrdU-labeled cells were located in the basal layer. Twelve days after the last injection, BrdU was detected in the intermediate or superficial layer in addition to the basal layer. These findings suggest that the basal cell is a progenitor cell giving rise to daughter cells that migrate upward to replace intermediate and superficial cells., This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Health Science Research Grants for Research on Environmental Health from the Ministry of Health, Labour and Welfare of Japan.