Serum Brevican as a Biomarker of Cerebrovascular Disease in an Elderly Cognitively Impaired Cohort.

In the brain, the extracellular matrix (ECM) composition shapes the neuronal microenvironment and can undergo substantial changes with cerebral pathology. Brevican is integral to the formation of the ECM's neuroprotective perineuronal nets (PNNs). Decreased brevican levels were reported in vascular dementia (VaD) but not in Alzheimer's disease (AD). However, the status of brevican in clinical cohorts with high concomitance of AD pathological burden and cerebrovascular disease (CeVD) is unclear. In this study, 32 non-cognitively impaired (NCI), 97 cognitively impaired no dementia (CIND), 46 AD, and 23 VaD participants recruited from memory clinics based in Singapore underwent neuropsychological and neuroimaging assessments, together with measurements of serum brevican. Association analyses were performed between serum brevican and neuroimaging measures of CeVDs, including white matter hyperintensities (WMHs), lacunes, cortical infarcts, and cerebral microbleeds. Using an aggregated score for CeVD burden, only CIND participants showed lower brevican levels with higher CeVD compared to those with lower CeVD burden (p = 0.006). Among the CeVD subtypes assessed, only elevated WMH burden was associated with lower brevican levels (OR = 2.7; 95% CI = 1.3-5.5). Our findings suggest that brevican deficits may play a role in early cerebrovascular damage in participants at risk of developing dementia.

Elevated Soluble TNF-Receptor 1 in the Serum of Predementia Subjects with Cerebral Small Vessel Disease.

Tumor necrosis factor-receptor 1 (TNF-R1)-mediated signaling is critical to the regulation of inflammatory responses. TNF-R1 can be proteolytically released into systemic blood circulation in a soluble form (sTNF-R1), where it binds to circulating TNF and functions to attenuate TNF-mediated inflammation. Increases of peripheral sTNF-R1 have been reported in both Alzheimer's disease (AD) dementia and vascular dementia (VaD). However, the status of sTNF-R1 in predementia subjects (cognitive impairment, no dementia, CIND) is unknown, and putative associations with cerebral small vessel disease (CSVD), as well as with longitudinal changes in cognitive functions are unclear. We measured baseline serum sTNF-R1 in a longitudinally assessed cohort of 93 controls and 103 CIND, along with neuropsychological evaluations and neuroimaging assessments. Serum sTNF-R1 levels were increased in CIND compared with controls (p < 0.001). Higher baseline sTNF-R1 levels were specifically associated with lacunar infarcts (rate ratio = 6.91, 95% CI 3.19-14.96, p < 0.001), as well as lower rates of cognitive decline in the CIND subgroup. Our data suggest that sTNF-R1 interacts with vascular cognitive impairment in a complex manner at predementia stages, with elevated levels associated with more severe CSVD at baseline, but which may subsequently be protective against cognitive decline.

Suppression of tumorigenicity in human ovarian carcinoma cell line SKOV-3 by microcell-mediated transfer of chromosome 11.

To determine the pathogenic role of chromosomes 11 and 17 in the carcinogenesis of human ovarian cancers, neo(R)-tagged chromosome 11 or 17 was transferred from cell lines A9H11 or A9H17, respectively, into the ovarian carcinoma cell line SKOV-3 using microcell-mediated chromosome transfer. The chromosome transfer was verified by polymerase chain reaction detection of the neo(R) gene, fluorescence in situ hybridization detection of an extra chromosome 11, and microsatellite polymorphism detection of an exogeneous chromosome 11. Five SKOV-3/A9H11 hybrids and five SKOV-3/A9H17 hybrid clones were generated. For the chromosome 11 transfer, complete suppression of tumorigenicity was observed in four clones, (11)9-8 and 11(H)7-2, 11(H)8-3, and 11(H)7-2, 100 days post implantation. For the chromosome 17 transfer, no complete suppression of tumorigenicity was observed. However, an increased latency period ranging from 25 to 49 days in contrast to 7 days for the SKOV-3 parental line, and a significant reduction in tumor size was observed. There was no correlation between the in vitro growth rate and the tumorigenicity or length of latency period. Our results demonstrate functionally that chromosome 11 may carry a tumor suppressor gene(s) while chromosome 17 may carry a tumor growth-inhibitor gene(s) for the ovarian carcinoma cell line, SKOV-3.

Development of rat pancreatic islet cells in vitro.

Islet cell lines were produced by retroviral transduction of SV-40 T antigen to monolayer cultures of neonatal rat islets. One line, RN0-11, showed evolution of phenotypes in vitro. It evolved from a non-hormone secreting cell line to an insulin secreting line. It further developed glucagon producing capability before it lost all hormone producing phenotypes. At passage 8, RN0-11 cells secreted a small amount of insulin 25 ng/10(6) cells/24 hours. They were unresponsive to glucose and secreted 1.7-1.9 ng of insulin/10(6) cells/2 hours under various levels of glucose. At passage 16, they secreted 2,577 ng of insulin/10(6) cells/24 hours and responded to glucose stimulation in static incubation. The insulin secreted by these cells at 0, 2.8, 5.5, 11.1, 16.7, and 27.7 mM of glucose was 6.46 +/- 2.56, 17.74 +/- 2.66, 32.24 +/- 0.58, 30.66 +/- 1.59, 33.55 +/- 4.83, and 20.95 +/- 2.17 ng/10(6) cells/2 hours respectively. The responsiveness to glucose and the ability to secrete insulin diminished as cells were passaged in culture, and by passage 35 no insulin was detectable in medium under any level of glucose tested. Northern blot analyses also showed corresponding changes of insulin expression in these cells at different passages. In addition, glucagon was detectable at passage 14 by immunocytochemistry and at passage 16 by Northern blot analysis. By passage 35, no insulin or glucagon expression was detectable by Northern blot analysis or immunocytochemistry. Immunocytochemical staining of these cells at passage 14 showed insulin-positive and glucagon-positive cells and cells positive for both insulin and glucagon. Presence of insulin and glucagon in the same cells suggests single clonality of the cell line. The evolution of RN0-11 cells in vitro provides an opportunity to study the development of islet cells.

Reduced insulin receptor signaling in the obese spontaneously hypertensive Koletsky rat.

Insulin resistance is associated with both obesity and hypertension. However, the cellular mechanisms of insulin resistance in genetic models of obese-hypertension have not been identified. The objective of the present study was to investigate the effects of genetic obesity on a background of inherited hypertension on initial components of the insulin signal transduction pathway and glucose transport in skeletal muscle and liver. Oral glucose tolerance testing in SHROB demonstrated a sustained postchallenge elevation in plasma glucose at 180 and 240 min compared with lean spontaneously hypertensive rat (SHR) littermates, which is suggestive of glucose intolerance. Fasting plasma insulin levels were elevated 18-fold in SHROB. The rate of insulin-stimulated 3-O-methylglucose transport was reduced 68% in isolated epitrochlearis muscles from the SHROB compared with SHR. Insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit and insulin receptor substrate-1 (IRS-1) in intact skeletal muscle of SHROB was reduced by 36 and 23%, respectively, compared with SHR, due primarily to 32 and 60% decreases in insulin receptor and IRS-1 protein expression, respectively. The amounts of p85 alpha regulatory subunit of phosphatidylinositol-3-kinase and GLUT-4 protein were reduced by 28 and 25% in SHROB muscle compared with SHR. In the liver of SHROB, the effect of insulin on tyrosine phosphorylation of IRS-1 was not changed, but insulin receptor phosphorylation was decreased by 41%, compared with SHR, due to a 30% reduction in insulin receptor levels. Our observations suggest that the leptin receptor mutation fak imposed on a hypertensive background results in extreme hyperinsulinemia, glucose intolerance, and decreased expression of postreceptor insulin signaling proteins in skeletal muscle. Despite these changes, hypertension is not exacerbated in SHROB compared with SHR, suggesting these metabolic abnormalities may not contribute to hypertension in this model of Syndrome X.

Growth dynamics of pancreatic islet cell populations during fetal and neonatal development of the rat.

Rats from 18 days fetal to 28 days neonatal ages were studied for the total population sizes and cell proliferation activities of insulin secreting B cells, glucagon secreting A cells, somatostatin secreting D cells, and pancreatic polypeptide secreting PP cells. Cell population sizes were assessed by morphometric quantitation of immunohistochemically stained cells by a linear scanning method and cell proliferation activities were estimated by [3H]-thymidine labeling indices of these cell populations. There was a continuous increase in population sizes for all 4 islet cell types, with the fastest increase occurring in the last 4 days of gestation. The accelerated growth of these islet cell populations during late gestation was accomplished by a high cell proliferative activity at 20-22 days of gestation and a large influx of undifferentiated epithelial cells differentiating into the specific islet cell populations during this period. There was a reduction of population growth and cell proliferation for all islet cell types during the first 3-4 days of life. Growth activities continued for all islet cell populations after the 4th postnatal day, with a renewed acceleration in growth activities for B and A cells at this time. After the 10th neonatal day, the cell proliferation and total population growth continued at slow rates for all 4 islet cell types. The contribution from undifferentiated epithelial cells into the specific islet cell populations was negligible for B and A cells but continued at a low rate for PP and D cells during the first 10 days after birth. For B and A cell populations, there was a possibility that some cell loss occurred during the first 10 days of neonatal life. These dynamic changes of the growth characteristics provide a basis for understanding the abnormal growth of the endocrine pancreas.

Long-term effects on pancreatic function of feeding a HC formula to rats during the preweaning period.

We have investigated pancreatic changes associated with hyperinsulinemia and an insulin secretory response to an oral glucose load in a new rat model for obesity. Male Sprague-Dawley pups were reared on a high-carbohydrate (HC) or high-fat (HF) formula by gastrostomy during the suckling period and were weaned onto a stock diet. These animals remained either nutritionally unchallenged or challenged with a high-sucrose diet during the postweaning period. The HC formula-fed animals showed increased insulin concentrations in the plasma and pancreas and also showed impaired insulin secretory response compared with mother-fed control or HF animals in adult life. Immunocytochemical and morphometric studies revealed that hyperinsulinemia in the HC animals during the preweaning period and also in adult life was associated with hypertrophy of beta-cells in the pancreas. The results show that consumption of a HC formula during the suckling period influences pancreatic islet morphology resulting in hyperinsulinemia which eventually leads to the development of obesity later in adult life.

Hormonal regulation of chimeric genes containing the phosphoenolpyruvate carboxykinase promoter regulatory region in hepatoma cells infected by murine retroviruses.

Hepatoma cells were infected with replication-incompetent murine retroviruses containing the selectable gene for amino-3'-glycosyl phosphotransferase (neo) and/or the nonselectable gene for bovine growth hormone (bGH). Expression of these genes was controlled by the promoter regulatory region of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from the rat, which contains hormone and tissue-specific regulatory elements. Expression of the transduced PEPCK-neo gene was stimulated by Bt2cAMP and glucocorticoids and inhibited by insulin. The amount of RNA which initiated within the retroviral 5' long terminal repeat (5' LTR) was inhibited when internal promoters were present in the retroviral vector. When no internal promoter was present, expression from the 5' LTR was higher and stimulated by glucocorticoids, due to the presence of a glucocorticoid regulatory element in the 5' LTR. Infection of cells with retroviruses altered the basal expression and hormonal regulation of the endogenous PEPCK gene, but had no effect on the expression of the tyrosine aminotransferase gene, which is regulated in a similar manner by cAMP and glucocorticoids. A segment of the PEPCK promoter acted as a hormonally regulated enhancer, bringing the SV40 early promoter under the control of Bt2cAMP. A second, nonselectable gene (PEPCK-bGH), contained in the retroviral vector together with PEPCK-neo, was expressed and regulated appropriately when introduced into hepatoma cells. The proviruses were initially integrated randomly into the host cell genome, but after prolonged selection for expression of the transduced PEPCK-neo gene, cells were selected which contain a predominant site(s) of integration. Among populations of cells, however, the predominant site(s) of proviral integration was different. The selection of cells with a specific site of integration from a population was accelerated by the presence of PEPCK promoter sequences in the provirus. Despite the need to better characterize their effects on the host cell, retroviruses appear to be versatile tools for the specific introduction of regulated genes into cells.

An immunohistochemical, ultrastructural, and physiologic study of pancreatic islets from copper-deficient, penicillamine-treated rats.

Murine copper deficiency induced by diet and supplemented with a copper chelator is known to produce a progressive atrophy of pancreatic acinar tissue largely replaced by noninflammatory lipomatosis, while the ductal and endocrine systems appear to remain unaffected. The islets were studied morphologically and physiologically in animals rendered copper deficient by diet and supplemented with D-penicillamine. Using immunohistochemistry, the distribution of islet cell types from copper-deficient animals exhibited a normal cellular complement for A-, B-, D-, and PP-cells. Ultrastructural analysis showed the islet tissue remains normal in appearance during the course of the metal-deficient state. Physiologic data based on the response of islets to a low- and high-glucose load in perfused, isolated pancreata as well as intravenous glucose tolerance tests indicated that insulin-secreting B-cells were functionally normal. Because of the accessibility of islets enhanced by atrophy of acini, this model may be adopted for the isolation of viable islets and for in situ physiologic studies of islet hormone secretion.

Immunocytochemical localization of insulin-immunoreactive cells in the pancreatic ducts of rats treated with trypsin inhibitor.

It is well known that soybean trypsin inhibitor exerts trophic effects on the exocrine pancreas, resulting in the hypertrophy of acinar cells. Some evidence also exists for hyperplasia in acinar tissue, the ductal epithelium and islet tissue. Rats maintained for 3 weeks on an oral administration of soybean trypsin inhibitor (200 mg/50 ml drinking water) were compared with untreated animals. Significant changes were noted in treated animals (p less than 0.01). Trypsin inhibitor-treated rats showed an increase in pancreatic weight (2.33 +/- 0.46 g). The volume ratio of acinar, islet and connective tissue as measured by the stereology point-count technique remained the same in both groups. Ductal tissue, however, exhibited an increase in volume ratio, 3.77 +/- 4.38% per 2714 micron2 area of tissue, in trypsin inhibitor-treated animals. All tissue components showed an increase in the experimental animals: acinar (125%), islet (144%), ductal (325%) and connective tissue (94%). Increased size of acinar cell nuclei, as measured by average cord length, 6.20 +/- 0.13 micron, and a decreased nuclear density of acinar cells, 28 +/- 4.74 per 150 micron2 area of tissue, indicated hypertrophic changes in these cells of the experimental animals. Using immunohistochemical localization and the point-count technique, a significant fraction of the total pancreatic volume in experimental animals was represented by ducts containing immunoreactive cells. The percent of volume ratio, 0.42 +/- 0.15% per 2714 micron2 area of tissue, was calculated for ducts containing insulin-immunoreactive cells within their epithelium.

Electron microscopic immunocytochemical localization of glucagon and pancreatic polypeptide in rat pancreas: characterization of a population of islet cells containing both peptides.

The distribution of glucagon and pancreatic polypeptide was studied immunocytochemically in rat pancreas at both light and electron microscopic levels. My earlier observation that these two peptides are distributed in three cell types--cells containing glucagon, cells containing pancreatic polypeptide, and cells containing both--was confirmed at the electron microscopic level. In the glucagon-pancreatic polypeptide cells, the immunoreactivities of the two peptides were present in the same secretion granules. In addition, these glucagon and pancreatic polypeptide-containing granules were morphologically distinct from glucagon granules but similar to pancreatic polypeptide granules and somatostatin granules.

Rat pancreatic beta-cells in protein deficiency: a study involving morphometric analysis and alloxan effect.

The possible cause(s) of impaired glucose tolerance in protein-calorie malnutrition was studied. The beta-cell mass was morphometrically determined and the sensitivity to alloxan was characterized in rats fed ad libitum a 4% protein diet, pair-fed or fed ad libitum a 20% protein diet. The percentage of beta-cells in the pancreas was neither affected by protein deficiency nor influenced by caloric intake. However, the diabetogenicity of alloxan was greatly reduced in protein-malnourished rats. This reduction in diabetogenicity by alloxan was partially reversed by feeding animals with sulfhydryl compound. These results suggest that decreased insulin secretion in protein malnutrition is not due to a reduction in beta-cell number.

Glucagon and pancreatic polypeptide immunoreactivities co-exist in a population of rat islet cells.

In mammalian pancreas, glucagon and pancreatic polypeptide have been shown to be present in distinct cell types. The present communication reports that, in rat pancreas, in addition to glucagon and pancreatic polypeptide cell populations, there is a small population of cells which contain both glucagon and pancreatic polypeptide immunoreactivities.

Effect of glucose on beta cell proliferation and population size in organ culture of foetal and neonatal rat pancreases.

The effect of glucose on growth of the beta cell population was characterized in rat pancreatic organ culture. The effect was monitored by measuring [3H]thymidine labelling indices of beta cells during the culture period and by quantitation of beta cell population size at the end of the culture period. Foetal and neonatal pancreases and different glucose levels were compared. Glucose was found to be effective in stimulating the beta cell proliferation and beta cell population increase at 300 mg/100 ml in 18-day foetal pancreatic explants, but not in 3-day neonatal explants, when compared to the control level of 100 mg/100 ml. A higher level of glucose (500 mg/100 ml) was ineffective and may even inhibit beta cell population growth. The higher than control levels of glucose (300 mg/100 ml and 500 mg/100 ml) were able to stimulate insulin secretion in neonatal tissue, but not in foetal tissue, although foetal tissue may develop such response later in culture. These results suggest that glucose stimulates beta cell proliferation and insulin secretion through different mechanisms. They further show that the potentiality for beta cell proliferation under glucose stimulation decreases with age of the explants and that the capacity for beta cell to proliferate as a function of glucose stimulation is limited.

Changes of pancreatic beta cell population during larval development of Rana pipiens.

The morphology and population size of immunohistochemically stained insulin-containing pancreatic beta cells are studied in Rana pipiens during larval development. These beta cells appear as single cells or form small clusters at early stages and gradually develop into large clusters arranged in cords surrounding the blood sinusoids in midlarval stages. The total quantity of these cells increases from stage II through midlarval stages, reaching maximum at stages XVIII and XIX. A significant decrease of total beta cell quantity is observed between stages XIX and XX. This decrease occurs slightly later than the onset of degeneration of exocrine tissue. Morphological indication of beta cell breakdown is also observed during this period. Although the population size of beta cells stays relatively constant from stages XXI to XXV, a further maturational change is observed in beta cell morphology between stages XXIV and XXV. During these stages, the polarization of stained immunoreactivity toward sinusoids is more obvious and there is a general decrease of staining intensity in the cytoplasm.

Chromosomal replication asynchrony of a human breast carcinoma cell line. I. studied by continuous BrdU incorporation and G-banding analysis.

Continuous BrdU incorporation and the Giemsa staining technique were used to study the cell cycle kinetics of a human breast tumor cell line. It was found that the interchromosomal replication pattern of the neoplastic cell was significantly different from that of normal cells in two respects. First, the pattern is highly asynchronous; within a single cell there are chromosomes at different replication cycles; that is, some chromosomes complete their DNA replication before others begin. Second, the replication schedule for the chromosomes, as identified by superimposing the BrdU-Giemsa technique on the trypsin G-banding technique, is relatively consistent within the cell line but differs from that of normal cells. Some chromosomes that replicate late in normal human lymphocytes and fibroblasts replicate early in this cell line. In contrast to the unusual interchromosomal replication pattern, gross analysis of the intrachromosomal replication schedule shows no apparent difference from that reported for normal cells. The asynchrony phenomenon reported here may be associated with the etiology of aneuploidy in neoplasia.

Distribution and morphometric quantitation of pancreatic endocrine cell types in the frog, Rana pipiens.

Cells reactive to anti-anglerfish insulin, anti-porcine glucagon, anti-synthetic somatostatin, and anti-bovine pancreatic polypeptide were identified in adult Rana pipiens male pancreases using peroxidase anti-peroxidase immunohistochemistry. Insulin positive cells are columnar shaped and arranged in cords. Glucagon positive and somatostatin positive cells are located around the core of insulin positive cells. Isolated cells and clusters of cells of only one cell type are also found. Adjacent sections stained with anti-glucagon and anti-bovine pancreatic polypeptide showed that glucagon positivity and pancreatic polypeptide positivity are found in the same cells. Comparison of double stained adjacent sections confirmed the presence of these two antigens in the same cells, and further showed the occasional presence of cells which are positive to only glucagon or pancreatic polypeptide. Staining of rat pancreas with these two antisera showed that glucagon and pancreatic polypeptide are present in two distinct cell populations. Morphometric quantitation of immunohistochemically stained sections of Rana pipiens pancreases showed that about 2% of the pancreas is endocrine tissue. Of this, 43% is comprised of insulin positive cells, and 43% is occupied by glucagon-pancreatic polypeptide positive cells. Somatostatin positive cell occupy about 14% of the total islet volume. The presence of glucagon and pancreatic polypeptide in the same cell population in the frog, but in different cell populations in mammals, may reflect special functional adaptation in this species, or a close relation of these two hormones and their cells of production during evolution.

Cell turnover in the beak of Rana pipiens.

Rana pipiens tadpoles were injected with 3H-thymidine at different stages to label basal cells of the horny beaks, the keratinized oral specializations of many anuran tadpoles. Cells in the column and sheath of the beak are derived from the basal layer. Movement of the labeled cells in beak columns can be traced, permitting calculation of the rate of cell addition into the columns. With the counts of the total number of column cells and cell addition rate, the rate of apical cell loss from a column throughout the larval stages can also be calculated. The rate of cell addition decreases steadily from stage III through stage XX, in all portions of both beaks. The rate of change of these column cell addition rates is similar among middle and lateral portions of both upper and lower beaks. There is a relatively constant rate of cell loss until stage XII or XIII. It then increases sharply, and the beaks are completely lost at stages XIX or XX. The longer, lower beak columns have a much faster cell loss rate than do the upper beak columns at late larval stages. Life spans for cells that move to the sheath may be about one-half those for cells that enter the beak columns, It is suggested that thyroid hormone accelerates the rate of column cell loss.