Angiostatin overexpression in Morris hepatoma results in decreased tumor growth but increased perfusion and vascularization.

Growth of malignant tumors is dependent on sufficient blood supply. Thus, inhibition of tumor angiogenesis is emerging as a promising target in the treatment of malignancies. Human angiostatin (hANG) is one of the most potent inhibitors of endothelial cell proliferation, angiogenesis, and tumor growth in vivo. However, its mechanisms operating in vivo are not well understood. METHODS: To obtain more information about functional changes in the angiogenic process, we established Morris hepatoma (MH3924A) cell lines expressing hANG (hANG-MH3924A). The effects of hANG expression on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) were measured in coculture experiments in vitro. To evaluate changes in tumor perfusion and blood volume, H2 15O and 68Ga-DOTA-albumin (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid) were used for PET studies in vivo. Additionally, immunohistologic quantification of vascularization, apoptosis, and proliferation as well as gene array analyses were performed. RESULTS: Our in vitro experiments demonstrate reduced proliferation and increased apoptosis in HUVECs when being cocultured with hANG-MH3924A. In support, tumor growth of hANG-MH3924A is diminished by 95% in vivo. However, tumor perfusion and blood volume are increased in hANG-MH3924A corresponding to an increased microvessel density. Furthermore, hANG-transfected tumors show changes in expression of genes related to apoptosis, stress, signal transduction, and metabolism. CONCLUSION: hANG expression leads to inhibition of tumor growth, increased apoptosis, and changes in the expression of multiple genes involved in stress reactions, signal transduction, and apoptosis, which indicates a multifactorial reaction of tumors. An enhanced microvessel density is seen as part of these reactions and is associated with increased perfusion as measured by PET.     

Total cortisol,free cortisol,and growth hormone associated with brief social separation experiences in young macaques

Many behavioral, immunological, and physiological consequences of brief maternal separation in bonnet (Macaca radiata) and pigtail monkeys (Macaca nemistrina) have been documented. However, the impact of social separation on plasma cortisol and growth hormone is unknown for these particular species. In the present study, the behavioral and endocrinological consequences of a 2-week maternal separation in socially housed infant bonnet and pigtail monkeys were followed. In seven pairs (separated and matched control) of bonnet and six pairs of pigtail infants, plasma was obtained under baseline, separated, and reunion conditions twice weekly for the duration of the study. Blood samples were obtained from both infants of the pair in approximately 10 min. Plasma total cortisol, free cortisol, and growth hormone were measured in these samples. Focal animal behavioral observations were made on all subjects twice daily throughout the study period. In both species, total cortisol and free cortisol rose immediately following maternal separation in comparison to the matched nonseparated controls and returned to basal levels (e.g., that of matched non-separted controls) following reunion with the mother. In contrast, plasma growth hormone rose only in the pigtail infants over a time course that peaked around the time of reunion. Multiple regression techniques indicated for the first week of separation, in the separated but not control subjects, that mean plasma free and total cortisol was positively related to distress behaviors (vocalization and postural slouch) observed during this week and negatively related to social behaviors (play and proximity to others) noted during the same period. In contrast, plasma growth hormone was related to both species and sex of the subjects but unrelated to behavioral variables. © 1995 John Wiley & Sons, Inc.     

Anosognosia in mild cognitive impairment: Relationship to activation of cortical midline structures involved in self-appraisal.

Awareness of cognitive dysfunction shown by individuals with Mild Cognitive Impairment (MCI), a condition conferring risk for Alzheimer's disease (AD), is variable. Anosognosia, or unawareness of loss of function, is beginning to be recognized as an important clinical symptom of MCI. However, little is known about the brain substrates underlying this symptom. We hypothesized that MCI participants' activation of cortical midline structures (CMS) during self-appraisal would covary with level of insight into cognitive difficulties (indexed by a discrepancy score between patient and informant ratings of cognitive decline in each MCI participant). To address this hypothesis, we first compared 16 MCI participants and 16 age-matched controls, examining brain regions showing conjoint or differential BOLD response during self-appraisal. Second, we used regression to investigate the relationship between awareness of deficit in MCI and BOLD activity during self-appraisal, controlling for extent of memory impairment. Between-group comparisons indicated that MCI participants show subtly attenuated CMS activity during self-appraisal. Regression analysis revealed a highly significant relationship between BOLD response during self-appraisal and self-awareness of deficit in MCI. This finding highlights the level of anosognosia in MCI as an important predictor of response to self-appraisal in cortical midline structures, brain regions vulnerable to changes in early AD.     

Muscarinic Receptors Activate Calcium Channels and Calcium Dependent Potassium Channels in NIE-115 Neuroblastoma Cells

Abstract: Responses of NIE-115 neuroblastoma cells to application of carbachol were studied using intracellular recording techniques. Activation of muscarinic cholinergic receptors by carbachol resulted in a depolarization of the cells. The response was blocked by pirenzepine (1 μM) and by CoCl₂ (5 mM), verapamil (10 μM) and gallopamil (10 μM), and prolonged by quinine (5 mM). It is suggested that muscarinic receptors increase the membrane calcium permeability, and that the influx of calcium activates calcium dependent potassium channels.     

Neutralization assay using a modified vaccinia virus Ankara vector expressing the green fluorescent protein is a high-throughput method to monitor the humoral immune response against vaccinia virus

Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.     

Rapid PCR Detection of Helicobacter pylori-Associated Virulence and Resistance Genes Directly from Gastric Biopsy Material

We have developed a PCR-based method to detect macrolide resistance and the virulence gene cagA in Helicobacter pylori within 24 h, thereby improving the lengthy process of culture-based approaches. Total DNA was prepared directly from stomach biopsy specimens. The procedure proved to be rapid and reliable and could be utilized for diagnostic purposes.     

Cytotoxicity in vitro of preleukaemic lymphoid cells on syngeneic monolayers of embryo or thymus cells

Lymphoid cells from preleukaemic AKR mice were cytotoxic for monolayers of syngeneic embryo and thymus target cells in tissue culture. This reactivity was detectable with cells from mice aged 3–36 weeks but was not present with cells from younger mice. Cytotoxic reactions to syngeneic embryo tissues were also seen with lymphoid cells from high leukaemia strain C3H mice carrying Moloney virus but not with lymphoid cells from normal low leukaemic strain C3H/Bi or DBA/2 mice. Lymphoid or lymphoma cells from leukaemic AKR mice showed reduced reactivity. Phytohaemagglutinin was not necessary for the reaction of preleukaemic AKR cells against AKR monolayers and cytotoxicity was inhibited by preincubation of target cells with an antiserum directed against AKR G+ cells.

The reactivity of preleukaemic AKR and C3H lymphoid cells against syngeneic monolayers may represent some type of allogeneic inhibition due to acquired antigenic differences between aggressor and target cell but the data fit best an interpretation that some lymphoid cells in preleukaemic AKR and C3H mice acquire immunological reactivity to virus-induced G+ or M+ antigens exhibited by the target cells.

     

Granulocyte Stimulating Factor in Patients on Peritoneal Dialysis and LPS Stimulated Peripheral Blood Mononuclear Cells

Dialysate and serum levels of granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) were analyzed in patients with continuous ambulatory peritoneal dialysis (CAPD). Samples from the peritoneal effluent and from serum were obtained during the first months of dialysis and during peritonitis from the first three dialysate bags drained on the day of admittance and from nightbags on days three and ten. Serum samples were drawn on days one and ten. On the first day of infection G-CSF was detected in twelve out of fifteen samples in the dialysate and reached its peak median level, 443 pg/ml, in the first drained bag and thereafter decreased significantly. Also in serum a peak, 190 pg/ml, was observed on the first day. LIF was found in six of ten analyzed dialysate samples, with a peak median level of 77 pg/ml on day one, while only four of ten patients had detectable GM-CSF. Peripheral blood mononuclear cells from non-infected CAPD patients were stimulated with lipopolysacharide and G-CSF levels in the supernatants increased significantly (P < 0.05) after 6 h stimulation. We conclude that G-CSF is produced locally in the dialysate during the acute stage of peritonitis and to a lesser extent also systemically. These findings are in line with G-CSF production after LPS stimulation of peripheral blood mononuclear cells.