Granulocyte colony-stimulating factor administration to healthy volunteers: analysis of the immediate activating effects on circulating neutrophils

In four healthy volunteers, we analyzed in detail the immediate in vivo effects on circulating neutrophils of subcutaneous administration of 300 micrograms of granulocyte colony-stimulating factor (G-CSF). Neutrophil activation was assessed by measurement of degranulation. Mobilization of secretory vesicles was shown by a decrease in leukocyte alkaline phosphatase content of the circulating neutrophils. Furthermore, shortly postinjection, Fc gamma RIII was found to be upregulated from an intracellular pool that we identified by immunoelectron microscopy as secretory vesicles. Intravascular release of specific granules was shown by increased plasma levels of lactoferrin and by upregulation of the expression of CD66b and CD11b on circulating neutrophils. Moreover, measurement of fourfold elevated plasma levels of elastase, bound to its physiologic inhibitor alpha 1- antitrypsin, indicated mobilization of azurophil granules. However, no expression of CD63, a marker of azurophil granules, was observed on circulating neutrophils. G-CSF--induced mobilization of secretory vesicles and specific granules could be mimicked in whole blood cultures in vitro, in contrast to release of azurophil granules. Therefore, we postulate that the most activated neutrophils leave the circulation, as observed shortly postinjection, and undergo subsequent stimulation in the endothelial microenvironment, resulting in mobilization of azurophil granules. Our data demonstrate that G-CSF should be regarded as a potent immediate activator of neutrophils in vivo.     

Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes

BACKGROUND/AIMS: Liver failure is a life threatening condition currently treated by palliative measures and, when applicable, organ transplantation. The use of a bioartificial organ capable of fulfilling the main functions of the liver would represent an attractive alternative. However, the shortage of suitable donor cells, and their limited growth ability have impeded the development of this strategy. We investigated whether lentiviral vectors allow for conditional immortalization of human hepatocytes and whether these immortalized hepatocytes could reverse lethal acute liver failure. METHODS: We exposed primary human hepatocytes to Cre-excisable lentiviral vectors coding for SV40T Antigen, telomerase, and/or Bmi-1 and tested the functionality of the resulting cell lines. Therapeutic potential of immortalized hepatocytes were tested in a murine model of acetaminophen-induced hepatic injury. RESULTS: The immortalized hepatocytes grew continuously yet were non-tumorigenic, stopped proliferating when exposed to Cre recombinase, and conserved defining properties of primary hepatocytes, including the ability to secrete liver-specific proteins and to detoxify drugs. The implantation of encapsulated immortalized human hepatocytes rescued mice from lethal doses of acetaminophen. CONCLUSIONS: Lentiviral vectors represent tools of choice for immortalization of non-dividing primary cells, and lentivirally immortalized human hepatocytes are promising reagents for cell-based therapy of acute liver failure.     

Model for estimation of clinically achievable plasma concentrations for investigational anticancer drugs in man

A major problem related to in vitro testing of new investigational anticancer compounds is the lack of accurate pharmacokinetic data for the drugs in man. Based on the concept of a relationship between certain toxicologic endpoints in animal systems and maximally tolerated doses in man, we hypothesized that a comparable agreement might exist between these experimental animal toxicology data and clinically achievable peak plasma concentrations (PPCs). A retrospective analysis of the known data pairs of 28 commonly used cytotoxic compounds supports the existence of a reasonably good correlation between ip LD50 values (Pearson test) in nontumored mice and PPCs in man (r2 = 0.501; P less than 0.0001). Our data suggest that by use of the resultant statistical regression model a rational starting point can be selected for in vitro screening of entirely new agents for which human PPCs are not available. Additionally, application of this approach may also prove useful in relation to selecting in vitro doses for chemosensitivity assays intended for predictive correlation with clinical response in cancer patients.     

Estrogens inhibit copper and cell-mediated modification of low density lipoprotein.

The effects of estrogens on LDL modification by copper ions, U 937 monocyte-like cells or endothelial cells was studied by determination of the lipid peroxidation product content and measurement of the relative electrophoretic mobility. The presence of estradiol, estriol and estrone inhibited LDL oxidation in a dose-dependent manner in the range of concentrations from 5 to 50 microM. In the case of oxidation by Cu2+, the decreasing order of efficiency was: estradiol, estriol, estrone. In monocyte-induced oxidation, the protective effect of estrogens was more marked, and the order of efficiency was the same, except that estrone was as active as estriol. Pretreatment of monocyte cells with estrogens also inhibited the subsequent modification of LDL by these cells, tested in the absence of the hormones. Testosterone had no effect in all the studied systems. Furthermore, the degradation by J774 macrophage like cells of LDL modified either by Cu2+ or monocytes was markedly reduced when modification has been performed in the presence of estrogens. Since oxidative modification of LDL is believed to be involved in the appearance of atherosclerotic plaques, this effect of estrogens might be related to their protective action against atherosclerosis.     

Pathogenic role of antiphospholipid antibodies

The antiphospholipid antibody syndrome (APS) is characterized by recurrent arterial and venous thrombosis and/or pregnancy in association with antiphospholipid (aPL) antibodies. The pathogenic mechanisms in APS that lead to in vivo injury are incompletely understood. Recent evidence suggests that APL antibodies alter regulation of haemostasis and induce activation of complement. We will discuss the current knowledge on how aPL antibodies trigger increased inflammation and enhanced thrombotic tendency, and thereby lead to tissue damage.     

Endothelial hyperplasia and endothelial galectin-3 expression are prognostic factors in primary central nervous system lymphomas

Recently, considerable attention has been focused on the identification of clinically relevant prognostic markers for primary central nervous system lymphomas (PCNSL). The present study investigated whether three morphological features, i.e. necrosis, reactive perivascular T-cell infiltrate and endothelial hyperplasia, and galectin-1 and galectin-3 immunohistochemical expression have prognostic roles in a series of 58 PCNSL samples from 44 immunocompetent and 14 immunocompromised patients. The presence of endothelial hyperplasia (identified in 21% of the assessable cases) was identified as a bad prognostic factor for immunocompetent PCNSL patients, whereas the other morphological features were not associated with any prognostic value. Lymphomatous cells of eight PCNSL cases expressed galectin-3 without any prognostic value, and lymphomatous cells did not express galectin-1. In contrast, endothelial expression of galectin-3 was identified (by means of uni- and multi-variate analyses) as a bad prognostic factor for immunocompetent PCNSL patients. In addition, a combination of endothelial hyperplasia and/or endothelial galectin-3 expression was shown to be an independent prognostic factor for immunocompetent PCNSL patients treated with methotrexate-based chemotherapy. In summary, this study suggests that endothelial-related markers can identify risk groups of PCNSL patients and indicates that galectin-3 could be involved in PCNSL angiogenesis.     

DNA amplification of HIV-1 in seropositive individuals and in seronegative at-risk individuals

We assessed the HIV-1 status of seropositive and seronegative at-risk individuals by the polymerase chain reaction. Fifty-four out of 55 HIV-1-seropositive samples scored positive. However, HIV-1 proviral DNA was not detected in 16 seronegative homosexuals, 20 seronegative polytransfused haemophiliacs and 20 seronegative thalassaemic children, 20 individuals with isolated and persistent anti-core antibodies and 74 seronegative blood donors. These data indicate that positive HIV-1 DNA is likely to be an exceptional phenomenon in HIV-seronegative people.     

Verbal learning in Alzheimer's disease and mild cognitive impairment: fine-grained acquisition and short-delay consolidation performance and neural correlates

The aim of this study was to examine correlations between acquisition and short-delay consolidation and brain metabolism at rest measured by fluorodeoxyglucose positron emission tomography (FDG-PET) in 44 Alzheimer's disease (AD) patients, 16 patients with mild cognitive impairment (MCI) who progressed to dementia (MCI-AD), 15 MCI patients who remained stable (MCI-S, 4–8 years of follow-up), and 20 healthy older participants. Acquisition and short-delay consolidation were calculated respectively as mean gained (MG) and lost (ML) access to items of the California Verbal Learning Task. MG performance suggests that acquisition is impaired in AD patients even at predementia stage (MCI-AD). ML performance suggests that short-delay consolidation is deficient only in confirmed AD patients. Variations in acquisition performance in control participants are related to metabolic activity in the anterior parietal cortex, an area supporting task-positive attentional processes. In contrast, the acquisition deficit is related to decreased activity in the lateral temporal cortex, an area supporting semantic processes, in patients at an early stage of AD and is related to metabolic activity in the hippocampus, an area supporting associative processes, in confirmed AD patients.