A new bioassay including a small scale hepatocyte bioreactor for hepato-mediated toxicity testing in a target cell line

New approaches for in vitro testing of hepato-mediated toxicity are undertaken to offer alternatives to in vivo animal testing. The described bioassay for hepato-mediated toxicity testing is based on a small scale hepatocyte-bioreactor with pig hepatocytes connected to a silicon sensor based microphysiometer system for monitoring of the extracellular acidification rate (EAR) of cells and the microphysiometer alone. EAR represents the metabolic activity of tested cells (hepatocytes and ZR 751 cells) under the influence of perfused media, compared to controls, which were set to 100%. Cyclophosphamide (CYCL), whose cytostatic effect is dependent on CYP 450 biotransformation was used as a model substrate. CYCL showed decrease of EAR in hepatocytes, but not in ZR 751 cells. Bioreactor supernatant including CYCL was pumped into the microphysiometer and EARs of the target ZR 751 cell line were recorded. After 7 h of bioreactor supernatant perfusion the ZR 751 cell line showed an EAR decrease of 18.68% +/- 10.18, as compared to controls (bioreactor supernatant from the identical set-up without CYCL). Thus the presented model of hepato-activated toxicity showed an EAR decrease in the ZR 751 cell line that reflected the toxic activation of CYCL by the bioreactor. This new bioassay serves as an example of future applications for hepatocyte bioreactors in automated toxicity testing devices, e.g. in preclinical drug studies or evaluation of hepato-mediated toxicity, not depending on cell destruction or further assays.     

Localization of the cell activator lipopeptide in bone marrow-derived macrophages by electron energy loss spectroscopy (EELS)

Synthetic lipopeptide analogues of bacterial lipoprotein constitute potent polyclonal activators for monocytes/macrophages and B lymphocytes. However, the fate of the lipopeptides after their interaction with target cells is as yet unknown. In order to follow the routes and to determine the distribution of the lipopeptide within macrophages after stimulation, we investigated lipopeptide-stimulated bone marrow-derived macrophages using the novel method of electron energy loss spectroscopy (EELS). Our results show that the lipopeptide was present in different compartments of the cell. The major amount of the activator was located within the cytoplasm and the plasma membrane, and minor quantities were detected within the nuclear membrane and the nucleus. The distribution of the lipopeptides varied depending on the duration of stimulation. Our results should help to elucidate the molecular mechanisms of macrophage stimulation by lipopeptides or other cell activators.     

Inhibition of leukotriene formation and IL-8 release by the PAF-receptor antagonist SM-12502

We analyzed the effect of the PAF receptor antagonist (+)-cis-3,5-dimethyl-2-(3-pyridyl) thiazolidin-4-one hydrochloride (SM-12502) on the release of leukotriene B₄ and IL-8 from human leukocytes. Peripheral blood from healthy donors was separated in two different fractions: polymorphonuclear leukocytes (PMN) and a lymphocyte, monocyte and basophil granulocyte cell fraction (LMB). After incubation of the cell population with different concentrations of SM-12502 the cells were subsequently stimulated with either the Ca ionophore A23187, the bacterial derived peptide fMLP, or with an activator of heterotrimetric G-proteins, the sodium fluoride (NaF, in the presence of Al³⁺). The PAF receptor antagonist led to a concentration and time dependent inhibition of LTB₄ formation and IL-8 release from PMN and LMB. Our data clearly indicate an inhibitory effect of the PAF receptor antagonist SM-12502 on the formation of mediators of the lipoxygenase pathway and on the release of IL-8.     

Negative pressure effects during the isothermal crystallization of isotactic poly(propylene) studied by light and atomic force microscopy

The formation of holes during the late stage of the isothermal crystallization in thin films of isotactic poly(propylene) between two cover glasses was observed by light microscopy and atomic force microscopy. This behavior can be described consistently by the well-known negative pressure effect. Light microscopy reveals the simultaneous and sudden occurrence of a large number of small holes at the liquid-solid interface after the liquid in front of the spherulites is completely confined by other spherulites for a certain time interval. In exceptional cases only a few holes appear and finally large cavities are formed. Atomic force microscopy measurements carried out in the height mode are able to prove the hole formation in front of the spherulites. Furthermore, a substantial thinning of the two-dimensional spherulites in thin films can be observed prior to the hole formation.     

Monosomy 7p in meningiomas: a rare constituent of tumor progression

We present karyotypes of 15 meningiomas with structural aberrations of chromosome 7, which were taken from a consecutive series of 400 cytogenetically characterized meningiomas. Twelve of these tumors (80%) displayed partial or complete monosomy 7p with a consensus deleted region of 7p12 approximately pter, in 6 of 15 cases arising from an unbalanced whole-arm t(1;7)(q11;p11), and in 4 of 15 cases from a whole-arm translocation involving other chromosomes. Other types of partial aneusomy 7 (3/15 cases) or balanced aberrations of chromosome 7 (2/15 cases) were relatively rare. In most cases (11/15), the centromeric region of chromosome 7 was involved in the rearrangements. We conclude that in meningiomas, the near-centromeric region of chromosome 7 is particularly prone to structural rearrangements most frequently resulting in monosomy 7p. The investigation of the histopathologic features of this rare cytogenetic subgroup of meningiomas showed no clear genotype/phenotype correlation. As 7 of 11 of the meningiomas with monosomy 7p belonged to World Health Organization grades II or III, which usually comprise less than 20% of all meningiomas, partial loss of 7p appears to be involved in tumor progression in meningiomas. Because monosomy 7p is typically associated with the strongly progression-associated monosomy 1p, however, monosomy 7p represents a cofactor more than a stand-alone feature of meningioma progression.     

The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures

The active derivative of vitamin A, retinoic acid (RA), is essential for normal embryonic development. The spatio-temporal distribution of embryonic RA results from regulated expression of RA-synthesizing retinaldehyde dehydrogenases and RA-metabolizing cytochrome P450s (CYP26). Excess RA administration or RA deficiency results in a complex spectrum of embryonic abnormalities. As a first step in understanding the developmental function of RA-metabolizing enzymes, we have disrupted the murine Cyp26A1 gene. We report that Cyp26A1-null mutants die during mid-late gestation and show a number of major morphogenetic defects. Spina bifida and truncation of the tail and lumbosacral region (including abnormalities of the kidneys, urogenital tract, and hindgut) are the most conspicuous defects, leading in extreme cases to a sirenomelia ("mermaid tail") phenotype. Cyp26A1 mutants also show posterior transformations of cervical vertebrae and abnormal patterning of the rostral hindbrain, which appears to be partially posteriorly transformed. These defects correlate with two major sites of Cyp26A1 expression in the rostral neural plate and embryonic tail bud. Because all of the Cyp26A1(-/-) abnormalities closely resemble RA teratogenic effects, we postulate that the key function of CYP26A1 is to maintain specific embryonic areas in a RA-depleted state, to protect them against the deleterious effect of ectopic RA signaling.     

Evaluation of enterovirus serological tests IgM-EIA and complement fixation in patients with meningitis, confirmed by detection of enteroviral RNA by RT-PCR in cerebrospinal fluid

An enzyme immunoassay (EIA) for detection of anti-enterovirus IgM antibodies was compared with complement fixation test in 43 patients with confirmed enterovirus meningitis by RT-PCR of cerebrospinal fluids (CSF). In 34% of patients with enterovirus meningitis, IgM antibodies could be found, whereas complement fixation tests were positive in only 20%. The specificity was determined with sera of 105 patients with non-enterovirus meningitis. Specificity of IgM EIA and of complement fixation was 94% and 85%, respectively. In four patients with meningitis but without enterovirus detection in CSF, RT-PCR and virus isolation from stools were positive. In three of these patients, IgM antibodies were detected, giving a strong indication of an enterovirus-associated disease. Because of the high specificity of IgM EIA, diagnosis of enterovirus-associated diseases can be carried out in a single serum sample, whereas by complement fixation tests, only fourfold increases in antibody titres in paired sera indicate an acute infection. The application of IgM EIA is especially important in cases of meningitis when CSF samples are not available and for diagnosis of enterovirus diseases with other clinical symptoms such as fever, enteritis, and hand-foot-and-mouth disease.     

Priming of Immunological Memory by Pneumococcal Conjugate Vaccine in Children Unresponsive to 23-Valent Polysaccharide Pneumococcal Vaccine

Pneumococcal polysaccharide vaccine (PPV) is of limited immunogenicity in infants and immunocompromised patients. Our prospective randomized controlled trial investigated whether priming with pneumococcal conjugate vaccine (PCV) induced specific immunological memory in previously nonresponders to PPV. Of a total of 33 children (2 to 18 years) with polysaccharide-specific immunodeficiency (PSI), group A (n = 16) received two doses of 7-valent PCV in a 4- to 6-week interval, and a booster dose of 23-valent PPV after one year. Group B (n = 17) received two doses of PPV in a 1-year interval exclusively. Specific antibody concentrations for serotypes 4, 5, 6B, 9V, 14, 18C, 19F, and 23F were determined (enzyme-linked immunosorbent assay) before and at 7 and 28 days after administration of the PPV booster and compared to an opsonophagocytosis assay. Of group A, 64 to 100% had antibody concentrations of ≥1 μg/ml on day 28 after the booster versus 25 to 94% of group B. Group A had significantly higher antibody concentrations for all PCV-containing serotypes already on day 7, indicating early memory response. Antibody concentrations were in accordance with functional opsonic activity, although opsonic titers varied among individuals. Pneumococcal vaccination was well tolerated. The incidence of airway infections was reduced after priming with PCV (10/year for group A versus 15/year for group B). Following a PPV booster, even patients primarily not responding to PPV showed a rapid and more pronounced memory response after priming with PCV.