NK cell development in bone marrow and liver: site matters.

The NKp46 protein is found on resting and activated natural killer (NK) cells and is involved in the recognition of malignant and infected cells. The expression of NKp46 is believed to precede that of DX5 in early NK cell development. We show that this is not the case in the bone marrow (BM). Here, NKp46 is predominantly expressed after DX5, whereas the liver harbors a subpopulation that expresses NKp46 but not DX5. NK cell precursors in the liver show much lower levels of Eomesodermin than NK cell precursors in the BM, although they express higher levels of granzymes and unlike the NK cell precursors in the BM are fully able to degranulate and produce interferon gamma (IFN-γ). The development of NK cells thus differs between the two organs. This needs to be considered when using NKp46 and DX5 as NK cell markers and when performing NK cell-specific gene deletion in Ncr1 transgenic mice.

Endoglandular trophoblast, an alternative route of trophoblast invasion? Analysis with novel confrontation co-culture models.

BACKGROUND: Routes of trophoblast invasion seem to be clear, whereas specific invasive pathways need further elucidation. Extravillous trophoblasts (EVTs) transform spiral arteries to guarantee appropriate blood flow to the placenta in the second trimester. Embryo nutrition during the first trimester is thought to be histiotrophic, whereas proof that EVTs also invade uterine glands is lacking. We developed novel three-dimensional confrontation co-culture models to elucidate invasion of EVTs into uterine glands. METHODS: First trimester decidua parietalis and placental villous explants were directly confronted and co-cultured for 72 h, or confronted indirectly after 72 h pre-culture for re-epithelialization of decidua pieces. Cryosections were stained by immunohistochemistry or immunofluorescent/immunohistochemical double labelling and compared with first trimester placentation sites in situ. RESULTS: EVTs deeply invaded decidual tissues in direct confrontation assays and were found between the decidual epithelial cells and epithelial basement membrane. EVTs were also detected in the decidual stroma in direct proximity to glands, sometimes even replacing glandular epithelial cells. Similar observations were made in sections from the first trimester decidua/placental bed. In the invaded parts of sections of decidua basalis, 55% +/- 7% (mean +/- SEM; n = 10, range 6-11 weeks) of glandular cross sections were associated with or infiltrated by EVTs. CONCLUSIONS: Using novel confrontation co-culture assays, a potential new route of EVT invasion was detected. EVTs appear to break through the basement membrane of uterine glands to open their lumen towards the intervillous space. These data support the hypothesis of histiotrophic nutrition of the embryo prior to onset of maternal blood flow within the placenta.

Impaired blood polymorphonuclear leukocyte migration and infection risk in severe trauma.

OBJECTIVES: We investigated the association of impaired blood polymorphonuclear leukocyte (PMN) migration with the incidence of bacterial infections in patients with severe trauma. METHOD: Twenty-six intensive-care patients with different injury severity scores were enrolled in a prospective study. PMN migration was measured daily using 300 microl fresh whole blood in a membrane filter assay. Migration was evaluated in an automated image analyzer that recorded numbers and distribution of the immigrant PMNs within a filter. The relevant parameter was the percentage of PMNs that migrated from the blood samples into the filters upon f-Met-Leu-Phe stimulation. RESULTS: Nine patients developed posttraumatic infections verified microbiologically. These patients showed a reduced PMN migratory capacity in comparison with the 17 patients without infections. A migrating portion of six per cent or less at least three days in succession preceded infections by one to 19 days and indicated infection in eight true positive versus three false positive cases, and 14 true negative versus one false negative case, i.e. specificity was 82.3% and sensitivity 88.8%, p=0.0008. Trauma severity had no influence on PMN migration. CONCLUSIONS: Trauma patients with impaired PMN migration are at risk for bacterial infections. Whole-blood migration tests can define the infection risk and thus may be useful predictive markers for infections.

A simple method for measuring the F-actin content of human polymorphonuclear leukocytes in whole blood.

We have developed an improved method for measuring the filamentous (F) actin content of human blood polymorphonuclear leukocytes (PMNs). The essential feature of the method is the immediate fixation of the F-actin cytoskeleton. Fresh whole blood (100 microliters) is shock-cooled by the addition of 1.0 ml of a mixture of 18.75% glycerol and 5% formaldehyde in phosphate buffer pre-cooled to -8 degrees C and subsequently fixed at 4 degrees C for 15 min. After lysis in distilled water and removal of the red blood cells by centrifugation, the F-actin cytoskeleton of the PMNs is stained with fluorescein isothiocyanate (FITC)-phalloidin and quantified by means of flow cytometry. In healthy test subjects, PMN stimulation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) for 20 s resulted in a significantly increased F-actin assembly, while in patients with multiple organ failure, two subpopulations arose: one with greater F-actin content and a second with lower F-actin content in comparison with the unstimulated blood sample. This simple and fast method may be a useful tool in basic and clinical research.

Impaired whole-blood polymorphonuclear leukocyte migration as a possible predictive marker for infections in preterm premature rupture of membranes.

OBJECTIVES: Steroids, used in preterm premature rupture of membranes (pPROM), to reduce the risk of morbidity and mortality of the preterm neonate, impair the maternal polymorphonuclear leukocyte (PMN)-based immune system. In spite of combination with antibiotics, prenatal and postnatal bacterial infections of mother and child are frequent. This pilot study focuses on the influence of steroids in pPROM on maternal PMN functional capacity and subsequent infections. METHODS: After opting for expectant management, eight women with pPROM and no signs of infection were treated by steroids (betamethasone 5.7 mg, i.m. every 24 hours, for three days) and antibiotic therapy with either amoxicillin and clavulanic acid, piperacillin or ampicillin i.v. up to delivery. The conventional inflammation parameters of PMN blood count and C-reactive protein (CRP) were measured daily in parallel with PMN migratory capacity towards N-formyl-methionyl-leucyl-phenylalanine stimulation and under blank conditions, estimated by a whole blood membrane filter assay. RESULTS: In all patients PMN migration decreased during the application of steroids. Three patients showed a decrease in PMN migration below critical values and in spite of antibiotic prophylaxis acute pyelonephritis developed 2-6 days later. PMN count and CRP were not predictive of maternal infection. CONCLUSION: Reduced PMN function, caused by steroid treatment in pPROM, is suggested to be a reason for serious bacterial infections in spite of antibiotic prophylaxis. PMN migration reflects individual PMN defensive capacity.

The affinity of MCF7 breast cancer cells to hyaluronan substrates of different molecular weight and concentrations in an in vitro model.

The affinity of MCF7 breast cancer cells to hyaluronan (HA) was investigated in an in vitro model. The cells form a tightly adhering monolayer on native HA with a concentration of 5 mg/ml. On native HA at higher concentrations the cells reduce their adhesion to the substrate in favor of increased intercellular bonds, resulting in a cluster-like aggregate that tends to detach from the substrate. Aggregate formation is accomplished after 12 h incubation. The phenomenon is independent of the CD44 receptor. Degradation of native HA by hyaluronidase abolishes aggregate formation even at high HA concentrations in favor of formation of a firmly adhering monolayer. This model may help to understand tumor spread on HA tissue structures and may explain therapy successes with hyaluronidase in tumor patients.

[ECG characteristics in epilepsy of homo- and heterospecific hereditary origin and schizophrenia with latent epileptic predisposition]. / Osobennosti EEG pri épilepsii s gomo- i geterospetsificheskoi nasledstvennoi otiagoshchennost'iu i shizofrenii so skrytoi epilepticheskoi predraspolozhennost'iu.

The EEG data were compared among 260 epileptic patients, including 94 patients loaded with schizophrenia, 71 patients loaded with epilepsy, 95 patients without revealed hereditary loading with mental diseases, and among 32 schizophrenics in whom epileptic seizures could be seen during insulin therapy. Hereditary loading with epilepsy or schizophrenia in epileptic patients together with latent epileptic schizophrenia ++predisposition influence the characteristics of the electroencephalogram.

A spectrophotometric method for the determination of copper ions and its application to the co-precipitation of copper in the crystallization of sodium.

The molar absorptivity of the cyanide complexes [Cu(CN)(3)](2-) and [Cu(CN)(4)](3-), at their isosbestic wavelength (235 nm) is 1.13 x 10(3) l.mole(-1)mm.(-1) and can be used for the quantitative determination of micro-amounts of copper in the ppm range. The determination of 1-10 mug of Cu(2+) per g of NaCl, or 0.25-2.5 mug ml , is described in detail. The co-precipitation of copper with NaCl crystallizing from aqueous solutions has been studied by this method.

Determination of bivalent cationic impurities in sodium chloride by atomic absorption, and their distribution in the recrystallized salt.

Bivalent cationic impurities present in NaCl crystals were determined quantitatively by atomic absorption, 1.71M NaCl solutions (10% w v ) being used without pretreatment. The sensitivity for several cations was 50-60% of that in pure water solutions. The analyses revealed a non-homogeneous distribution of the impurities, due to differences in their activity during the process of crystallization.