CD24 expression on human keratinocytes

Abstract: CD24 or Nectadrin is a cell surface glycoprotein expressed in pre-B lymphocytes, T lymphocytes, neurons, muscle cells and carcinoma cells. Its function is not completely known, but it has been suggested that it is involved in cell adhesion and signalling. CD24 has recently been identified as the human molecule homologous to the murine heat-stable antigen (HSA). HSA is expressed by murine keratinocytes and delivers costimulatory signals in T-cell activation. Long-term cultures of normal human keratinocytes (HKC) were obtained from skin of human female breast sections and either left untreated or were treated with phorbol-12-myristate-13-acetate (PMA) at 10–100 ng/ml, calcium 0.5–2 mM or IFN-γ 100–1000 U/ml, for 24–48 h. Using RT-PCR and flow cytometry we showed that HKC express low levels of CD24 even under basal conditions, and the treatment with calcium, PMA or IFN-γ increased levels of CD24 mRNA and protein. To the best of our knowledge, this is the first report to measure CD24 expression and production by cultured HKC in basal conditions and after stimulation. Further studies are needed to determine biological and therapeutical relevance of these findings.     

Purification and characterization of a UDP-glucosyltransferase produced by Legionella pneumophila

Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.     

Light scattering from cervical cells throughout neoplastic progression: influence of nuclear morphology,DNA content,and chromatin texture

A number of noninvasive fiber optic optical technologies are under development for real-time diagnosis of neoplasia. We investigate how the light scattering properties of cervical cells are affected by changes in nuclear morphology, DNA content, and chromatin texture, which occur during neoplastic progression. We used a Cyto-Savant computer-assisted image analysis system to acquire quantitative nuclear features measurements from 122 Feulgen-thionin-stained histopathologic sections of cervical tissue. A subset of the measured nuclear features was incorporated into a finite-difference time-domain (FDTD) model of cellular light scattering. The magnitude and angular distribution of scattered light was calculated for cervical cells as a function of pathologic grade. The nuclear atypia strongly affected light scattering properties. The increased size and elevated DNA content of nuclei in high-grade lesions caused the most significant changes in scattering intensity. The spatial dimensions of chromatin texture features and the amplitude of refractive index fluctuations within the nucleus impacted both the angular distribution of scattering angles and the total amount of scattered light. Cellular scattering is sensitive to changes in nuclear morphology that accompany neoplastic progression. Understanding the quantitative relationships between nuclear features and scattering properties will aid in the development of noninvasive optical technologies for detection of precancerous conditions.     

Comparison of the cytotoxicity of two nitroheterocyclic drugs (NHCD) towards transformed and non-transformed cells.

The cytotoxicity of two nitroheterocyclic compounds (NHCD), Nitracrine, 1-nitro-9(3'3'-dimethylaminopropylamino) acridine (Polfa, Poland) and Quinifuryl, 2-(5'-nitro-2'-furanyl) ethenyl-4-[N-[4-(N,N-diethylamino)-1'-methylbutyl] carbamoyl] quinoline (Dr. N. M. Sukhova, Institute of Organic Synthesis, Riga, Latvian Republic), towards two lines of leukaemic cells and a line of non-transformed cells, was determined under normoxia conditions. Although both drugs showed significant cytotoxicity to all cell lines (LC(50) for 24h, < or = 2 microM) with that of Nitracrine exceeding Quinifuryl, their toxicity towards murine leukaemia P388 was substantially higher, compared to murine fibroblasts NIH3T3. In addition, the rate of cell death was also two- to three-fold higher in case of P388 cells versus NIH3T3. Interestingly, human erythroleukaemia K562 cells were shown to uptake the drugs 10 min after their addition to the tissue culture medium, while the LC(50) values were reached after a substantial delay of 3h. This delay might be due to the intracellular transformation of drugs required for cell killing.     

Impact of the CYP3A5 genotype on midazolam pharmacokinetics and pharmacodynamics during intensive care sedation

Objective Information is lacking on whether the CYP3A5 genotype affects the disposition and effects of midazolam during the long-term intensive care sedation of patients. This study was undertaken to estimate whether the CYP3A5 genotype can explain a relevant portion of pharmacokinetic interindividual variability. Methods We determined the CYP3A5 genotype in 71 Caucasian patients who underwent long-term sedation during intensive care treatment. We then assessed the relation between the genotype and both the plasma concentrations of midazolam and 1′-OH-midazolam in 645 plasma samples and the simultaneously estimated Ramsay sedation score, both of which were recorded during routine midazolam drug monitoring. Results Eight patients had the CYP3A5*1/*3 genotype and 63 patients the CYP3A5*3/*3 genotype. The concentration–dose ratio [C/D; plasma concentration of midazolam (ng/ml) divided by the rate of infusion (mg/h); expressed as the mean (95% confidence interval)] was 87.4 (70.8, 108.9) for the *3/*3 patients and 79.0 (48.9, 129.0) for *1/*3 patients. The corresponding data for infusion rate (IR; in mg/h), Ramsay score (RS) and the ratio 1′-OH-midazolam concentration/midazolam concentration (ROH) for *3/*3 and *1/*3 patients were IR 7.4 (6.2, 8.6) vs. 11.4 (4.9, 17.9), RS 5.4 (5.2, 5.6) vs. 5.3 (4.2, 6.0) and ROH 0.11 (0.09, 0.13) vs. 0.17 (0.11, 0.26), respectively. Conclusions The CYP3A5*1/*3 genotype did not lead to an apparently lower midazolam concentration/dose ratio or Ramsay score values. As the present sedation procedure during intensive care therapy may be described as a physician closed-loop titration towards Ramsay scores of 4 ± 1, our data do not indicate that prior determination of the genotype will result in better care or economic savings.     

Particulate air pollution from bushfires: human exposure and possible health effects

Toxicological studies have implicated trace metals adsorbed onto airborne particles as possible contributors to respiratory and/or cardiovascular inflammation. In particular, the water-soluble metal content is considered to be a harmful component of airborne particulate matter. In this work, the trace metal characteristics of airborne particulate matter, PM2.5, collected in Singapore from February to March 2005 were investigated with specific reference to their bioavailability. PM2.5 mass concentrations varied between 20.9 mug/m3 and 46.3 microg/m3 with an average mass of 32.8 microg/m3. During the sampling period, there were several bushfires in Singapore that contributed to sporadic increases in the particulate air pollution, accompanied by an acrid smell and asthma-related allergies. The aerosol samples were subjected to analysis of trace elements for determining their total concentrations as well as their water soluble fractions. Our results showed an increase in concentration of several water-soluble trace metals during bushfires compared to their urban background levels in Singapore. In order to measure the human exposure to particulate air pollution, the daily respiratory uptake (DRU) of several trace metals was calculated and compared between haze and nonhaze periods. The DRU values were significantly higher for several metals, including Zn, Cu, and Fe, during bushfires. Electron paramagnetic resonance (EPR) measurements showed that the particulate samples collected during bush fires generate more toxic hydroxyl radicals (OH.) than those in the background air, due to the presence of more soluble iron ions.     

α-MSH regulates interleukin-10 expression by human keratinocytes

Abstract Normal human keratinocytes (HKC) are able to synthesize α-MSH. Because the production of α-MSH by HKC is induced significantly by ultraviolet B radiation, the involvement of keratinocyte-derived α-MSH in UV-induced immunosuppression has been suggested. The induction of the antiinflammatory cytokine IL-10 in monocytes is a major mechanism in the antiinflammatory actions of α-MSH. In the present study, HKC were investigated for their ability to produce IL-10 after α-MSH stimulation. HKC were obtained from the skin of human female breast sections and either left untreated or treated with 0.01 or 0.1 μg/ml α-MSH for different times. Using RT-PCR, HKC were shown to express IL-10 mRNA even under basal conditions, and treatment with α-MSH increased expression. Only minimal concentrations of IL-10 protein were detected in supernatants from the α-MSH-stimulated cultures. To the best of our knowledge this is the first report of IL-10 expression by cultured HKC after α-MSH stimulation. Further studies are needed to determine the biological and therapeutic relevance of these findings. Received: 11 November 1997