Treatment with aflatoxin B1 for immortalization of human fibroblasts from Li-Fraumeni patients

Immortalization of normal human fibroblasts is a very rare event. Multiple genes such as p53 and cellular senescence genes are possibly involved in immortalization of human fibroblasts, suggesting that multiple treatments with carcinogens are required for the immortalization. We describe here the procedure for immortalization of human fibroblasts (MDAH 087) from Li-Fraumeni cancer syndrome with a germ-line p53 mutation. The cells were subjected to multiple treatments with aflatoxin B₁ (AFB₁) in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), and 3 of 9 MDAH 087 cell cultures treated 1–3 times with 0.1–1 µg/ml AFB₁ became immortal, defined as continuous growth for over 300 population doublings after the first treatment. However, cultures of human fibroblasts from a normal embryo treated under the same conditions failed to escape senescence. The results indicate that the model of human fibroblasts with a mutated p53 allele exposed to AFB₁ is potentially useful for studying mechanisms of chemically induced immortalization.     

MCP-1-dependent signaling in CCR2(-/-) aortic smooth muscle cells

Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a mediator of inflammation that has been implicated in the pathogenesis of a wide variety of human diseases. CCR2, a heterotrimeric G-coupled receptor, is the only known receptor that functions at physiologic concentrations of MCP-1. Despite the importance of CCR2 in mediating MCP-1 responses, several recent studies have suggested that there may be another functional MCP-1 receptor. Using arterial smooth muscle cells (SMC) from CCR2(-/-) mice, we demonstrate that MCP-1 induces tissue-factor activity at physiologic concentrations. The induction of tissue factor by MCP-1 is blocked by pertussis toxin and 1,2-bis(O-aminophenyl-ethane-ethan)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, suggesting that signal transduction through the alternative receptor is G(alphai)-coupled and dependent on mobilization of intracellular Ca(2+). MCP-1 induces a time- and concentration-dependent phosphorylation of the mitogen-activated protein kinases p42/44. The induction of tissue factor activity by MCP-1 is blocked by PD98059, an inhibitor of p42/44 activation, but not by SB203580, a selective p38 inhibitor. These data establish that SMC possess an alternative MCP-1 receptor that signals at concentrations of MCP-1 that are similar to those that activate CCR2. This alternative receptor may be important in mediating some of the effects of MCP-1 in atherosclerotic arteries and in other inflammatory processes.     

Image analysis derived ploidy and proliferation indices in soft tissue sarcomas: comparison with clinical outcome.

AIMS--To compare prognostic information obtained by image analysis cytometry of paraffin wax embedded soft tissue sarcomas with conventional assessment. METHODS--A CAS 200 image analyser was used to determine DNA content of Feulgen stained cytology preparations and tissue sections and to quantify immunostaining by Ki67 and PC10 antibodies. A mitotic count in 50 high power fields was undertaken and histological grade assigned by the Trojani system. Clinical details including follow up and outcome were obtained by case note review. The Kruskal-Wallis one way analysis test, Spearman rho significance test, Kaplan-Meier method, and log-rank test were applied in statistical analysis. RESULTS--Ploidy status, DNA index, 2.5c exceeding rate, 5c exceeding rate, mitotic count and Trojani grade all correlated significantly with clinical outcome. The relation between Ki67 index and outcome did not reach significance. The PC10 index and outcome were not related. Only 2.5c exceeding rate, 5c exceeding rate, and mitotic count correlated significantly with Trojani grade. CONCLUSIONS--DNA content determination of soft tissue sarcomas by image analysis provides quantifiable information of benefit in prediction of outcome. Larger series are required to determine the independent value of ploidy. In this study quantification of anti-Ki67 and anti-PC10 immunostaining was not of prognostic benefit) by contrast with mitotic count and Trojani grade.     

Local and systemic antibody class responses to an infectious laryngotracheitis virus vaccine strain

Chickens infected with infectious laryngotracheitis virus (ILTV) responded by producing virus-specific IgG in their sera, which increased steadily in concentration, but with slight fluctuations, until peak titres were reached 40 days post-inoculation (pi), immediately prior to the second challenge. Thereafter, following an initial lag, concentrations continued to increase for 21 days before falling slightly at the end of the experiment. In contrast, peak concentrations of ILTV-specific IgM were reached 6 days pi falling to their lowest levels by day 16, before increasing to a second peak and trough on days 26 and 32, respectively. This cyclical production of ILTV-specific IgM was confirmed in a second experiment. The pattern of production of ILTV-specific IgG, IgM and IgA, detected in tracheal washings, occurred in the same cyclical manner. IgM was produced first, peak concentrations being detected 5 days pi, whereas IgG and IgA did not peak until 10 days pi, with second peaks of each class being detected 25-30 days pi. The possibility that the cyclical antibody class response to ILTV infection is related to the previously reported intermittent pattern of re-excretion of the virus is discussed.     

Evaluation of four methods for detection of group B streptococcal colonization.

Four methods (streak plate, pour plate, selective broth, and direct fluorescent-antibody staining) were evaluated for their ability to detect group B streptococcal colonization in parturient women and their offspring. When colonization was defined as a positive culture by any method from any site, selective broth was the most sensitive method, detecting 100% of colonized mothers and infants at birth and 48 h of age. This method failed to detect only one colonized individual (infant at 24 h of age). The other three methods detected from 20 to 56% of colonized individuals.     

Candidemia in pediatric outpatients receiving home total parenteral nutrition.

This is a cohort study of pediatric outpatients receiving total parenteral nutrition (TPN) and follow-up care in a Tennessee hospital between January and June 1999. The study was conducted following an increase in the incidence of candidemia. Of 13 children receiving home TPN, five had candidemia; three were due to Candida parapsilosis. Case patients were more likely to have an underlying hematologic disease (P = 0.02) as well as previous history of fungemia (P = 0.02). Two case patients had successive candidemia episodes 3 months apart; karyotypes and RAPD profiles of each patient's successive C. parapsilosis isolates were similar. Candida spp. were frequently detected in hand cultures from cohort members (four of 10) and family member caregivers (nine of 11); C parapsilosis was isolated from five caregivers. Our findings underscore the challenges of maintaining stringent infection control practices in the home health care setting and suggest the need for more intensive follow-up and coordination of home TPN therapy among pediatric patients.     

Mast cell heterogeneity: Evidence and implications

Mast cells and basophils play a fundamental role in the pathogenesis of allergic disease, although their physiologic role is largely unknown. A large body of evidence now indicates that the properties of mast cells are dependent on the tissue and species from which they are derived. Such mast cell heterogeneity encompasses differences in morphology, development, cytochemistry, and function. The evidence for such heterogeneity, and some of its clinical implications, is discussed.     

Rinderpest virus lineage differentiation using RT-PCR and SNAP-ELISA

An RT-PCR/ELISA system has been developed that detects and differentiates Rinderpest virus (RPV) from the other closely related morbillivirus of ruminants, Peste des petits Ruminants virus (PPRV). In addition, using lineage specific probes, it is possible to determine whether the virus sample is wild-type or vaccine, and the likely origin of the outbreak if it is wild-type. It involves carrying out a RT-PCR with one digoxygenin (Dig)-labelled primer followed by a hybridisation step with a virus-specific, biotin-labelled, probe. The hybridisation step is carried out in an ELISA format on a streptavidin-coated plate. The DIG-labelled products are detected using a specific anti-DIG monoclonal antibody and an anti-mouse horseradish peroxidase conjugate. The hybridisation step replaces nucleotide sequencing or nested PCR for confirmation of the identity of DNA product. The assay is fast and easy to carry out and can give semi-quantitative estimates of the virus content of samples.