The cytokines NAP-1 (IL-8), MCP-1, IL-1 beta,and GRO in rabbit inflammatory skin lesions produced by the chemical irritant sulfur mustard

Developing and healing dermal inflammatory lesions were produced in rabbits by the topical application of dilute sulfur mustard (SM),⁹ the military vesicant. In tissue sections of such lesions, cells containing the mRNA of important cytokines were identified with in situ hydridization techniques. These cytokines were neutrophil attractant/activation protein-1 (NAP-1 (also called IL-8)), monocyte chemoattractant (activating) protein 1 (MCP-1), interleukin 1 (beta) (IL-1 (beta)), and GRO (a growth factor and chemokine). Mononuclear cells (mainly macrophages and activated fibroblasts) contained the mRNA of all four of these cytokines. A higher percentage of cytokine-producing mononuclear cells (macrophages and activated fibroblasts) was present in lesions at 2 days (their peak size) than at 6 days, when they were almost healed. Granulocytes emigrated from the bloodstream, passed through the lesions, and were the major constituent of the protective crust. This sequence correlated with the distribution of cells able to produce NAP-1: At 2 days and 6 days, the mononuclears that contained messenger RNA for this granulocyte chemoattractant were found mainly in the upper part of the dermis. At 2 days and 6 days, cells containing the mRNA of IL-1, a primary cytokine, were also found predominantly in the upper dermis, i.e., nearest the site of injury. In contrast, mononuclears containing the mRNA of MCP-1 (a monocyte chemoattractant), and the mRNA of GRO (a granulocyte chemoattractant) were more equally distributed throughout the dermis. SM stimulated hair follicle epithelial cells to up-regulate GRO mRNA and, to a lesser degree, NAP-1 mRNA. Apparently, the irritation produced by SM directly or indirectly induces such epithelial cells to manufacture these growth factors. In the rabbit, hair follicles are known to be the main source of new epithelial cells after the covering epithelium has been destroyed. Therefore, GRO is probably a major autocrine-paracrine stimulus for such repair. A brief review of the role of cytokines in dermal inflammation is presented.Abbreviations SM Sulfur mustard: bis(2-chloroethyl)sulfide - GM-CSF Granulocyte-Macrophage Colony Stimulating Factor - GRO A member of the CXC subfamily of chemokines that promotes the multiplication of cells, formerly called melanoma growth stimulating activity (MGSA) - IFN (gamma)-Interferon-gamma - IL-1 Interleukin 1 - IL-8 Interleukin 8 (same as NAP-1)-a CXC chemokine - MCP-1 Monocy te Chemoattractant (Activating) Protein-1-a C-C chemokine - NAP-1 Neutrophil Attractant/Activating Protein-1 (same as IL-8)-a C-X-C chemokine - TGF (beta) Transforming Growth Factor (beta) - TNF (alpha) Tumor Necrosis Factor (alpha) - EDTA Ethylenediamine tetraacetate - DEPC Diethylpyrocarbonate - PBS Phosphate-buffered saline solution - PGE₂ Prostaglandin E₂ - PGI₂ Prostaglandin I₂ (prostacyclin) - SSC Sodium chloride-sodium citrate solution On leave of absence from the Institute for Medical Immunology, Kumamoto University School of Medicine, Kumamoto, Japan.On leave of absence from the Department of Internal Medicine, Oita Medical University, Oita, Japan.     

Generation of B lymphocytes from a single hemopoietic progenitor cell in vitro

The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed in 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells.     

Chemoprevention of heterocyclic amine-induced mammary carcinogenesis in rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most prevalent carcinogenic heterocyclic amines in the environment, targeting the colon, prostate, pancreas, and mammary gland in rodents. Chemopreventive effects of synthetic and naturally occurring compounds on PhIP-induced rat mammary carcinogenesis were investigated in a series of experiments. In a PhIP feeding model, groups of 20-21 female F344 rats each, were treated with 0.02% PhIP alone or PhIP plus 0.5% 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), 1% green tea catechins, 1% alpha-tocopherol, 0.1% ellagic acid, or 1% chlorophyllin, each in the diet, or 0.1% caffeine in drinking water for 52 weeks. To assess the mechanism of HTHQ and caffeine inhibition of PhIP-induced carcinogenesis, effects of these compound on the in vitro metabolic activation of PhIP were examined in the presence of S9 mix. In the next series of experiments, the PhIP intragastric dose model was applied to allow separate investigation of the effects of chemicals during the initiation and postinitiation periods. In these experiments, female Sprague-Dawley rats were given eight intragastric doses of 100 mg/kg body weight during the first 4-8 weeks for initiation. Either during initiation or after initiation, or only after initiation, animals were treated with either corn or perilla oil at doses of 5 and 20%, conjugated fatty acid derived from safflower oil (CFA-S) or perilla oil (CFA-P) at a dose of 1%, arctiin at doses of 0.02 and 0.2% in the diet, or sodium nitrite (NaNO(2)) at a dose of 0.2% in drinking water. In the PhIP feeding model, administration of PhIP alone for 52 weeks induced adenocarcinomas in 40% of rats, but the incidence was remarkably reduced to 5% by the simultaneous treatment with 0.5% HTHQ, a strong lipophilic phenolic antioxidant, or to 10% by 0.1% caffeine. Administration of 1% chlorophyllin exerted similar, albeit weaker, effects. alpha-Tocopherol at a dose of 0.5% only reduced the multiplicity of carcinomas, and 1% green tea catechins only the mean size of mammary tumors. In a metabolic activation study of PhIP, HTHQ and caffeine clearly inhibited the formation of metabolites. In the PhIP gastric dose model, among the naturally occurring compounds examined, a plant lignan arctiin, perilla oil, which contains a large amount of n-6 alpha-linolenic acid, and CFA-S or CFA-P inhibited mammary tumor development, particularly in the postinitiation period, although a clear dose response was not observed. Treatment with 0.2% NaNO(2) in the initiation period was found to lower the volume of mammary tumors. The present results indicate that a number of compounds may be candidate chemopreventive agents against PhIP-induced mammary carcinogenesis, acting through different mechanisms and depending on the stage of carcinogenesis.     

Primary osteosarcoma of the uterine corpus: case report and review of the literature

A rare case of rapidly growing osteosarcoma that developed in the uterine corpus of a 62-year-old woman is presented. The tumor occupied almost the entire pelvic cavity and extended into the abdominal cavity, with marked involvement of the intestines. Histopathologically, the tumor was composed of an osteoblastic component, accompanied by conspicuous bone formation, and a fibroblastic component. The tumor cells were positive for vimentin and osteocalcin, as well as desmin, alpha-smooth muscle actin and muscle-specific actin, but negative for h-caldesmon. The results indicated myofibroblastic differentiation in a part of the tumor. A review of 14 reported cases and our case of uterine osteosarcoma revealed that this tumor has a biologically aggressive nature, although its histopathological and immunohistochemical features are similar to those of osteosarcomas in soft tissue and bone. As the prognosis of patients with this tumor is poor, it is of importance to differentiate this tumor from other types of tumors arising from the uterine corpus.     

Maximum likelihood fitting of FROC curves under an initial-detection-and-candidate-analysis model

We have developed a model for FROC curve fitting that relates the observer's FROC performance not to the ROC performance that would be obtained if the observer's responses were scored on a per image basis, but rather to a hypothesized ROC performance that the observer would obtain in the task of classifying a set of "candidate detections" as positive or negative. We adopt the assumptions of the Bunch FROC model, namely that the observer's detections are all mutually independent, as well as assumptions qualitatively similar to, but different in nature from, those made by Chakraborty in his AFROC scoring methodology. Under the assumptions of our model, we show that the observer's FROC performance is a linearly scaled version of the candidate analysis ROC curve, where the scaling factors are just given by the FROC operating point coordinates for detecting initial candidates. Further, we show that the likelihood function of the model parameters given observational data takes on a simple form, and we develop a maximum likelihood method for fitting a FROC curve to this data. FROC and AFROC curves are produced for computer vision observer datasets and compared with the results of the AFROC scoring method. Although developed primarily with computer vision schemes in mind, we hope that the methodology presented here will prove worthy of further study in other applications as well.     

Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51

BACKGROUND: Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S-phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear. RESULTS: Using immunocytochemistry, we found that WRNp forms distinct nuclear foci in response to DNA damaging agents, including camptothecin (CPT), etoposide, 4-nitroquinolin-N-oxide and bleomycin. The presence of aphidicolin inhibited CPT-induced WRNp foci strongly but not bleomycin-induced foci. These WRNp foci overlapped with the foci of replication protein A (RPA) almost entirely and with the foci of Rad51 partially, implicating cooperative functions of these proteins in response to DNA damage. We also found that WRNp foci partially co-localize with sites of 5-bromo-2'-deoxy-uridine incorporation. CONCLUSIONS: These findings suggest that WRNp form nuclear foci in response to aberrant DNA structures, including DNA double-strand breaks and stalled replication forks. We propose that WRNp takes part in the homologous recombinational repair and in the processing of stalled replication forks.     

Radial gradient-based segmentation of mammographic microcalcifications: observer evaluation and effect on CAD performance

Precise segmentation of microcalcifications is essential in the development of accurate mammographic computer-aided diagnosis (CAD) schemes. We have designed a radial gradient-based segmentation method for microcalcifications, and compared it to both the region-growing segmentation method currently used in our CAD scheme and to the watershed segmentation method. Two observer studies were conducted to subjectively evaluate the proposed segmentation method. The first study (A) required observers to rate the segmentation accuracy on a 100-point scale. The second observer evaluation (B) was a preference study in which observers selected their preferred method from three displayed segmentation methods. In study A, the observers gave an average accuracy rating of 88 for the radial gradient-based and 50 for the region-growing segmentation method. In study B, the two observers selected the proposed method 56% and 62% of the time. We also investigated the effect of the proposed segmentation method on the performance of computerized classification scheme in differentiating malignant from benign clustered microcalcifications. The performances of the classification scheme using a linear discriminant analysis (LDA) or a Bayesian artificial neural network classifier both showed statistically significant improvements when using the proposed segmentation method. The areas under the receiver-operating characteristic curves for case-based performance when using the LDA classifier were 0.86 with the proposed segmentation method, 0.80 with the region-growing method, and 0.83 with the watershed method.     

Tubular aggregates in the skeletal muscle of the senescence-accelerated mouse; SAM

We investigated the skeletal muscles of nine strains of senescence accelerated mouse (SAM), DDD, AKR/J, C57BL/6J, A/J and BALB/c mice. We found that male SAMP8, SAMP7, C57BL/6J, A/J and BALB/c mice expressed tubular aggregates (TAs) in their skeletal muscle. Among these strains, the SAMP8 strain, which exhibits a short life span and various age-associated neurodegenerative disorders plus mitochondrial dysfunction, showed TAs more markedly than the others. Thus, we compared SAMP8 mice against SAMR1 mice, an accelerated senescence-resistant strain. Light- and electron micrographs showed that male SAMP8 mice exhibited an age-dependent aggravation of TA accumulation. There were no significant differences in the serum lactate/pyruvate levels between the SAMP8 and SAMR1 mice. However, the serum creatine kinase (CK) levels of the 3 and 6-month-old SAMP8 mice were higher than that of the corresponding SAMR1 mice. Considering the serum CK levels and the mitochondrial dysfunction of SAMP8 mice, we conclude that the TAs may be involved in the homeostasis of energy metabolism that is not appropriately regulated in the SAMP8 mouse mitochondrion.