Human melanoma progression in skin reconstructs : biological significance of bFGF

Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression.     

Use of an immunoperoxidase method for identification of Bacteroides fragilis.

An indirect immunoperoxidase (IP) slide test was evaluated for the laboratory identification of Bacteroides fragilis. Antigen-antibody complexes were detected with goat anti-rabbit immunoglobulin G-peroxidase conjugate with 3-amino-9-ethyl-carbazole as the peroxidase substrate. Ninety-one percent of 44 B. fragilis strains tested were IP positive (3+ to 4+ reactions) with greater than or equal to 1:160 dilutions of rabbit antiserum produced against whole cells of B. fragilis ATCC 23745. The antiserum was species specific. No cross-reactions were observed with 35 Bacteroides strains of other species or with a variety of facultative or aerobic gram-negative bacilli. Four B. fragilis strains were IP negative. One of these (VPI 2393) was the deoxyribonucleic acid (DNA) homology group II reference strain. The other three were clinical isolates. IP-negative and representative IP-positive strains were tested for DNA homology with the type strains for DNA homology groups I and II (VPI 2553 and VPI 2393). Two of the three clinical isolates were classified as DNA homology group II, and the remaining strain was classified as a group I. Capsular material known to be unique to B. fragilis was common to both DNA homology groups as indicated by reactions with purified anticapsular antiserum. The IP technique provides a suitable alternative to fluorescent microscopy for the rapid immunological identification of B. fragilis.     

Bacteria induce osteoclastogenesis via an osteoblast-independent pathway

Bacteria or their products may cause chronic inflammation and subsequent bone loss. This inflammation and bone loss may be associated with significant morbidity in chronic otitis media, periodontitis, endodontic lesions, and loosening of orthopedic implants caused by lipopolysaccharide (LPS)-contaminated implant particles. Currently, it is not clear how bacteria or endotoxin-induced bone resorption occurs and what cell types are involved. Here we report that Porphyromonas gingivalis, a periodontal pathogen, and Escherichia coli LPS induce osteoclastic cell formation from murine leukocytes in the absence of osteoblasts. In contrast, stimulation with parathyroid hormone had no effect. These multinucleated, tartrate-resistant acid phosphatase-positive cells were positive for receptor activator of NF-kappaB (RANK), the receptor for osteoprotegerin ligand (OPGL), also known as RANK ligand (RANKL). Blocking antibodies demonstrated that their formation was dependent upon expression of OPGL and, to a lesser extent, on tumor necrosis factor alpha. Mononuclear cells represented a significant source of OPGL production. In vivo, P. gingivalis injection stimulated OPGL expression in both mononuclear leukocytes and osteoblastic cells. Thus, these findings describe a pathway by which bacteria could enhance osteolysis independently of osteoblasts and suggest that the mix of cells that participate in inflammatory and physiologic bone resorption may be different. This may give insight into new targets of therapeutic intervention.     

Size fractions of ambient particulate matter induce granulocyte macrophage colony-stimulating factor in human bronchial epithelial cells by mitogen-activated protein kinase pathways

Environmental pollutants, including ambient particulate matter (PM), increase respiratory morbidity. Studies of model PM particles, including residual oil fly ash and freshly generated diesel exhaust particles, have demonstrated that PM affects inflammatory airway responses. Neither of these particles completely represents ambient PM, and therefore questions remain about ambient particulates. We hypothesized that ambient PM of different size fractions collected from an urban environment (New York City air), would activate primary culture human bronchial epithelial cells (HBECs). Because of the importance of granulocyte-macrophage colony-stimulating factor (GM-CSF) on inflammatory and immunomodulatory processes, we focused our studies on this cytokine. We demonstrated that the smallest size fraction (ultrafine/fine; < 0.18 micro m) of ambient PM (11 micro g/cm(2)), upregulated GM-CSF production (2-fold increase). The absence of effect of carbon particles of similar size, and the day-to-day variation in response, suggested that the chemical composition, but not the particle itself, was necessary for GM-CSF induction. Activation of the extracellular signal-regulated kinase and the p38 mitogen-activated protein kinase was associated with, and necessary for, GM-CSF release. These studies serve to corroborate and extend those on model particles. Moreover, they emphasize the role of the smallest size ambient particles in airway epithelial cell responses.     

Growth and nutritional status of the Evenki reindeer herders of Siberia

This study examines physical growth and nutritional status in a sample of 478 (247 males; 231 females) Evenki herders of Central Siberia. The Evenki display slow growth in stature and body weight, particularly during late childhood and adolescence. Adult males fall below the U.S. 5th percentiles for both stature and body weight. Adult females are below the 5th percentile for stature and approximate the 15th percentile for weight. Despite their diminutive size, the Evenki appear to have adequate energy reserves, as indicated by their skinfold measurements, which range between the U.S. 15th and 50th percentiles. Among adults, women are relatively heavier and fatter than men and tend to increase in weight and fatness with age. Poor growth in the Evenki does not appear to be directly attributable to limited food availability. Rather, it is hypothesized that elevated metabolic requirements, associated with adaptation to a high latitude ecosystem, are responsible for limiting the amount of energy that is allocated to growth. Relatively high levels of adiposity in adult females appear to be the product of changes in activity patterns and fertility levels that resulted after the collectivization of the Evenki. © 1994 Wiley-Liss, Inc.     

Expression of interleukin-1 in Reed-Sternberg cells and neoplastic cells from true histiocytic malignancies.

Interleukin-1, a peptide produced by monocytes, histiocytes, and interdigitating reticulum cells, plays an important role in the regulation of immune function. In this styde, we examined the production of interleukin-1 in 115 patients with a variety of human lymphomas by using a rabbit anti-interleukin-1 antibody and the immunoperoxidase technique. Interleukin-1 was detected in Reed-Sternberg cells from 20 patients with Hodgkin's disease as well as in neoplastic cells from 9 patients with true histiocytic lymphoma or malignant histiocytosis. In the other 86 cases, which included T- and B-cell lymphomas, no interleukin-1 could be detected. This result indicates a close relationship between Hodgkin's disease and true histiocytic malignancies and provides additional evidence to support our hypothesis that Reed-Sternberg cells are related to interdigitating reticulum cells.     

Point mutations in the P30 domain of the gag gene of Moloney murine leukemia virus

A series of point mutations in the P30 domain of the Moloney murine leukemia virus gag gene was generated by bisulfite treatment of heteroduplex DNAs containing a single-stranded region in the gag gene. One virus bearing such a mutation exhibited a coordinate defect in gag and pol function, and was similar to previously described deletion mutants with alterations in this gene. One mutant virus displayed a different phenotype: it could assemble virion particles and provide pol function, but the particles were defective in the early stages of infection. The continued concordance of the mutants' failure or ability to both assemble virions and provide pol lends further support to the proposal that similar parts of the gag gene are required for these two processes.     

Etiological diagnosis of influenza A virus by enzymatic radioimmunoassay.

An enzymatic radioimmunoassay for influenza A virus was developed by using polystyrene beads coated with rabbit immunoglobulin G to capture viral hemagglutinins (H1 and H3). Captured hemagglutinin was detected with goat immunoglobulin G followed by affinity-purified rabbit anti-goat immunoglobulin G labeled with alkaline phosphatase. [3H]AMP was added to quantify alkaline phosphatase activity, and free [3H]adenosine was measured with a scintillation counter. The assay detected as little as 0.1 ng of purified hemagglutinin. It was specific for hemagglutinin subtype and, depending on the source of the goat immunoglobulin G used, detected either H1 or H3. There was no reaction with neuraminidase or core antigens of influenza strain WSN-33. The clinical efficacy of the assay was evaluated with sequential nasal washes from 33 patients with naturally acquired H1N1 influenza. In the first 3 days of infection, the assay was consistently less sensitive than the viral culture, although detectable antigen persisted in secretions longer than did the infectious virus. Testing of multiple samples greatly increased the number of individuals in whom an etiological diagnosis could be made by immunoassay (81% of patients were positive for viral antigens at some point in their illness), and such testing was necessary to achieve the sensitivity of a single culture. Mean antigen levels were highest in nasal washes with the highest titers of infectious virus.     

Matrix vesicle biogenesis in vitro by rachitic and normal rat chondrocytes.

Calcifying matrix vesicles (MVs) are released from chondrocytes and osteoblasts in monolayer culture. In the present studies, we tested the ability of rachitic versus normal rat growth plate chondrocytes in micromass or monolayer primary cultures to produce MVs. Unlike earlier reports of in vitro MV biogenesis by chicken chondrocytes in which most MVs were released into the medium, we found that most of the released rat matrix vesicles were entrapped in a newly formed cartilaginous matrix enveloping the cells. These matrix-associated MVs could be isolated by mild collagenase treatment and concentrated by differential centrifugation. Vesicle production slowed in the older 2- to 4-week-old cultures and, unlike vesicle release from cultured chicken chondrocytes, active vesicle production did not show a second burst of activity at 3 to 4 weeks. Alkaline phosphatase (ALP) activity diminished with time in culture in cells and matrix vesicles, suggesting a decrease in differentiative expression. Protein profiles on SDS polyacrylamide gels of native matrix vesicles and culture-derived MVs from rachitic and normal cells were quite similar and showed a typical simplified protein pattern as compared to chondrocyte plasma membrane proteins. There were distinctive proteins migrating at 130, 80 to 95, 66, 43, 20, and 14 kd. Culture-derived MVs showed vigorous in vitro calcifying activity that was ALP related. We conclude that 1) rachitic chondrocytes are essentially normal in their matrix vesicle production; 2) matrix entrapment of MVs is a characteristic of rat chondrocyte cultures; and 3) culture-produced MVs are similar to native MVs in protein profile and calcifiability, and thus can be studied as a model for normal MV composition and calcification.     

Phenotypes and phorbol ester-induced differentiation of human histiocytic lymphoma cell lines (U-937 and SU-DHL-1) and Reed-Sternberg cells.

Hodgkin's mononuclear cells, Reed-Sternberg (H-RS) cells, and U-937 and SU-DHL-1 histocytic cell lines were induced to differentiate by phorbol ester in cultures. The phenotypes of cells were determined by a panel of antibodies specific for monocytes, histiocytes, and interdigitating reticulum cells. Before induction, SU-DHL-1 cells and H-RS cells expressed similar markers, such as HeFi-1, 2H9, 1A2, and 1E9. In addition, SU-DHL-1 cells were also stained by Tac and Leu M5. Other monocyte markers, including OK M1, Co Mo2, BRL Mo1, BRL Mo2, and Leu M3 were consistently negative in both types of cells. After induction, SU-DHL-1 cells conserved the same phenotype, but H-RS cells became negative for HeFi-1, 1A2, and 2H9. The U-937 cells expressed Leu M1 and Co Mo2 and became positive for Leu M5, OK M1, Co Mo2, BRL Mo2, 2H9, and 1E9 after phorbol ester induction. The U-937 cells did not express HeFi-1 or 1A2. The marker expression of H-RS cells, SU-DHL-1 cells, and U-937 cells were compared with those of histiocytes or interdigitating reticulum cells in lymphoid tissues and with neoplastic cells in true histiocytic lymphoma and malignant histiocytosis. It is concluded that SU-DHL-1, U-937, and H-RS cells are derived from or most closely related to fixed histiocytes, free histiocytes, and interdigitating reticulum cells, respectively. Our study further confirms the diagnosis of SU-DHL-1 as true histiocytic lymphoma but reveals that U-937 is a case of malignant histiocytosis rather than the previously diagnosed histiocytic lymphoma. The phenotypes and induction properties of SU-DHL-1 cells are quite different from those of U-937 cells, which suggests that true histiocytic lymphoma and malignant histiocytosis are two distinct disease entities.