Orthotopic Wilms tumor xenografts derived from cell lines reflect limited aspects of tumor morphology and clinical characteristics.

BACKGROUND: Wilms tumor (WT) is a pediatric tumor of the kidney, the treatment of which includes heavy chemotherapy. Affected children would likely benefit from more targeted therapies with limited side effects. Establishment of relevant orthotopic WT xenografts is important to better understand mechanisms of WT growth and for preclinical drug testing. PROCEDURE: Here we established and characterized orthotopic xenografts from WT cell lines WiT49, CCG-99-11, and WT-CLS1 to ascertain in what aspects each of them recapitulated WT histology, immunophenotype, invasion, and metastatic spread. RESULTS: WiT49 xenografts recapitulated near triphasic WTs with clear WT1 staining and anaplastic features, but with tumor restricted to the kidney. On the contrary both CCG-99-11 and WT-CLS1 xenografts conveyed metastatic disease. CCG-99-11 showed a blastemal phenotype whereas WT-CLS1 xenografts did not properly reflect any specific WT subtype. CONCLUSIONS: From the three tested cell lines, orthotopic WiT49 xenografts best reflect the triphasic pattern of classical WT.

Evaluation of CITED1, SIX1, and CD56 protein expression for identification of blastemal elements in Wilms tumor.

OBJECTIVES: Successful further treatment of Wilms tumors (WTs) after preoperative chemotherapy and surgery depends on correct histopathologic risk stratification, including quantification of remaining blastemal elements. In the present study, we assessed the usefulness of protein markers for the detection of WT blastema. METHODS: Expression of the candidate blastemal protein markers CITED1, SIX1, and CD56 was evaluated by immunofluorescence regarding sensitivity and specificity for staining blastema in a tissue microarray containing cores from 30 WTs, a small number of rarer pediatric renal neoplasms, and normal postnatal kidney. RESULTS: CITED1, SIX1, and CD56 were expressed in blastema in 100%, 89%, and 74%, respectively, of the WTs with this component present. However, they were also expressed in 64%, 25%, and 79%, respectively, of epithelial WT elements and 48%, 52%, and 62%, respectively, of stromal WT elements. CONCLUSIONS: SIX1 showed the highest specificity, CITED1 the highest sensitivity, and CD56 low specificity and sensitivity for detection of postchemotherapy WT blastema. Cytokeratin staining proved to be a useful way to determine rudimentary tubular elements not readily recognized by routine staining.

Clear cell sarcoma of the kidney demonstrates an embryonic signature indicative of a primitive nephrogenic origin.

Clear cell sarcoma of the kidney (CCSK) is a tumor affecting children with a median age of 3 years at diagnosis. The cell of origin of CCSK is unknown and data on the molecular changes giving rise to CCSK is scarce. This has hindered the identification of positive diagnostic markers and development of molecularly targeted treatment protocols for CCSK. We have characterized a panel of CCSK to gain information regarding its molecular profile and possible origin. High-resolution genomic analysis with single nucleotide polymorphism array of 37 tumors did not reveal any clues to the mechanisms behind tumor development as remarkably few genetic imbalances were found. Gene expression analysis revealed a highly characteristic gene signature, enriched for pathways involved in embryonic development, including kidney formation. The presence of markers for two different developmental lineages in the embryonic kidney was therefore investigated in the tumor cells. FOXD1 which identifies cells giving rise to stromal elements, and CITED1, a marker for cells primed for nephrogenic epithelial differentiation, were both highly expressed in CCSK. In addition, the early embryonic marker OSR1 was expressed at higher levels in CCSK than in Wilms tumor, normal fetal kidney or adult kidney. As this marker discriminates the intermediate mesoderm from other mesodermal structures, our study could suggest that CCSK arises from a mesodermal cell type that retains the capacity to initiate differentiation towards both nephrons and stroma, but remains locked in a primitive state.

Biofilm-forming Pseudomonas aeruginosa bacteria undergo lipopolysaccharide structural modifications and induce enhanced inflammatory cytokine response in human monocytes.

To determine whether growth of bacteria in biofilms triggers a specific immune response, we compared cytokine induction in human monocytes and mouse macrophages by planktonic and biofilm bacteria. We compared Pseudomonas aeruginosa and Staphylococcus aureus, two bacteria often colonizing the airways of cystic fibrosis patients. Planktonic and biofilm S. aureus induced equivalent amounts of cytokine in human monocytes. In contrast, biofilm-forming P. aeruginosa induced a higher production of tumor necrosis factor and interleukin-6 than their planktonic counterpart, both for clinical isolates and laboratory strains. This increased cytokine production was partly dependent on phagocytosis. In contrast, no difference in cytokine induction was observed with mouse macrophages. We investigated the structures of the lipopolysaccharides (LPSs) of these Gram-negative bacteria in biofilm and planktonic cultures of P. aeruginosa. Switch between the two life-styles was shown to cause several reversible LPS structure modifications affecting the lipid A and polysaccharide moieties of both clinical isolates and laboratory strains. In addition, LPS isolated from biofilm-grown bacteria induced slightly more inflammatory cytokines than that extracted from its planktonic counterpart. Our results, therefore, show that P. aeruginosa biofilm LPS undergoes structural modifications that only partially contribute to an increased inflammatory response from human monocytes.

Human antimicrobial peptide LL-37 is present in atherosclerotic plaques and induces death of vascular smooth muscle cells: a laboratory study.

BACKGROUND: Death of smooth muscle cells in the atherosclerotic plaques makes the plaques more prone to rupture, which can initiate an acute ischemic event. The development of atherosclerosis includes the migration of immune cells e.g. monocytes/macrophages and T lymphocytes into the lesions. Immune cells can release antimicrobial peptides. One of these, human cathelicidin antimicrobial peptide hCAP-18, is cleaved by proteinase 3 generating a 4.5 kDa C-terminal fragment named LL-37, which has been shown to be cytotoxic. The aim of the study was to explore a potential role of LL-37 in the pathophysiology of atherosclerosis. METHODS: We investigated the presence of LL-37 in human atherosclerotic lesions obtained at autopsy using immunohistochemistry. The direct effects of LL-37 on cultured vascular smooth muscle cells and isolated neutrophil granulocytes were investigated with morphological, biochemical and flow cytometry analysis. RESULTS: The neointima of atherosclerotic plaques was found to contain LL-37-like immunoreactivity, mainly in macrophages. In cultured smooth muscle cells, LL-37 at 30 mug/ml caused cell shrinkage, membrane blebbing, nuclear condensation, DNA fragmentation and an increase in caspase-3 activity as studied by microscopy, ELISA and enzyme activity assay, respectively. Flow cytometry demonstrated that LL-37 in a subset of the cells caused a small but rapidly developing increase in membrane permeability to propidium iodide, followed by a gradual development of FITC-annexin V binding. Another cell population stained heavily with both propidium iodide and FITC-annexin V. Neutrophil granulocytes were resistant to these effects of LL-37. CONCLUSION: This study shows that LL-37 is present in atherosclerotic lesions and that it induces death of vascular smooth muscle cells. In a subset of cells, the changes indicate the development of apoptosis triggered by an initial mild perturbation of plasma membrane integrity. The findings suggest a role for LL-37 as a mediator of immune cell-induced death of vascular smooth muscle cells in atherosclerosis.

Antimicrobial and chemoattractant activity, lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37.

Antimicrobial peptides have been evaluated in vitro and in vivo as alternatives to conventional antibiotics. Apart from being antimicrobial, the native human cathelicidin-derived peptide LL-37 (amino acids [aa] 104 to 140 of the human cathelicidin antimicrobial peptide) also binds and neutralizes bacterial lipopolysaccharide (LPS) and might therefore have beneficial effects in the treatment of septic shock. However, clinical trials have been hampered by indications of toxic effects of LL-37 on mammalian cells and evidence that its antimicrobial effects are inhibited by serum. For the present study, LL-37 was compared to two less hydrophobic fragments obtained by N-terminal truncation, named 106 (aa 106 to 140) and 110 (aa 110 to 140), and to a previously described more hydrophobic variant, the 18-mer LLKKK, concerning antimicrobial properties, lipopolysaccharide neutralization, toxicity against human erythrocytes and cultured vascular smooth muscle cells, chemotactic activity, and inhibition by serum. LL-37, fragments 106 and 110, and the 18-mer LLKKK inhibited the growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans in a radial diffusion assay, inhibited lipopolysaccharide-induced vascular nitric oxide production, and attracted neutrophil granulocytes similarly. While fragments 106 and 110 caused less hemolysis and DNA fragmentation in cultured cells than did LL-37, the 18-mer LLKKK induced severe hemolysis. The antibacterial effect of fragments 106 and 110 was not affected by serum, while the effect of LL-37 was reduced. We concluded that the removal of N-terminal hydrophobic amino acids from LL-37 decreases its cytotoxicity as well as its inhibition by serum without negatively affecting its antimicrobial or LPS-neutralizing action. Such LL-37-derived peptides may thus be beneficial for the treatment of patients with sepsis.