A Novel Organotypic Model Mimics the Tumor Microenvironment

Carcinoma cell invasion is traditionally studied in three-dimensional organotypic models composed of type I collagen and fibroblasts. However, carcinoma cell behavior is affected by the various cell types and the extracellular matrix (ECM) in the tumor microenvironment. In this study, a novel organotypic model based on human uterine leiomyoma tissue was established and characterized to create a more authentic environment for carcinoma cells. Human tongue squamous cell carcinoma cells (HSC-3) were cultured on top of either collagen or myoma. Organotypic sections were examined by immunohistochemistry and in situ hybridization. The maximal invasion depth of HSC-3 cells was markedly increased in myomas compared with collagen. In myomas, various cell types and ECM components were present, and the HSC-3 cells only expressed ECM molecules in the myoma model. Organotypic media were analyzed by radioimmunoassay, zymography, or Western blotting. During carcinoma cell invasion, matrix metalloprotease-9 production and collagen degradation were enhanced particularly in the myoma model. To evaluate the general applicability of the myoma model, several oral carcinoma, breast carcinoma, and melanoma cell lines were cultured on myomas and found to invade in highly distinct patterns. We conclude that myoma tissue mimics the native tumor microenvironment better than previous organotypic models and possibly enhances epithelial-to-mesenchymal transition. Thus, the myoma model provides a promising tool for analyzing the behavior of carcinoma cells.Tumor growth and invasion are not just determined by the malignant tumor cells, but instead various cell types and the extracellular matrix (ECM) of the tumor microenvironment affect the outcome.¹ Particularly, fibroblasts have many prominent roles in the cancer progression. In fact, in many carcinomas, the majority of the stromal cells are fibroblasts that possess myofibroblastic characteristics and are called cancer-associated fibroblasts. They produce ECM molecules, proteases, growth factors, and chemokines that crucially affect the carcinoma cell behavior.^2,3 In this context, the organotypic three-dimensional skin model developed by Fusenig et al⁴ replicates the in vivo situation more closely in vitro than the two-dimensional cell culture experiments. The model allows studying of carcinoma cell invasion in three-dimensional collagen gel embedded with fibroblasts. The degree of invasion can also be quantitatively analyzed.^5,6 However, this kind of organotypic model remains somewhat artificial due to the lack of other cell types besides fibroblasts and ECM components that are present in vivo. In addition to the carcinoma cells and fibroblasts, endothelial and inflammatory cells, as well as several ECM molecules, are known to contribute to the tumor growth. The induction of angiogenesis, recruitment of inflammatory cells, and increased turnover of ECM components result in tumor progression.^7,8 Therefore, we wished to determine whether real human tissue can be used in the organotypic method to provide a more natural stroma-like environment for studying carcinoma cell invasion. We used uterine leiomyoma tissue, which mainly consists of smooth muscle actin (SMA)-positive cells and collagens.⁹ The existence of various additional cell types and proteins in the myoma tissue was characterized, and the invasiveness of malignant human tongue squamous cell carcinoma cells (HSC-3) into this novel myoma organotypic culture was measured by different methods and compared with the traditional collagen organotypic model. To test the general applicability of the myoma model, the invasion patterns of various cell lines were examined in myoma and collagen organotypic cultures.     

Matrilysin-1 (MMP-7) and MMP-19 are expressed by Paget's cells in extramammary Paget's disease

BACKGROUND: Extramammary Paget's disease (EMPD) is a rare malignant neoplasm of apocrine gland bearing skin characterized by intraepidermal proliferation of adenocarcinoma cells. Tumor growth depends on the ability of tumor cells to migrate by proteolysis and on angiogenesis. The matrix metalloproteinase (MMP) enzymes have been implicated in both of these processes in other types of skin cancer. METHODS: The expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, and MMP-19 was analyzed by immuno- histochemistry and/or in situ hybridization in 27 EMPD and five mammary PD (MMPD) specimens. The distribution of laminin-5 (LN-5) and tenascin-C, two extracellular matrix proteins associated with tumor invasion, was studied by immunohistochemistry. RESULTS: MMP-7 (matrilysin-1) and MMP-19 were the most frequently expressed MMPs in Paget's cells. Overexpression of MMP-2, MMP-9, or MMP-13, which is seen in many cancers, was not evident in EMPD. LN-5 and tenascin-C positivity did not correlate with the level of invasion. MMP-7, MMP-13, and MMP-19 were detected abundantly in MMPD, while MMP-9 was absent. CONCLUSIONS: MMP expression did not generally associate with the level of invasion of EMPD. In three samples positive for MMP-7 and four samples positive for MMP-19, an underlying carcinoma was detected, suggesting the importance of these two MMPs as predictors of secondary EMPD or the putative origin of Paget's cells from the dermal adenocarcinoma cells of apocrine duct origin.     

A disaster preparedness and response project in Afghanistan: participants' perceptions

Title. A disaster preparedness and response project in Afghanistan: participants' perceptions. Aim. This paper is a report of a study to describe Emergency Mobile Unit team members’ and healthcare professionals’ perceptions of a disaster preparedness and response project and to explore the elements of participation that could support its sustainability. Background. Many developing countries have limited preparedness for disaster response. There is a need to better understand the role of national and local participation, and their interplay. We also need to consider the moderation and eventual planned withdrawal of international humanitarian organizations’ support. Afghanistan is one example of a country in postdisaster development where the opportunity arose for an in‐depth study of disaster preparedness and response. Method. Data were collected in Finland and Afghanistan in 2004 using an ethnographic approach, with seven thematic interviews (n = 8) and two focus groups (n = 7). The participants were Afghan healthcare professionals and expatriates who had facilitated Emergency Mobile Unit training. Findings. Constraints in the project arose from uncoordinated implementation and poor job satisfaction, in addition to intrinsic characteristics of the situation. A second theme to emerge was that participation was a positive response to health emergencies. Thirdly, a need for further development and overall support for Emergency Mobile Units was clearly evident. Conclusion. Improved coordination and measures to increase job satisfaction for national aid workers are needed, and a more positive knowledge‐based response system and continued overall support for emergency mobile teams. Involving disaster‐affected people and the local community, especially women, in health development projects will help to ensure both success and sustainability.     

CNS-active drugs in aging population at high risk of cerebrovascular events: evidence from preclinical and clinical studies

The recovery process following cerebral insults such as stroke is affected by aging and pharmacotherapy. The use of medication including CNS-active drugs has increased in the elderly during recent years. However, surprisingly little is known about how safe they are with respect to severity of sensorimotor and cognitive impairments or recovery of function following possible cerebrovascular accidents. This review examines the experimental and clinical literature, primarily from 1995 onwards, concerning medication in relation to cerebrovascular events and functional recovery. Special attention is directed to polypharmacy and to new CNS-active drugs, which the elderly are already taking or are prescribed to treat emerging, stroke-induced psychiatric symptoms. The neurobiological mechanisms affected by these drugs are discussed.     

P75 nerve growth factor receptor is expressed in regenerating human nerve grafts

BACKGROUND: The purpose of this study was to elucidate the expression of p75 nerve growth factor receptor (p75NGFR) in human cross-facial nerve grafts and to compare the immunohistological findings with patient data and the functional outcome in facial reanimation. MATERIALS AND METHODS: The study comprised 37 sural nerve graft specimens. All of the patients had long-lasting complete facial paralysis and were operated on by the standard two-stage procedure involving cross-facial nerve grafts and microneurovascular muscle transfer. Nerve biopsies were taken 4 to 20 months (mean, 8 months) after the cross-facial nerve grafting. Immunohistochemistry for p75NGFR as well as for Schwann cells (S-100; Dako, Glostrup, Denmark) and for Neurofilament-200 (NF-200; Boehringer, Mannheim, Germany) was performed. RESULTS: In graft biopsies, the mean number of NF-200-positive axons amounted to 38% (range, 6-81%) of that in control samples. Further, regenerated axons were thinner than in control samples. Morphologically, the grafted nerves were characterized by fibrosis and invasion of inflammatory cells. A longer time between cross-facial nerve grafting and biopsy sampling correlated with a higher number of viable axons (NF-200) (P = 0.002). In all cases, expression of p75NGF receptor was clearly higher at the distal end of the grafted nerve. Expression of p75NGFR was lower in older than in younger patients (P = 0.003). A high expression of p75NGFR was often seen with better function of the transplanted muscle. CONCLUSION: Increased expression of p75NGFR in human nerve grafts was noted, especially in younger patients. We suggest that p75NGFR expression might be a contributing factor in a successful axonal regeneration and eventual recovery of muscle function.     

Lymphocyte homing into the gut

Conclusions Lymphocyte-HEV cell interaction that is a prerequisite for lymphocyte homing is mediated by receptors on the lymphocyte surface and their ligand molecules, addressins, on the endothelial cells. In humans, the Hermes-defined 90-kDa glycoprotein class (CD44) includes the homing receptor for mucosal HEV. In addition, members of the integrin family, VLA-4 and LFA-1 are involved in lymphocyte binding to HEV in mucosa-associated lymphatic tissues. Furthermore, a third member of the integrin family, LPAM-1, functions as a mucosal-homing receptor in the mouse. Since there is remarkable conservation in the mechanisms mediating lymphocyte-HEV interaction, it is likely that the human equivalent for LPAM-1 will be found in near future.The only addressin molecule on mucosal HEV described so far is the MECA-367 in mouse. However, isolated Hermes antigen from human lymphocytes binds to MECA-367 antigen suggesting that the human mucosal addressin is, at the least, functionally closely identical with MECA-367. All the molecules described above, together with molecules still to be identified, co-operate and establish stable contacts between lymphocytes and mucosal HEV. The molecules and mechanisms operating between lymphocytes and mucosal HEV that differ from those in peripheral lymph node and synovial HEV, direct organ-specific lymphocyte traffic to mucosa-associated lymphatic tissues and are responsible for the physiological and pathological characteristics of immune responses in the mucosal lymphoid system.     

Comparison of CYP1A1 induction and genotoxicity in vitro as indicators of potentially harmful effects of environmental samples

Cytochrome P450IA1 (CYP1A1) induction of Hepa-1 mouse and H4IIE rat hepatoma cell lines was compared using selected environmental samples. The results were in agreement for both cell lines: no induction was observed for the fly ash extract from peat combustion, an intermediate induction was found for the fly ash extract from biosludge combustion, and a strong induction was detected for natural peat extract. However, Hepa-1 responded to the samples more sensitively than did H4IIE: the half maximal induction (ED₅₀) values for Hepa-1 were smaller than those for H4IIE. In a bacterial DNA repair assay without metabolic activation and in a mammalian sister chromatid exchange test in the presence of metabolic activation the samples were virtually non-genotoxic. Thus the CYP1A1-inducing potency and genotoxicity of the samples were not correlated. In light of these results, the CYP1A1 induction test might be a useful addition to conventional genotoxicity tests, which may fail to detect potentially harmful compounds/mixtures.     

The gene for human lymphocyte homing receptor is located on chromosome 11

The mouse monoclonal antibody Hermes-3 recognizes the human lymphocyte homing receptor. A panel of mouse-human T lymphocyte hybrids, carrying all mouse chromosomes and a limited number of human chromosomes, was analyzed for expression of human homing receptor by indirect immunofluorescence and immunoprecipitation of radiolabeled cell lysates with Hermes-3 antibody. Karyotypic analysis of the tested clones showed that the expression of human homing receptor correlated to the presence of human chromosome 11 in all but one clone. However, concanavalin A induced a weak to moderate expression of the homing receptor in this clone, but not in a chromosome 11- clone. Another clone, heterogeneous for the expression of homing receptor, was separated into a Hermes-3+ and a Hermes-3- fraction with a fluorescence-activated cell sorter. Karyotypic analysis performed after sorting showed human chromosome 11 to segregate with the Hermes-3 antigen. To confirm these data we correlated the expression of two chromosome 11-coded antigens, Trop-4 and Leu-7, with the expression of the homing receptor. In our hybrid clones these three antigens were expressed concordantly. The gene coding for the human lymphocyte homing receptor recognized by Hermes-3 is thus assigned to chromosome 11.