Quantitative morphometric examination of the human pubic symphyseal surface of Australian individuals with age using computed tomography

Current forensic practice in age estimation relies on the application of morphological standards as a means to characterize complex threedimensional skeletal surfaces. Research in our laboratory has demonstrated that the application of the morphologically based Suchey-Brooks method to a contemporary Queensland, Australian population demonstrated significant inaccuracy in age-estimation. Consequently, this study presents preliminary results to quantify age-related skeletal changes of the pubic symphysis in Queensland individuals using novel geometric and micro-architectural protocols that have the potential of improving age estimation in the forensic context. Computed tomography scans of the right and left pubis were obtained from Caucasian individuals aged 15–70 years (n=195) from the Queensland Health Forensic and Scientific Services. Morphometric variables including surface area, circumference, maximum height and width of the symphyseal surface, and micro-architectural assessment of cortical and trabecular bone structure were conducted in Rapidform XOS and Osteomeasure, respectively. Morphometric analysis demonstrated increases in maximum height and width of the surface with age independent of gender, with most significant (P<0.05) changes between the 25–34 and 55–64 year subsets. Sexual dimorphism and bilateral asymmetry were prominent features in the Queensland population. Micro-architectural analysis demonstrated degradation of cortical composition with age, with differential bone resorption between the medial, ventral and dorsal aspects of the symphysis. The ability to quantitatively model age-related changes to the pubic symphysis provides potential for future methodological refinement, where rigor and robust geometric assessment of the surface may remove the subjectivity associated with aging the pubic symphysis.

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

Background Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. Methods We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. Results In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. Conclusion We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.

Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

Background Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

Molecular and cellular analysis of basement membrane invasion by human breast cancer cells in Matrigel-based in vitro assays

In vitro analyses of basement membrane invasiveness employing Matrigel (a murine tumor extract rich in basement membrane components) have been performed on human breast cancer model systems. Constitutive invasiveness of different human breast cancer (HBC) cell lines has been examined as well as regulation by steroid hormones, growth factors, and oncogenes. Carcinoma cells exhibiting a mesenchymal-like phenotype (vimentin expression, lack of cell border associated uvomorulin) show dramatically increased motility, invasiveness, and metastatic potential in nude mice. These findings support the hypothesis that epithelial to mesenchymal transition (EMT)-like events may be instrumental in the metastatic progression of human breast cancer. The MCF-7 subline MCF-7ADR appears to have undergone such a transition. The importance of such a transition may be reflected in the emergence of vimentin expression as an indicator of poor prognosis in HBC. Matrix degradation and laminin recognition are highlighted as potential targets for antimetastatic therapy, and analyses of laminin attachment and the matrix metalloproteinase (MMP) family in HBC cell lines are summarized. Matrigel-based assays have proved useful in the study of the molecular mechanisms of basement membrane invasiveness, their regulation in HBC cells, and their potential as targets for antimetastatic therapy.

Collagen-induced activation of the Mr. 72,000 type IV collagenase in normal and malignant human fibroblastoid cells

Although the Mr. 72,000 type IV collagenase (matrix metalloproteinase 2) has been implicated in a variety of normal and pathogenic processes, its activation mechanism in vivo is unclear. We have found that fibroblasts from normal and neoplastic human breast, as well as the sarcomatous human Hs578T and HT1080 cell lines, activate endogenous matrix metalloprotease 2 when cultured on type I collagen gels, but not on plastic, fibronectin, collagen IV, gelatin, matrigel, or basement membrane-like HR9 cell matrix. This activation is monitored by the zymographic detection of Mr 59,000 and/or Mr 62,000 species, requires 2-3 days of culture on vitrogen to manifest, is cycloheximide inhibitable, and correlates with an arborized morphology. A similar activation pattern was seen in these cells in response to Concanavalin A but not transforming growth factor β or 12-O-tetradecanoylphorbol-13-acetate. The interstitial matrix may thus play an important role in regulating matrix degradation in vivo.

Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M(r) in equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M(r) zymographic activities at 50, 46 and 37 kDa M(r) are collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M(r) are increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (~2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

Phagocytosis of cross-linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity

During invasion and metastasis, cancer cells interact closely with the extracellular matrix molecules by attachment, degradation, and migration. We demonstrated previously the local degradation of fluorescently labeled gelatin matrix by cancer cells at invasive membrane protrusions, called invadopodia. Using the newly developed quantitative fluorescence-activated cell sorting-phagocytosis assay and image analysis of localized degradation of fluorescently labeled matrix, we document here that degradation and site- specific removal of cross-linked gelatin matrix is correlated with the extent of phagocytosis in human breast cancer cells. A higher phagocytic capacity is generally associated with increasing invasiveness, documented in other invasion and motility assays as well. Gelatin phagocytosis is time and cell density dependent, and it is mediated by the actin cytoskeleton. Most of the intracellular gelatin is routed to actively acidified vesicles, as demonstrated by the fluorescent colocalization of gelatin with acidic vesicles, indicating the intracellular degradation of the phagocytosed matrix in lysosomes. We show here that normal intracellular routing is blocked after treatment with acidification inhibitors. In addition, the need for partial proteolytic degradation of the matrix prior to phagocytosis is demonstrated by the inhibition of gelatin phagocytosis with different serine and metalloproteinase inhibitors and its stimulation by conditioned medium containing the matrix metalloproteinases MMP-2 and MMP-9. Our results demonstrate that phagocytosis of extracellular matrix is an inherent feature of breast tumor cells that correlates with and may even directly contribute to their invasive capacity. This assay is useful for screening and evaluating potential anti-invasive agents because it is fast, reproducible, and versatile.

Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP- 2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA- MB-435, MCF-7(ADR)) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.

The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by αVβ3 integrin in human breast cancer cells

The influence of αVβ3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing β3 integrin status. Overexpression of β3 integrin caused increased cell surface expression of αV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. β3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, αVβ3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of β3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with β3 integrin expression. Although our studies confirm important biological effects of αVβ3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, β3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by αVβ3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.

Climate variability, climate change and land degradation

Effective response by government and individuals to the risk of land degradation requires an understanding of regional climate variations and the impacts of climate and management on condition and productivity of land and vegetation resources. Analysis of past land degradation and climate variability provides some understanding of vulnerability to current and future climate changes and the information needs for more sustainable management. We describe experience in providing climate risk assessment information for managing for the risk of land degradation in north-eastern Australian arid and semi-arid regions used for extensive grazing. However, we note that information based on historical climate variability, which has been relied on in the past, will now also have to factor in the influence of human-induced climate change. Examples illustrate trends in climate for Australia over the past decade and the impacts on indicators of resource condition. The analysis highlights the benefits of insights into past trends and variability in rainfall and other climate variables based on extended historic databases. This understanding in turn supports more reliable regional climate projections and decision support information for governments and land managers to better manage the risk of land degradation now and in the future.

Differential substrate behaviours of ethylene oxide and propylene oxide towards human glutathione transferase theta hGSTT1-1

The transformation of ethylene oxide (EO), propylene oxide (PO) and 1- butylene oxide (1-BuO) by human glutathione transferase theta (hGSTT1-1) was studied comparatively using 'conjugator' (GSTT1 + individuals) erythrocyte lysates. The relative sequence of velocity of enzymic transformation was PO > EO >> 1-BuO. The faster transformation of PO compared to EO was corroborated in studies with human and rat GSTT1-1 (hGSTT1-1 and rGSTT1-1, respectively) expressed by Salmonella typhimurium TA1535. This sequence of reactivities of homologous epoxides towards GSTT1-1 contrasts to the sequence observed in homologous alkyl halides (methyl bromide, MBr; ethyl bromide, EtBr; n-propyl bromide, PrBr) where the relative sequence MeBr >> EtBr > PrBr is observed. The higher reactivity towards GSTT1-1 of propylene oxide compared to ethylene oxide is consistent with a higher chemical reactivity. This is corroborated by experimental data of acid-catalysed hydrolysis of a number of aliphatic epoxides, including ethylene oxide and propylene oxide and consistent with semi-empirical molecular orbital modelings.

Human amnion epithelial cell transplantation abrogates lung fibrosis and augments repair

Rationale: Chronic lung disease characterized by loss of lung tissue,inflammation, and fibrosis represents a major global health burden. Cellular therapies that could restore pneumocytes and reduce inflammation and fibrosis would be a major advance in management. Objectives: To determine whether human amnion epithelial cells (hAECs), isolated from term placenta and having stem cell–like and antiinflammatory properties, could adopt an alveolar epithelial phenotype and repair a murine model of bleomycin-induced lung injury. Methods: Primary hAECs were cultured in small airway growth medium to determine whether the cells could adopt an alveolar epithelial phenotype. Undifferentiated primary hAECs were also injected parenterally into SCID mice after bleomycin-induced lung injury and analyzed for production of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Mouse lungs were also analyzed for inflammation and collagen deposition. Measurements and Main Results: hAECs grown in small airway growth medium developed an alveolar epithelial phenotype with lamellar body formation, production of SPs A–D, and SP-D secretion. Although hAECs injected into mice lacked SPs, hAECs recovered from mouse lungs 2 weeks posttransplantation produced SPs. hAECs remained engrafted over the 4-week test period. hAEC administration reduced inflammation in association with decreased monocyte chemoattractant protein-1, tumor necrosis factor-a, IL-1 and -6, and profibrotic transforming growth factor-b in mouse lungs. In addition,lung collagen content was significantly reduced by hAEC treatment as a possible consequence of increased degradation by matrix metalloproteinase-2 and down-regulation of the tissue inhibitors f matrix metalloproteinase-1 and 2. Conclusions: hAECs offer promise as a cellular therapy for alveolar restitution and to reduce lung inflammation and fibrosis.

Anti-Inflammatory and antiosteoclastogenic activities of parthenolide on human periodontal ligament cellsIn vitro

Periodontitis is an inflammatory disease that causes osteolysis and tooth loss. It is known that the nuclear factor kappa B (NF-κB) signalling pathway plays a key role in the progression of inflammation and osteoclastogenesis in periodontitis. Parthenolide (PTL), a sesquiterpene lactone extracted from the shoots of Tanacetum parthenium, has been shown to possess anti-inflammatory properties in various diseases. In the study reported herein, we investigated the effects of PTL on the inflammatory and osteoclastogenic response of human periodontal ligament-derived cells (hPDLCs) and revealed the signalling pathways in this process. Our results showed that PTL decreased NF-κB activation, I-κB degradation, and ERK activation in hPDLCs. PTL significantly reduced the expression of inflammatory (IL-1β, IL-6, and TNF-α) and osteoclastogenic (RANKL, OPG, and M-CSF) genes in LPS-stimulated hPDLCs. In addition, PTL attenuated hPDLC-induced osteoclastogenic differentiation of macrophages (RAW264.7 cells), as well as reducing gene expression of osteoclast-related markers in RAW264.7 cells in an hPDLC-macrophage coculture model. Taken together, these results demonstrate the anti-inflammatory and antiosteoclastogenic activities of PTL in hPDLCs in vitro. These data offer fundamental evidence supporting the potential use of PTL in periodontitis treatment.

Impact of agricultural chemicals on the environment and human health in Asia: Are there solutions?

Modern commercial agricultural practices in Asia during the last three to four decades involving chemicals (fertilisers and pesticides) have been associated with large increases in food production never witnessed before, especially under the Green Revolution technology in South Asia. This also involves large-scale increases in commercial vegetable crops. However, the high reliance on chemical inputs to bring about these increases in food production is not without problems. A visible, parallel correlation between higher productivity, high artificial input use and environmental degradation and human ill-health is evident in many countries where commercial agriculture is widespread. In this chapter, we focus on the impact of chemical inputs, in particular the impact of pesticides on the environment and on human health in South Asia with special reference to Sri Lanka...

Environmental change and human history in the Jabal Harun area, Jordan

The research is related to the Finnish Jabal Harun Project (FJHP), which is part of the research unit directed by Professor Jaakko Frösén. The project consists of two interrelated parts: the excavation of a Byzantine monastery/pilgrimage centre on Jabal Harun, and a multiperiod archaeological survey of the surrounding landscape. It is generally held that the Near Eastern landscape has been modified by millennia of human habitation and activity. Past climatic changes and human activities could be expected to have significantly changed also the landscape of the Jabal Harun area. Therefore it was considered that a study of erosion in the Jabal Harun area could shed light on the environmental and human history of the area. It was hoped that it would be possible to connect the results of the sedimentological studies either to wider climatic changes in the Near East, or to archaeologically observable periods of human activity and land use. As evidence of some archaeological periods is completely missing from the Jabal Harun area, it was also of interest whether catastrophic erosion or unfavourable environmental change, caused either by natural forces or by human agency, could explain the gaps in the archaeological record. Changes in climate and/or land-use were expected to be reflected in the sedimentary record. The field research, carried out as part of the FJHP survey fieldwork, included the mapping of wadi terraces and cleaning of sediment profiles which were recorded and sampled for laboratory analyses of facies and lithology. To obtain a chronology for the sedimentation and erosion phases also OSL (optically stimulated luminescence) dating samples were collected. The results were compared to the record of the Near Eastern palaeoclimate, and to data from geoarchaeological studies in central and southern Jordan. The picture of the environmental development was then compared to the human history in the area, based on archaeological evidence from the FJHP survey and the published archaeological research in the Petra region, and the question of the relationship between human activity and environmental change was critically discussed. Using the palaeoclimatic data and the results from geoarchaeological studies it was possible to outline the environmental development in the Jabal Harun area from the Pleistocene to the present.It is appears that there was a phase of accumulation of sediment before the Middle Palaeolithic period, possibly related to tectonic movement. This phase was later followed by erosion, tentatively suggested to have taken place during the Upper Palaeolithic. A period of wadi aggradation probably occurred during the Late Glacial and continued until the end of the Pleistocene, followed by significant channel degradation, attributed to increased rainfall during the Early Holocene. It seems that during the later Holocene channel incision has been dominant in the Jabal Harûn area although there have been also small-scale channel aggradation phases, two of which were OSL-dated to around 4000-3000 BP and 2400-2000 BP. As there is no evidence of tectonic movements in the Jabal Harun area after the early Pleistocene, it is suggested that climate change and human activity have been the major causes of environmental change in the area. At a brief glance it seems that many of the changes in the settlement and land use in the Jabal Harun area can be explained by climatic and environmental conditions. However, the responses of human societies to environmental change are dependent on many factors. Therefore an evaluation of the significance of environmental, cultural, socio-economic and political factors is needed to decide whether certain phenomena are environmentally induced. Comparison with the wider Petra region is also needed to judge whether the phenomena are characteristic of the Jabal Harun area only, or can they be connected to social, political and economic development over a wider area.