[Show abstract][Hide abstract] ABSTRACT: The move away from animal models for skin safety testing is inevitable. It is a question of when, not if. As skin safety studies move away from traditional animal-based approaches, a number of replacement technologies are becoming available. Human skin in organ culture is one such technology. Organ-cultured skin has several features that distinguish it from other technologies. First and foremost, organ-cultured skin is real skin. Almost by definition, therefore, it approximates the intact skin better than other alternative models. Organ culture is an easy-to-use and relatively inexpensive approach to preclinical safety assessment. Although organ culture is not likely to replace high-throughput enzyme assays or monolayer culture/skin equivalent cultures for initial compound assessment, organ culture should find use when the list of compounds to be evaluated is small and when simpler models have narrowed the dose range. Organ-cultured skin also provides a platform for mechanistic studies.
[Show abstract][Hide abstract] ABSTRACT: In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca(2+) (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3-5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1-3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3-4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic epithelium.Laboratory Investigation advance online publication, 23 December 2013; doi:10.1038/labinvest.2013.145.
[Show abstract][Hide abstract] ABSTRACT: Progressive bone mineral loss and increasing bone fragility are hallmarks of osteoporosis. A combination of minerals isolated from the red marine algae, Lithothamnion sp. was examined for ability to inhibit bone mineral loss in female mice maintained on either a standard rodent chow (control) diet or a high-fat western diet (HFWD) for 5, 12, and 18 months. At each time point, femora were subjected to μ-CT analysis and biomechanical testing. A subset of caudal vertebrae was also analyzed. Following this, individual elements were assessed in bones. Serum levels of the 5b isoform of tartrate-resistant acid phosphatase (TRAP) and procollagen type I propeptide (P1NP) were also measured. Trabecular bone loss occurred in both diets (evident as early as 5 months). Cortical bone increased through month 5 and then declined. Cortical bone loss was primarily in mice on the HFWD. Inclusion of the minerals in the diet reduced bone mineral loss in both diets and improved bone strength. Bone mineral density was also enhanced by these minerals. Of several cationic minerals known to be important to bone health, only strontium was significantly increased in bone tissue from animals fed the mineral diets, but the increase was large (5-10 fold). Serum levels of TRAP were consistently higher in mice receiving the minerals, but levels of P1NP were not. These data suggest that trace minerals derived from marine red algae may be used to prevent progressive bone mineral loss in conjunction with calcium. Mineral supplementation could find use as part of an osteoporosis-prevention strategy.
Biological trace element research 10/2013; · 1.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The extracellular Ca(2+)-sensing receptor (CaSR) is a robust promoter of differentiation in colonic epithelial cells and functions as a tumor suppressor in colon cancer. CaSR mediates its biologic effects through diverse mechanisms. Loss of CaSR expression activates a myriad of stem cell-like molecular features that drive and sustain the malignant and drug-resistant phenotypes of colon cancer. This CaSR-null phenotype, however, is not irreversible and induction of CaSR expression in CaSR-null cells promotes cell death mechanisms and restores drug sensitivity. The CaSR also functions as a tumor suppressor in breast cancer and promotes cellular sensitivity to cytotoxic drugs. BRCA1 and CaSR functions intersect in breast cancer cells, and CaSR activation can rescue breast cancer cells from the deleterious effect of BRCA1 mutations.
Best practice & research. Clinical endocrinology & metabolism. 06/2013; 27(3):455-63.
[Show abstract][Hide abstract] ABSTRACT: The goal of this study was to determine if a multimineral natural product derived from red marine algae could reduce colon polyp formation in mice on a high-fat diet. C57BL/6 mice were maintained for up to 18 mo either on a high-fat "Western-style" diet or on a low-fat diet (AIN 76A), with or without the multimineral-supplement. To summarize, colon polyps were detected in 22 of 70 mice (31%) on the high-fat diet but in only 2 of 70 mice (3%) receiving the mineral-supplemented high-fat diet (P < 0.0001). Colon polyps were detected in 16 of 70 mice (23%) in the low-fat group; not significantly different from high-fat group but significantly higher than the high-fat-supplemented group (P = 0.0006). This was in spite of the fact that the calcium level in the low-fat diet was comparable to the level of calcium in the high-fat diet containing the multimineral-product. Supplementation of the low-fat diet reduced the incidence to 8 of 70 mice (11% incidence). Taken together, these findings demonstrate that a multimineral natural product can protect mice on a high-fat diet against adenomatous polyp formation in the colon. These data suggest that increased calcium alone is insufficient to explain the lower incidence of colon polyps.
Nutrition and Cancer 10/2012; 64(7):1020-8. · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gadolinium, a member of the lanthanoid family of transition metals, interacts with calcium-binding sites on proteins and other biological molecules. The overall goal of the present investigation was to determine if gadolinium could enhance calcium-induced epithelial cell growth inhibition in the colon. Gadolinium at concentrations as low as 1-5 μM combined with calcium inhibits proliferation of human colonic epithelial cells more effectively than calcium alone. Gadolinium had no detectable effect on calcium-induced differentiation in the same cells based on change in cell morphology, induction of E-cadherin synthesis, and translocation of E-cadherin from the cytosol to the cell surface. When the colon epithelial cells were treated with gadolinium and then exposed to increased calcium concentrations, movement of extracellular calcium into the cell was suppressed. In contrast, gadolinium treatment had no effect on ionomycin-induced release of stored intracellular calcium into the cytoplasm. Whether these in vitro observations can be translated into an approach for reducing abnormal proliferation in the colonic mucosa (including polyp formation) is not known. These results do, however, provide an explanation for our recent findings that a multi-mineral supplement containing all of the naturally occurring lanthanoid metals including gadolinium are more effective than calcium alone in preventing colon polyp formation in mice on a high-fat diet.
Biological trace element research 09/2012; · 1.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently show that CCN3 is a counter-regulatory molecule for the pro-fibrotic protein CCN2, and a potentially novel fibrosis therapy. The goal of this study was to assess the role of CCN3 in fibroproliferative/fibrotic responses in human dermal fibroblasts exposed to Omniscan, one of the gadolinium-based contrast agents associated with development of nephrogenic systemic fibrosis (NSF) a rare but life-threatening disease thought to be complication of NMR diagnostics in renal impaired patients. Human dermal fibroblasts were exposed to Omniscan; or to platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) as controls. Proliferation was assessed along with matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1 and type 1 procollagen in the absence and presence of CCN3. In parallel, CCN3 production was assessed in control and Omniscan-treated cells. The results showed that PDGF stimulated fibroblast proliferation, production of Timp-1 and MMP-1 whereas exogenous CCN3 inhibited, in a dose response manner, cell proliferation (approx. 50 % max.) and production of MMP-1 (approx 35 % max.) but had little effect on TIMP-1. TGF-β stimulated type 1 procollagen production but not proliferation, Timp-1 or MMP-1 compared to non-TGF-ß treated control cells, and CCN3 treatment blocked (approx. 80 % max.) this up-regulation. Interestingly, untreated, control fibroblasts produced high constitutive levels of CCN3 and concentrations of Omniscan that induced fibroproliferative/fibrogenic changes in dermal fibroblasts correspondingly suppressed CCN3 production. The use of PDGF and TGF-β as positive controls, and the study of differential responses, including that to Omniscan itself, provide the first evidence for a role of fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes, elucidating possible mechanism(s). In conclusion, these data support our hypothesis of a role for fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes in these cells, and suggest that CCN3 may be an important regulatory molecule in NSF and a target for treatment in this and other fibrotic diseases.
Journal of Cell Communication and Signaling 05/2012; 6(2):97-105.
[Show abstract][Hide abstract] ABSTRACT: Goal: The goal of this study was to determine if a multi-mineral natural product derived from the skeletal remains of the red marine algae, Lithothamnion calcareum, could inhibit gastro-intestinal tumor formation. Several dietary features of the western style diet are known to contribute to liver and colon tumorigenesis, but a role for a deficiency in essential minerals has not been explored. Methods: To address this question, C57BL/6 mice were maintained for 5, 12 and 18 months on either a low fat diet or a high fat "Western-style" diet with or without a mineral-rich product, as a dietary supplement. At the end of incubation period, animals were sacrificed and colon polyp formation was assessed by stereomicroscopy and confirmed histologically. Liver masses were also identified grossly and characterized histologically. Results: Liver: While all the livers appeared normal at 5 months, numerous liver masses were visible in the high fat diet mice sacrificed after 12 and 18 months. The majority of masses were in male mice (20 out of 100 males versus 3 out of 100 females, p=0.0002) and in male animals on high fat diet than on low fat diet (15 out of 50 versus 5 out of 50, p=0.0228).The multi-mineral supplement reduced the number of liver masses in male mice (4 of 50 mice in the mineral-supplemented diet groups compared to 16 of 50 mice without the supplement, p=0.0050). Histological evaluation revealed a total of 17 neoplastic lesions (9 adenomas and 8 hepatocellular carcinomas). Out of 8 hepatocellular carcinomas, 7 were found in unsupplemented diet groups and only one in mice on supplemented high fat diet. Colon: High fat diet induced colon polyps in two female mice at 5 months. Several polyps were detected at 12 and 18 months, most of these lesions were found in female mice (34 out of 100 females versus 11 out of 100 males, p=0.0002). Supplement reduced the incidence of polyps in the presence of high-fat from 18 of 50 female mice to 2 of 50 (p<0.0001). Colon polyps were detected in 15 of 50 mice in the low-fat unsupplemented diet group; not significantly different from the non-supplemented high fat group or supplemented low fat group but significantly higher than the high fat supplemented group (p=0.0009). This was in spite of the fact that the calcium level in the low fat diet was comparable to the level of calcium in the high fat supplemented diet. Histologically, there were 8 tumors identified as adenocarcinomas, 7 as adenomas, and 7 as foci of hyperplasia. Out of 8 adenocarcinomas, 7 were found in unsupplemented diet groups, one found in mice on supplemented low fat diet and zero in mineral-rich high fat diet group. Conclusion: These findings demonstrate that a multi-mineral natural product can protect mice on a high fat diet against adenomatous polyp formation in the colon and liver injury. These data suggest that a multi-mineral approach to colon polyp chemoprevention may be more effective than supplementation with calcium alone and the protective role for dietary minerals in liver cancer.
AACR 103rd Annual Meeting 2012-- Mar 31-Apr 4, 2012; 03/2012
[Show abstract][Hide abstract] ABSTRACT: C57BL/6 mice were maintained for up to 18 months on high-fat and low-fat diets with or without a multi-mineral supplement derived from the skeletal remains of the red marine algae Lithothamnion calcareum. Numerous grossly observable liver masses were visible in animals on the "western-style" high-fat diet sacrificed at 12 and 18 months. The majority of the masses were in male mice (20 out of 100 males versus 3 out of 100 females; p = 0.0002). There were more liver masses in animals on the high-fat diet than on the low-fat diet (15 out of 50 on high-fat versus 5 out of 50 on low-fat; p = 0.0254). The multi-mineral supplement reduced the number of liver masses in mice on both diets (3 out of 25 male mice in the low-fat diet group without the supplement versus 1 out of 25 mice with supplement; 12 of 25 male mice in the high-fat diet group without the supplement versus 3 of 25 mice with supplement [p = 0.0129]). Histological evaluation revealed a total of 17 neoplastic lesions (9 adenomas and 8 hepatocellular carcinomas), and 18 pre-neoplastic lesions. Out of eight hepatocellular carcinomas, seven were found in unsupplemented diet groups. Steatosis was widely observed in livers with and without grossly observable masses, but the multi-mineral supplement had no effect on the incidence of steatosis or its severity. Taken together, these findings suggest that a multi-mineral-rich natural product can protect mice against neoplastic and pre-neoplastic proliferative liver lesions that may develop in the face of steatosis.
Biological trace element research 01/2012; 147(1-3):267-74. · 1.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ulcerative dermatitis (UD) is a common, spontaneous condition in mice with a C57BL/6 background. Although initial lesions may be mild, UD is a progressive disease that often results in ulcerations or debilitating fibrotic contractures. In addition, lesions typically are unresponsive to treatment. Euthanasia is often warranted in severe cases, thereby affecting study outcomes through the loss of research subjects. Because the clinical assessment of UD can be subjective, a quantitative scoring method and documentation of the likely time-frame of progression may be helpful in predicting when animals that develop dermatitis should be removed from a study. Such a system may also be helpful in quantitatively assessing success of various treatment strategies and be valuable to clinical laboratory animal veterinarians. In this 1.5-y, prospective cohort study, we followed 200 mice to monitor the development and course of UD. Mice were examined every 2 wk. A clinical sign (alopecia, pruritus, or peripheral lymphadenopathy) was not identified that predicted development of UD lesions in the subsequent 2-wk period. Once UD developed, pruritus, the character of the lesion (single or multiple crust, coalescing crust, erosion, or ulceration), and the size of the lesion were the only parameters that changed (increased) over the course of the disease. Pruritus was a factor in the rapid progression of UD lesions. We used these findings to develop a quantitative scoring system for the severity of UD. This enhanced understanding of the progression of UD and the quantitative scoring system will enhance the monitoring of UD.
Journal of the American Association for Laboratory Animal Science: JAALAS 01/2012; 51(5):586-93. · 0.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In diabetes, foot ulceration may result from increased skin fragility. Retinoids can reverse some diabetes-induced deficits of skin structure and function, but their clinical utility is limited by skin irritation. The effects of diabetes and MDI 301, a nonirritating synthetic retinoid, and retinoic acid have been evaluated on matrix metalloproteinases (MMPs), procollagen expression, and skin structure in skin biopsies from nondiabetic volunteers and diabetic subjects at risk of foot ulceration using organ culture techniques.
Zymography and enzyme-linked immunosorbent assay were utilized for analysis of MMP-1, -2, and -9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) and immunohistochemistry for type I procollagen protein abundance. Collagen structure parameters were assessed in formalin-fixed, paraffin-embedded tissue sections.
The % of active MMP-1 and -9 was higher and TIMP-1 abundance was lower in subjects with diabetes. Type 1 procollagen abundance was reduced and skin structural deficits were increased in diabetes. Three μM MDI 301 reduced active MMP-1 and -9 abundance by 29% (P < .05) and 40% (P < .05), respectively, and increased TIMP-1 by 45% (P = .07). MDI 301 increased type 1 procollagen abundance by 40% (P < .01) and completely corrected structural deficit scores. Two μM retinoic acid reduced MMP-1 but did not significantly affect skin structure.
These data indicate that diabetic patients at risk of foot ulceration have deficits of skin structure and function. MDI 301 offers potential for repairing this skin damage complicating diabetes.
Journal of diabetes and its complications 11/2011; 25(6):398-404. · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to assess insoluble salts containing gadolinium (Gd(3+)) for effects on human dermal fibroblasts. Responses to insoluble Gd(3+) salts were compared to responses seen with Gd(3+) solubilized with organic chelators, as in the Gd(3+)-based contrast agents (GBCAs) used for magnetic resonance imaging. Insoluble particles of either Gd(3+) phosphate or Gd(3+) carbonate rapidly attached to the fibroblast cell surface and stimulated proliferation. Growth was observed at Gd(3+) concentrations between 12.5 and 125 μM, with toxicity at higher concentrations. Such a narrow window did not characterize GBCA stimulation. Proliferation induced by insoluble Gd(3+) salts was inhibited in the presence of antagonists of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways (similar to chelated Gd(3+)) but was not blocked by an antibody to the platelet-derived growth factor receptor (different from chelated Gd(3+)). Finally, high concentrations of the insoluble Gd(3+) salts failed to prevent fibroblast lysis under low-Ca(2+) conditions, while similar concentrations of chelated Gd(3+) were effective. In conclusion, while insoluble Gd(3+) salts are capable of stimulating fibroblast proliferation, one should be cautious in assuming that GBCA dechelation must occur in vivo to produce the profibrotic changes seen in association with GBCA exposure in the subset of renal failure patients that develop nephrogenic systemic fibrosis.
Biological trace element research 09/2011; 145(2):257-67. · 1.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A role for calcium in epithelial growth control is well-established in the colon and other tissues. In the colon, Ca²+ "drives" the differentiation process. This results in sequestration of β-catenin in the cell surface / cytoskeletal complex, leaving β-catenin unavailable to serve as a growth-promoting transcription enhancer in the nucleus. The signaling events that lead from Ca²+ stimulation to differentiation are not fully understood. A critical role for the extracellular calcium-sensing receptor (CaSR) is assumed, based on CaSR localization to the differentiating epithelial cells in the normal colonic mucosa (upper half of the crypt and crypt surface), decreased CaSR expression in colon carcinoma, and the results from in vitro studies with colonic epithelial cell lines. While Ca²+ is well-accepted as a growth-regulating agent in the colon, suppression of cell proliferation is not complete. At least part of the reason for this is the inherent variability in Ca²+ responsiveness among individual epithelial cells. Of interest, colon epithelial cells that are resistant to the growth-regulating activity of Ca²+ alone are still responsive to Ca²+ in conjunction with other transition metals. Whether a multi-mineral approach will, ultimately, prove to be more effective than Ca²+ alone as a colon cancer chemopreventive agent remains to be seen, but certainly worth investigating.
Histology and histopathology 06/2011; 26(6):769-79. · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to compare each of the 14 naturally occurring lanthanoid metal ions for ability to stimulate pro-fibrotic responses in human dermal fibroblasts. When fibroblasts were exposed to individual lanthanoids over the concentration range of 1-100 μM, increased proliferation was observed with each of the agents as compared with control cells that were already proliferating rapidly in a growth factor-enriched culture medium. Dose-response differences were observed among the individual metal ions. Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 levels were also increased in response to lanthanoid exposure but type I procollagen production was not. A dose-response relationship between induction of proliferation and increased MMP-1 was observed. Non-lanthanoid transition metal ions (aluminum, copper, cobalt, iron, magnesium, manganese, nickel, and zinc) were examined in the same assays; there was little stimulation with any of these metals. When epidermal keratinocytes were examined in place of dermal fibroblasts, there was no growth stimulation with any of the lanthanoids. Several of the lanthanoid metals inhibited keratinocyte proliferation at higher concentrations (50-100 μM).
Biological trace element research 04/2011; 144(1-3):621-35. · 1.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to assess the effects of gadolinium (Gd3+), provided as gadolinium chloride, on fibroblast function.
Human dermal fibroblasts in monolayer culture and intact skin in organ culture were exposed to the lanthanide metal (1-20 μM).
Increased proliferation was observed, in association with upregulation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1, without an apparent increase in production of type I procollagen. A platelet-derived growth factor (PDGF) receptor-blocking antibody inhibited fibroblast proliferation in response to Gd3+ as did inhibitors of signaling pathways--that is, mitogen-activated protein kinase and phosphatidylinositol-3 kinase pathways--that are activated by PDGF.
The responses to gadolinium chloride are similar to responses previously seen with chelated Gd3+ in clinically used magnetic resonance imaging contrast agents. Fibroblast responses appear to reflect Gd3+-induced PDGF receptor activation and downstream signaling. Increased dermal fibroblast proliferation in conjunction with effects on matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 could contribute to the fibroplastic/fibrotic changes seen in the lesional skin of individuals with nephrogenic systemic fibrosis.
[Show abstract][Hide abstract] ABSTRACT: Age-related changes and environmental stresses affect all of the cellular elements in the skin, but changes that occur in stromal fibroblasts are particularly important, because it is these cells that maintain the connective tissue of the skin. From the standpoint of maintaining connective tissue, the most important phenotypic changes include a progressive increase in connective tissue damage, mediated by matrix metalloproteinases (MMPs) and a reduction in new collagen synthesis. Intrinsic changes in the aged fibroblast as well as exposure to environmental insults (e.g. ultraviolet radiation) alter the intracellular signaling events that regulate both collagen synthesis and MMP elaboration. Furthermore, as the connective tissue becomes progressively damaged, it eventually becomes a stimulator of reduced fibroblast synthetic function. The result is a downward spiral that ends, if not checked, in the cosmetic and medical consequences that define aged skin.
Drug Discovery Today Therapeutic Strategies 12/2010; 7(3):65-70.