The Effect of Cold Ischemia Time and/or Formalin Fixation on Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor-2 Results in Breast Carcinoma

Aims. To compare the results of estrogen and progesterone receptors (ER, PR), and human epidermal growth factor receptor-2 (HER2) expression status on biopsy and excision specimens and to evaluate the effect of cold ischemia time and/or formalin fixation on these biomarkers. Methods. Breast carcinomas that were diagnosed between 2007 and 2009 by core needle biopsy, and subsequently excised in our institution, were included in the study. Data regarding the tumor morphology, grade, and ER, PR, and HER2 status were retrospectively collected from the pathology reports. Results. Five out of 149 (3.4%) cases with ER-positive receptor status in the biopsy specimen became ER-negative in the subsequent excision specimen. Nine out of 126 (7.1%) cases with PR-positive receptor status in the biopsy specimen became PR-negative in the excision specimen. Receptor status change was predominantly seen in tumors with low ER and PR receptor expression. HER2 results were consistent between biopsy and excision specimens in all cases tested. Conclusions. Cold ischemia time and/or formalin fixation affect mainly ER and PR testing with low Allred scores and support the implementation of the ASCO/CAP guidelines. HER2 results, however, were not affected in our limited number of patients

Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCysuPA(19-31)) directed against cysteine proteases and the urokinasetype plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the Nterminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCysuPA(19-31) or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OVMZ-6\#8). Additionally, OVMZ 6\#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitorproducing cells was significantly reduced compared to vectortransfected control cells. Thus, these novel, compact, and smallsize inhibitors directed against up to three different tumorassociated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis

Raman spectroscopy monitoring of the cellular activities of a tissue-engineered ex vivo produced oral mucosal equivalent

To ensure quality control and assurance in tissue engineering, noninvasive, real-time and aseptic evaluation of cell-based devices is required before product release. In this study, Raman spectroscopy was applied to monitor the cellular activities of an oral mucosa equivalent (EVPOME) produced ex vivo from cultured autogenous oral keratinocytes and acellular dermis—AlloDerm. Raman spectra showed a positive correlation of the peak area ratio of amide I (1655 cm −1 )/phenylalanine (1004 cm −1 ) with a negative linear regression ( R 2 > 0.95) according to the number of cultured days, especially on the 14thand 21st day. This work demonstrates the successful application of Raman spectroscopy for quantitatively monitoring and evaluating the maturity of EVPOME. Copyright © 2010 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/83170/1/2688_ftp.pd

Zoledronic acid inhibits macrophage SOCS3 expression and enhances cytokine production

Suppressor of cytokine signaling‐3 (SOCS3) has multiple functions including inhibition of Janus kinase (Jak) activity, regulation of protein degradation, and suppression of cytokine signaling. SOCS3 modulates macrophage response to cytokines such as IL‐6 and leptin that are systemically induced in obesity. Obesity is a suspected risk factor for SOCS3‐related pathology such as rheumatoid arthritis and Crohn's disease as well as zoledronic acid (ZA)‐induced osteonecrosis of the jaw (ONJ). Thus, understanding the ability of bisphosphonates to modulate SOCS3 is necessary to qualify their contribution to these disorders. ONJ occurs in up to 10% of patients using intravenous bisphosphonates and has an unknown pathogenesis that may be linked to decreased bone turnover, altered vascularity, bacterial invasion, and compromised wound healing. Given the increased risk of ONJ with obesity and importance of macrophages in wound healing, we hypothesized that amino‐bisphosphonates could contribute to the pathogenesis of ONJ by regulating macrophage responses to cytokines such as leptin and IL‐6. We report that ZA is a novel inhibitor of SOCS3 in primary macrophages and human ONJ biopsy specimens. Inhibition of SOCS3 by ZA resulted in significant increases in IL‐6 production. SOCS3 transcription is regulated by nuclear accumulation of phosphorylated‐Stat3 (P‐Stat3). We found that ZA decreased phosphorylation of Stat3 in a mevalonate‐pathway dependent manner. However, restoration of P‐Stat3 was not sufficient to correct SOCS3 inhibition. We propose that disruption of macrophage SOCS3 expression by amino‐bisphosphonates such as ZA may be a novel contributor to inflammatory phenotypes in obesity and the pathogenesis of ONJ. J. Cell. Biochem. 112: 3364–3372, 2011. © 2011 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87102/1/23267_ftp.pd

Tissue regeneration in dentistry: can salamanders provide insight?

The ability to regenerate damaged tissues would be of tremendous benefit for medicine and dentistry. Unfortunately, humans are unable to regenerate tissues such teeth, fingers or to repair injured spinal cord. With an aging population, health problems are more prominent and dentistry is no exception as loss of bone tissue in the orofacial sphere from periodontal disease is on the rise. Humans can repair oral soft tissues exceptionally well, however hard tissues, like bone and teeth, are devoid of the ability to repair well or at all. Fortunately, Mother Nature has solved nearly every problem that we would like to solve for our own benefit and tissue regeneration is no exception. By studying animals that can regenerate, like Axolotls (Mexican salamander), we hope to find ways to stimulate regeneration in humans. We will discuss the role of the transforming growth factor beta cytokines as they are central to wound healing in humans and regeneration in Axolotls. We will also compare wound healing in humans (skin and oral mucosa) to Axolotl skin wound healing and limb regeneration. Finally, we will address the problem of bone regeneration and present results in salamanders which indicate that in order to regenerate bone you need to recruit non-bone cells. Fundamental research, such as the work being done in animals that can regenerate, offers insight to help understand why some treatments are successful while others fail when it comes to specific tissues such as bones

Blockade of Mast Cell Activation Reduces Cutaneous Scar Formation

Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1a, IL-1b, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase b1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound

Cystatin C Deficiency Promotes Epidermal Dysplasia in K14-HPV16 Transgenic Mice

Cysteine protease cathepsins are important in extracellular matrix protein degradation, cell apoptosis, and angiogenesis. Mice lacking cathepsins are protected from tumor progression in several animal models, suggesting that the regulation of cathepsin activities controls the growth of various malignant tumors.We tested the role of cathepsins using a mouse model of multistage epithelial carcinogenesis, in which the human keratin-14 promoter/enhancer drove the expression of human papillomavirus type 16 (HPV16) early region E6/E7 transgenes. During the progression of premalignant dysplasia, we observed increased expression of cysteine protease cathepsin S, but concomitantly reduced expression of cathepsin endogenous inhibitor cystatin C in the skin tissue extract. Absence of cystatin C in these transgenic mice resulted in more progression of dysplasia to carcinoma in situ on the face, ear, chest, and tail. Chest and ear skin extract real time PCR and immunoblot analysis, mouse serum sample ELISA, tissue immunohistological analysis, and tissue extract-mediated in vitro elastinolysis and collagenolysis assays demonstrated that cystatin C deficiency significantly increased cathepsin expression and activity. In skin from both the chest and ear, we found that the absence of cystatin C reduced epithelial cell apoptosis but increased proliferation. From the same tissue preparations, we detected significantly higher levels of pro-angiogenic laminin 5-derived γ2 peptides and concurrently increased neovascularization in cystatin C-deficient mice, compared to those from wild-type control mice.Enhanced cathepsin expression and activity in cystatin C-deficient mice contributed to the progression of dysplasia by altering premalignant tissue epithelial proliferation, apoptosis, and neovascularization

Cystatin E/M suppresses legumain activity and invasion of human melanoma

<p>Abstract</p> <p>Background</p> <p>High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied.</p> <p>Methods</p> <p>A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay.</p> <p>Results</p> <p>Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay.</p> <p>Conclusions</p> <p>These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.</p