In situ zymography: topographical considerations

In situ gelatin zymography is a simple technique providing valuable information about the cellular and tissue localization of gelatinases. Until recently, the use of this technique has been confined to soft, relatively homogeneous tissue. In this report in situ zymography has been utilized to assess the sub-lamellar location of gelatinases in the hard, semi-keratinized epidermal layer and the adjacent soft connective tissue matrix of the dermis of the equine hoof. We show that alterations in the orientation at which the tissue is dipped and withdrawn from the emulsion cause profound alterations in emulsion thickness. Microscopic Variations in the surface topography of frozen tissue sections also influence emulsion thickness making interpretation of the results difficult. Given these results, researchers must be aware of potential variations in zymographic analysis may be influenced by physical tissue parameters in addition to suspected gelatinase activity. (C) 2001 Elsevier Science B.V. All rights reserved.

Lyme Disease: antibodies against Borrelia burgdorferi in farm workers in Argentina

Lyme Disease is a tick-borne (specially by Ixodes ticks) immune-mediated inflammatory disorder caused by a newly recognize spirochete, Borrelia burgdorferi. Indirect fluorescent antibody (IF) staining methods and enzyme-linked immunosorbent assay are frequently relied upon to confirm Lyme borreliosis infections. Although serologic testing for antibodies has limitations, it is still the only practical means of confirming B. burgdorferi infections. Because we have no previous report of Lyme disease in human inhabitants in Argentina, a study was designed as a seroepidemiologic investigation of the immune response to B. burgdorferi in farm workers of Argentina with arthritis symptoms. Three out of 28 sera were positive (#1,5 and 9). Serum # 1 was positive for Immunoglobulin G at dilution 1:320, serum # 5 and # 9 both to dilution 1:160; while for Immunoglobulin M all (#1, 5 and 9) were positive at low dilution (1:40) using IF. The results showed that antibodies against B. burgdorferi are present in an Argentinian population. Thus caution should be exercised in the clinical interpretation of arthritis until the presence of B. burgdorferi be confirmed by culture in specific media.

Iron Overload in a Murine Model of Hereditary Hemochromatosis Is Associated with Accelerated Progression of Osteoarthritis Under Mechanical Stress

OBJECTIVE: Hereditary hemochromatosis (HH) is a disease caused by mutations in the Hfe gene characterised by systemic iron overload and associated with an increased prevalence of osteoarthritis (OA) but the role of iron overload in the development of OA is still undefined. To further understand the molecular mechanisms involved we have used a murine model of HH and studied the progression of experimental OA under mechanical stress. DESIGN: OA was surgically induced in the knee joints of 10-week-old C57BL6 (wild-type) mice and Hfe-KO mice. OA progression was assessed using histology, micro CT, gene expression and immunohistochemistry at 8 weeks after surgery. RESULTS: Hfe-KO mice showed a systemic iron overload and an increased iron accumulation in the knee synovial membrane following surgery. The histological OA score was significantly higher in the Hfe-KO mice at 8 weeks after surgery. Micro CT study of the proximal tibia revealed increased subchondral bone volume and increased trabecular thickness. Gene expression and immunohistochemical analysis showed a significant increase in the expression of matrix metallopeptidase 3 (MMP-3) in the joints of Hfe-KO mice compared with control mice at 8 weeks after surgery. CONCLUSIONS: HH was associated with an accelerated development of OA in mice. Our findings suggest that synovial iron overload has a definite role in the progression of HH-related OA

Exploring motor neuron degeneration in ALS - prevention by glycoursodeoxycholic acid and signaling to microglia

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Relative adrenal insufficiency and hemodynamic status in cardiopulmonary bypass surgery patients. A prospective cohort study

BACKGROUND: The objectives of this study were to determine the risk factors for relative adrenal insufficiency in cardiopulmonary bypass patients and the impact on postoperative vasopressor requirements. METHODS: Prospective cohort study on cardiopulmonary bypass patients who received etomidate or not during anesthetic induction. Relative adrenal insufficiency was defined as a rise in serum cortisol

High prevalence of anti-apolipoprotein/A-1 autoantibodies in maintenance hemodialysis and association with dialysis vintage.

Autoantibodies to apolipoprotein/A-1 (anti-ApoA-1 IgG) have pro-atherogenic properties in patients at high cardiovascular risk, but its prevalence in patients with end-stage kidney disease is unknown. The aims of this single-center, cross-sectional study were to assess the prevalence of anti-ApoA-1 antibodies in patients on maintenance hemodialysis (MHD), and to examine its correlation with inflammatory biomarkers related to atherosclerotic plaque vulnerability and dialysis vintage. To this purpose, anti-ApoA-1 IgG levels and the concentrations of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), metalloproteinase-9 (MMP-9), tumor necrosis factor-α, and C-reactive protein (CRP) were assessed in the sera of 66 MHD patients (mean age: 68 ± 14 years, 36% women, 32% diabetics). Anti-ApoA-1 IgG positivity (defined as a blood value ≥ 97.5(th) percentile of the normal distribution as assessed in healthy blood donors) was 20%. Circulating levels of anti-ApoA-1 IgG correlated positively with dialysis vintage, but not with cardiovascular risk factors or previous cardiovascular events; no significant correlations were found between the anti-ApoA1 IgG levels and circulating levels of IL-6, IL-8, MCP-1, MMP-9, CRP, or low-density lipoprotein-cholesterol. In multivariable linear regression, adjusted for age and sex, only dialysis vintage remained positively and independently associated with anti-ApoA-1 titers (β = 0.05, 95% CI: 0.006; 0.28, P = 0.049). In conclusion, the prevalence of anti-ApoA-1 IgG is raised in the MHD-population, and positively associated with dialysis vintage, a major determinant of cardiovascular outcome. Whether antiApoA-1 antibodies play a role in the pathophysiology of accelerated atherosclerosis in the MHD-population merits further study.

Deletion of delta-opioid receptor in mice alters skin differentiation and delays wound healing.

In addition to their well-known antinociceptive action, opioids can modulate non-neuronal functions, such as immune activity and physiology of different cell types. Several findings suggest that the delta-opioid receptor (DOR) and its endogenous ligands (enkephalins) are important players in cell differentiation and proliferation. Here we show the expression of DOR in mouse skin and human skin cultured fibroblasts and keratinocytes using RT-PCR. In DOR knock-out (KO) mice, a phenotype of thinner epidermis and higher expression of cell differentiation marker cytokeratin 10 (CK 10) were observed compared with wild type (WT). Using a burn wound model, significant wound healing delay (about 2 days) and severe epidermal hypertrophy were shown at the wound margin of DOR KO mice. This wound healing delay was further investigated by immunohistochemistry using markers for proliferation, differentiation, re-epithelialization, and dermal repair (CK 6, CK 10, and collagen IV). The levels of all these markers were increased in wounds of KO mice compared with WT. During the wound healing, the epidermal thickness in KO mice augments faster and exceeds that of the WT by day 3. These results suggest an essential role of DOR in skin differentiation, proliferation, and migration, factors that are important for wound healing.

Shedding light on proteolytic cleavage of CD44: the responsible sheddase and functional significance of shedding.

CD44 is the major cell-surface receptor for hyaluronan, which is implicated in cell-cell and cell-matrix adhesion, cell migration, and signaling. Studies have shown that CD44-dependent migration requires CD44 to be shed from the cell surface and that matrix metalloproteinase-mediated cleavage may provide an underlying mechanism. However, the full spectrum of proteases that may participate in CD44 shedding has yet to be defined. In this issue, Anderegg et al. demonstrate that ADAM10, but not ADAM17 or MMP14, mediates constitutive shedding of CD44 in human melanoma cells and that knockdown of ADAM10 blocks the antiproliferative activity of the soluble proteolytic cleavage product of CD44.

MMP13 mutations are the cause of recessive metaphyseal dysplasia, Spahr type.

Metaphyseal dysplasia, Spahr type (MDST; OMIM 250400) was described in 1961 based on the observation of four children in one family who had rickets-like metaphyseal changes but normal blood chemistry and moderate short stature. Its molecular basis and nosologic status remained unknown. We followed up on those individuals and diagnosed the disorder in an additional member of the family. We used exome sequencing to ascertain the underlying mutation and explored its consequences on three-dimensional models of the affected protein. The MDST phenotype is associated with moderate short stature and knee pain in adults, while extra-skeletal complications are not observed. The sequencing showed that MDST segregated with a c.619T>G single nucleotide transversion in MMP13. The predicted non-conservative amino acid substitution, p.Trp207Gly, disrupts a crucial hydrogen bond in the calcium-binding region of the catalytic domain of the matrix metalloproteinase, MMP13. The MDST phenotype is associated with recessive MMP13 mutations, confirming the importance of this metalloproteinase in the metaphyseal growth plate. Dominant MMP13 mutations have been associated with metaphyseal anadysplasia (OMIM 602111), while a single child homozygous for a MMP13 mutation had been previously diagnosed as "recessive metaphyseal anadysplasia," that we conclude is the same nosologic entity as MDST. Molecular confirmation of MDST allows distinction of it from dominant conditions (e.g., metaphyseal dysplasia, Schmid type; OMIM # 156500) and from more severe multi-system conditions (such as cartilage-hair hypoplasia; OMIM # 250250) and to give precise recurrence risks and prognosis. © 2014 Wiley Periodicals, Inc.

Disrupting the EMMPRIN (CD147)-cyclophilin A interaction reduces infarct size and preserves systolic function after myocardial ischemia and reperfusion.

Objective-Inflammation and proteolysis crucially contribute to myocardial ischemia and reperfusion injury. The extracellular matrix metalloproteinase inducer EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) may be involved in both processes. The aim of the study was to characterize the role of the CD147 and CyPA interplay in myocardial ischemia/reperfusion (I/R) injury.Methods and Results-Immunohistochemistry showed enhanced expression of CD147 and CyPA in myocardial sections from human autopsies of patients who had died from acute myocardial infarction and from mice at 24 hours after I/R. At 24 hours and 7 days after I/R, the infarct size was reduced in CD147(+/-) mice vs CD147(+/+) mice (C57Bl/6), in mice (C57Bl/6) treated with monoclonal antibody anti-CD147 vs control monoclonal antibody, and in CyPA(-/-) mice vs CyPA(+/+) mice (129S6/SvEv), all of which are associated with reduced monocyte and neutrophil recruitment at 24 hours and with a preserved systolic function at 7 days. The combination of CyPA(-/-) mice with anti-CD147 treatment did not yield further protection compared with either inhibition strategy alone. In vitro, treatment with CyPA induced monocyte chemotaxis in a CD147-and phosphatidylinositol 3-kinase-dependent manner and induced monocyte rolling and adhesion to endothelium (human umbilical vein endothelial cells) under flow in a CD147-dependent manner.Conclusion-CD147 and its ligand CyPA are inflammatory mediators after myocardial ischemia and reperfusion and represent potential targets to prevent myocardial I/R injury.

Cancer Cell-Associated MT1-MMP Promotes Blood Vessel Invasion and Distant Metastasis in Triple-Negative Mammary Tumors.

Functional roles for the cancer cell-associated membrane type I matrix metalloproteinase (MT1-MMP) during early steps of the metastatic cascade in primary tumors remain unresolved. In an effort to determine its significance, we determined the in vivo effects of RNAi-mediated downregulation in mammary cancer cells on the migration, blood and lymphatic vessel invasion (LVI), and lymph node and lung metastasis. We also correlated the expression of cancer cell MT1-MMP with blood vessel invasion (BVI) in 102 breast cancer biopsies. MT1-MMP downregulation in cancer cells decreased lung metastasis without affecting primary tumor growth. The inhibition of lung metastasis correlated with reduced cancer cell migration and BVI. Furthermore, cancer cell-expressed MT1-MMP upregulated the expression of MT1-MMP in vascular endothelial cells, but did not affect MT1-MMP expression in lymphatic endothelial cells, LVI, or lymph node metastasis. Of clinical importance, we observed that elevated MT1-MMP expression correlated with BVI in biopsies from triple-negative breast cancers (TNBC), which have a poor prognosis and high incidence of distant metastasis, relative to other breast cancer subtypes. Together, our findings established that MT1-MMP activity in breast tumors is essential for BVI, but not LVI, and that MT1-MMP should be further explored as a predictor and therapeutic target of hematogenous metastasis in TNBC patients. Cancer Res; 71(13); 4527-38. ©2011 AACR.

Water-filtered infrared-A radiation (wIRA) is not implicated in cellular degeneration of human skin.

BACKGROUND: Excessive exposure to solar ultraviolet radiation is involved in the complex biologic process of cutaneous aging. Wavelengths in the ultraviolet-A and -B range (UV-A and UV-B) have been shown to be responsible for the induction of proteases, e. g. the collagenase matrix metalloproteinase 1 (MMP-1), which are related to cell aging. As devices emitting longer wavelengths are widely used in therapeutic and cosmetic interventions and as the induction of MMP-1 by water-filtered infrared-A (wIRA) had been discussed, it was of interest to assess effects of wIRA on the cellular and molecular level known to be possibly involved in cutaneous degeneration. OBJECTIVES: Investigation of the biological implications of widely used water-filtered infrared-A (wIRA) radiators for clinical use on human skin fibroblasts assessed by MMP-1 gene expression (MMP-1 messenger ribonucleic acid (mRNA) expression).Methods: Human skin fibroblasts were irradiated with approximately 88% wIRA (780-1400 nm) and 12% red light (RL, 665-780 nm) with 380 mW/cm(2) wIRA(+RL) (333 mW/cm(2) wIRA) on the one hand and for comparison with UV-A (330-400 nm, mainly UV-A1) and a small amount of blue light (BL, 400-450 nm) with 28 mW/cm(2) UV-A(+BL) on the other hand. Survival curves were established by colony forming ability after single exposures between 15 minutes and 8 hours to wIRA(+RL) (340-10880 J/cm(2) wIRA(+RL), 300-9600 J/cm(2) wIRA) or 15-45 minutes to UV-A(+BL) (25-75 J/cm(2) UV-A(+BL)). Both conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and quantitative real-time RT-PCR techniques were used to determine the induction of MMP-1 mRNA at two physiologic temperatures for skin fibroblasts (30 degrees C and 37 degrees C) in single exposure regimens (15-60 minutes wIRA(+RL), 340-1360 J/cm(2) wIRA(+RL), 300-1200 J/cm(2) wIRA; 30 minutes UV-A(+BL), 50 J/cm(2) UV-A(+BL)) and in addition at 30 degrees C in a repeated exposure protocol (up to 10 times 15 minutes wIRA(+RL) with 340 J/cm(2) wIRA(+RL), 300 J/cm(2) wIRA at each time). RESULTS: Single exposure of cultured human dermal fibroblasts to UV-A(+BL) radiation yielded a very high increase in MMP-1 mRNA expression (11 +/-1 fold expression for RT-PCR and 76 +/-2 fold expression for real-time RT-PCR both at 30 degrees C, 75 +/-1 fold expression for real-time RT-PCR at 37 degrees C) and a dose-dependent decrease in cell survival. In contrast, wIRA(+RL) did not produce cell death and did not induce a systematic increase in MMP-1 mRNA expression (less than twofold expression, within the laboratory range of fluctuation) detectable with the sensitive methods applied. Additionally, repeated exposure of human skin fibroblasts to wIRA(+RL) did not induce MMP-1 mRNA expression systematically (less than twofold expression by up to 10 consecutive wIRA(+RL) exposures and analysis with real-time RT-PCR). CONCLUSIONS: wIRA(+RL) even at the investigated disproportionally high irradiances does not induce cell death or a systematic increase of MMP-1 mRNA expression, both of which can be easily induced by UV-A radiation. Furthermore, these results support previous findings of in vivo investigations on collagenase induction by UV-A but not wIRA and show that infrared-A with appropriate irradiances does not seem to be involved in MMP-1 mediated photoaging of the skin. As suggested by previously published studies wIRA could even be implicated in a protective manner.

Inhibitors of human collagenase, MMP1

Different common drugs (Meloxicam, Tenoxicam and Piroxicam, and sodium alendronate) were tested both experimental and theoretically as inhibitors of interstitial human collagenase, also known as matrix metalloproteinase 1 (MMP-1). The in vitro collagenase activity, alone and in the presence of inhibitors, was quantified by the reaction with a fluorescent synthetic substrate and measuring the change of emission. Collagenase-inhibitor interaction was studied theoretically by computational calculations. Three among the four tested substances showed moderate inhibiting activity against the human collagenase.

The regulation and function of collagenase-3 (MMP-13) in cutaneous wound healing and squamous cell carcinoma

Matrix metalloproteinase-13 (MMP-13) is a potent proteolytic enzyme, whose expression has been previously associated with fetal bone development and postnatal bone remodeling and with adult gingival wound healing. MMP-13 is also known to be involved in the growth and invasion of various cancers including squamous cell carcinoma (SCC) of the skin. The aim of this study was to further elucidate the function and regulation of MMP-13 in wound repair and cancer. In this study, it was shown that fetal skin fibroblasts express MMP-13 in response to transforming growth factor-β in a p38 MAP kinase dependent manner. In addition, MMP-13 was found to be expressed in vivo by wound fibroblasts in human fetal skin grafted on SCID mice. Adenovirally delivered expression of MMP-13 enhanced collagen matrix contraction by fibroblasts in vitro in association with altered cytoskeletal structure, enhanced proliferation and survival. These results indicate that MMP-13 is involved in cell-mediated collagen matrix remodeling and suggest a role for MMP-13 in superior matrix remodeling and scarless healing of fetal skin wounds. Using an MMP-13 deficient mouse strain, it was shown that MMP-13 is essential for the normal development of experimental granulation tissue in mice. MMP-13 was implicated in the regulation of myofibroblast function and angiogenesis and the expression of genes involved in cellular proliferation and movement, immune response, angiogenesis and proteolysis. Finally, epidermal mitogen, keratinocyte growth factor (KGF) was shown to suppress the malignant properties of skin SCC cells by downregulating the expression of several target genes with potential cancer promoting properties, including MMP-13, and by reducing SCC cell invasion. These results provide evidence that MMP-13 potently regulates cell viability, myofibroblast function and angiogenesis associated with wound healing and cancer. In addition, fibroblasts expressing MMP-13 show high collagen reorganization capacity. Moreover, the results suggest that KGF mediates the anti-cancer effects on skin SCC

Arsenic trioxide reduces the invasive and metastatic properties of nasopharyngeal carcinoma cells in vitro

Nasopharyngeal carcinoma (NPC) is notorious for the metastases, which are in close association with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Arsenic trioxide (As2O3) has been shown to induce apoptosis and differentiation in NPC xenografts. Then, can it repress the cancer cells' metastasis potential? To elucidate this issue, the present study was performed. LMP1-negative cell line HNE1 and LMP1-positive cell line HNE1-LMP1 were used as in vitro model. Cells (1 x 10(5)/mL) were cultured with or without 3 µM As2O3 for 48 h. Then the survival cells were collected to investigate their potential of colony formation, attachment, invasion, and migration. Both confocal immunofluorescence staining and Western blot were used to detect the changes of LMP1 expression. The changes of MMP-9 were examined by RT-PCR assay and Western blot. The results were as follow: i) the colony formation inhibition rate (75.41 ± 3.9% in HNE1-LMP1 cells vs 37.89 ± 4.9% in HNE1 cells), the rate of attachment (HNE1-LMP1 vs HNE1: 56.40 ± 3.5 vs 65.87 ± 5.9%), the invasion inhibitory rate (HNE1-LMP1 vs HNE1: 56.50 ± 3.7 and 27.91 ± 2.1%), and the migration inhibitory rate (HNE1-LMP1 vs HNE1: 48.70 ± 3.9 vs 29.19 ± 6.27%) were all significantly different between the two cell lines (P < 0.01). ii) LMP1 was down-regulated in As2O3-treated HNE1-LMP1 cells. iii) The reduction of MMP-9 was found in As2O3-treated groups, more evident in HNE1-LMP1 cells. Thus, we conclude that As2O3 can reduce metastasis potential of NPC cells, involving inhibition of MMP-9 expression. LMP1 were also reduced in this process and seemed to enhance anti-metastasis activity of As2O3.