Translating molecular medicine into clinical tools: doomed to fail by neglecting basic preanalytical principles

This commentary discusses a study on measurements of matrix metalloproteinase 9 (MMP-9) in serum of pseudoxanthoma elasticum patients recently published in Journal of Molecular Medicine. This study can be considered the typical "obstacle" to effective translational medicine as previously documented in JTM journal. Although serum has been frequently proven as inappropriate sample for determining numerous circulating MMPs, among them MMP-9, there are over and over again studies, as in this case, that measure MMP-9 in serum. Comparative measurements in serum and plasma samples demonstrated higher concentrations for MMP-9 in serum due to the additional release from leukocytes and platelets following the coagulation/fibrinolysis process. From this example it can be concluded that translating basic research discoveries into clinical tools needs a more intensive exchange between basic biomedical research and clinical scientists already in an early stage. Otherwise a lost of translation, as discussed in JTM journal, seems to be inevitable

Are matrix metalloproteinases the missing link?

none3openMANNELLO F; TONTI G.A.; PAPA SMannello, Ferdinando; Tonti, GAETANA ANNA MARIA; Papa, Stefan

Effects of Blood Collection Methods on Gelatin Zymography of Matrix Metalloproteinases

none1noopenMANNELLO F.Mannello, Ferdinand

Comparison of Enzymatic and Non-Enzymatic Means of Dissociating Adherent Monolayers of Mesenchymal Stem Cells

The dissociation of adherent mesenchymal stem cell (MSC) monolayers with trypsin and enzyme-free dissociation buffer was compared. A significantly lower proportion of viable cells were obtained with enzyme-free dissociation buffers compared to trypsin. Subsequently, the dissociated cells were re-seeded on new cell culture dishes and were subjected to the MTT assay 24 h later. The proportion of viable cells that reattached was significantly lower for cells obtained by dissociation with enzyme-free dissociation buffer compared to trypsin. Frozen–thawed MSC displayed a similar trend, yielding consistently higher cell viability and reattachment rates when dissociated with trypsin compared to enzyme-free dissociation buffer. It was also demonstrated that exposure of trypsin-dissociated MSC to enzyme-free dissociation buffer for 1 h had no significant detrimental effect on cell viability

Multiplex Matrix Metalloproteinases Analysis in the Cerebrospinal Fluid Reveals Potential Specific Patterns in Multiple Sclerosis Patients.

Background: Matrix metalloproteinases (MMPs) are pleiotropic enzymes involved in extracellular protein degradation and turnover. MMPs are implicated in the pathogenesis of many neurological diseases, including multiple sclerosis (MS). Objective: To search the level of MMPs in the cerebrospinal fluid (CSF) of MS patients and detect possible disease-specific patterns. Methods: CSF samples from 32 MS patients and, from 15 control subjects with other inflammatory neurological diseases (OIND) were analyzed. The Bio-Plex Pro Human MMP 9-Plex Panel (Bio-Rad) was used for the quantification of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, and MMP-13. Results: CSF MMP-1 and MMP-12 levels were significantly reduced in MS as compared with OIND. In MS patients' CSF: (i) MMP-1 levels were significantly higher in women vs. men; (ii) MMP-10 concentrations were higher in patients with CSF-restricted IgG oligoclonal bands, and (iii) MMP-7 levels were increased in patients with longer disease duration. In the OIND group MMP-7 and MMP-12 levels significantly and directly correlated with age. Conclusions: Our study contributes to investigating the role of MMPs in MS, with regard to CSF immunological features and disease duration. Sex-specific differences were also detected in MMPs CSF levels

Aluminum induces inflammatory and proteolytic alterations in human monocytic cell line

none4noThe increasing exposure to aluminum has been linked with the development of different human pathologies (e.g., breast cancer, myofasciitis, neurodegenerative diseases), probably due to the consistent presence of aluminum salts in widely diffused cosmetic products and vaccines. However, the mechanisms underlying immunologic and proliferative alterations still remain unknown. In the present study we investigated the ability of different aluminum compounds (i.e., aluminum chloride vs Imject® Alum, a mixture of aluminum and magnesium hydroxide) to trigger both inflammatory and proteolytic responses in U-937 human monocytic cell line. We demonstrated, by multiplex immunoassay analyses, that monocytic cells treated with both Imject Alum and aluminum chloride showed different and peculiar expression profiles of 27 inflammatory mediators and 5 matrix metalloproteinases, with respect to untreated control cells. In particular, we found dose-dependent significantly increased levels of pro-inflammatory cytokines, growth factors, and chemoattractant chemokines; whereas among metalloproteinases, only collagenolytic protease showed a significant dose-dependent increase in Imject-treated cells with respect to controls and Al-chloride treated cells. Noteworthy, we found only in Imject Alum-treated cells the significant positive correlations among collagenolytic metalloproteinase and increased expression of pro-inflammatory chemokines, suggesting a possible involvement of aluminum in regulating the acute inflammatory responses. In agreement to emerging evidences, for the first time we demonstrated that the treatment of monocyte cells with aluminum-based adjuvant is able to induce an inflammatory status and a proteolytic cascade activation. In fact, the cell treatment with Imject Alum induced increased levels of several cytokines and proteinases, suggesting these monocyte mediators as possible biomarkers for aluminum-linked diseases. The identification of the biochemical pathways involved in Al-induced cell injury pave the way for improving the knowledge on the potential impact of aluminum in human physio-pathology.openLigi, D; Santi, M; Croce, L; Mannello, FLigi, Daniela; Santi, Martina; Croce, L; Mannello, Ferdinand

Gender-dependent differences in plasma matrix metalloproteinase-8 elevated in pulmonary tuberculosis.

Tuberculosis (TB) remains a global health pandemic and greater understanding of underlying pathogenesis is required to develop novel therapeutic and diagnostic approaches. Matrix metalloproteinases (MMPs) are emerging as key effectors of tissue destruction in TB but have not been comprehensively studied in plasma, nor have gender differences been investigated. We measured the plasma concentrations of MMPs in a carefully characterised, prospectively recruited clinical cohort of 380 individuals. The collagenases, MMP-1 and MMP-8, were elevated in plasma of patients with pulmonary TB relative to healthy controls, and MMP-7 (matrilysin) and MMP-9 (gelatinase B) were also increased. MMP-8 was TB-specific (p<0.001), not being elevated in symptomatic controls (symptoms suspicious of TB but active disease excluded). Plasma MMP-8 concentrations inversely correlated with body mass index. Plasma MMP-8 concentration was 1.51-fold higher in males than females with TB (p<0.05) and this difference was not due to greater disease severity in men. Gender-specific analysis of MMPs demonstrated consistent increase in MMP-1 and -8 in TB, but MMP-8 was a better discriminator for TB in men. Plasma collagenases are elevated in pulmonary TB and differ between men and women. Gender must be considered in investigation of TB immunopathology and development of novel diagnostic markers

uPA is upregulated by high dose celecoxib in women at increased risk of developing breast cancer

<p>Abstract</p> <p>Background</p> <p>While increased urokinase-type plasminogen activator (uPA) expression in breast cancer tissue is directly associated with poor prognosis, recent evidence suggests that uPA overexpression may suppress tumor growth and prolong survival. Celecoxib has been shown to have antiangiogenic and antiproliferative properties. We sought to determine if uPA, PA inhibitor (PAI)-1 and prostaglandin (PG)E₂expression in nipple aspirate fluid (NAF) and uPA and PGE₂expression in plasma were altered by celecoxib dose and concentration in women at increased breast cancer risk.</p> <p>Methods</p> <p>NAF and plasma samples were collected in women at increased breast cancer risk before and 2 weeks after taking celecoxib 200 or 400 mg twice daily (bid). uPA, PAI-1 and PGE₂were measured before and after intervention.</p> <p>Results</p> <p>Celecoxib concentrations trended higher in women taking 400 mg (median 1025.0 ng/mL) compared to 200 mg bid (median 227.3 ng/mL), and in post- (534.6 ng/mL) compared to premenopausal (227.3 ng/mL) women. In postmenopausal women treated with the higher (400 mg bid) celecoxib dose, uPA concentrations increased, while PAI-1 and PGE₂decreased. In women taking the higher dose, both PAI-1 (r = -.97, p = .0048) and PGE₂(r = -.69, p = .019) in NAF and uPA in plasma (r = .45, p = .023) were correlated with celecoxib concentrations.</p> <p>Conclusion</p> <p>Celecoxib concentrations after treatment correlate inversely with the change in PAI-1 and PGE₂in the breast and directly with the change in uPA in the circulation. uPA upregulation, in concert with PAI-1 and PGE₂downregulation, may have a cancer preventive effect.</p

Effect of Sulindac Sulfide on Metallohydrolases in the Human Colon Cancer Cell Line HT-29

Matrix metalloproteinase 7 (MMP7), a metallohydrolase involved in the development of several cancers, is downregulated in the ApcMin/+ colon cancer mouse model following sulindac treatment. To determine whether this effect is relevant to the human condition, HT-29 human colon cancer cells were treated with sulindac and its metabolites, and compared to results obtained from in vivo mouse studies. The expression of MMP7 was monitored. The results demonstrated that sulindac sulfide effectively downregulated both MMP7 expression and activity. Furthermore, activity-based proteomics demonstrated that sulindac sulfide dramatically decreased the activity of leukotriene A4 hydrolase in HT-29 cells as reflected by a decrease in the level of its product, leukotriene B4. This study demonstrates that the effect of sulindac treatment in a mouse model of colon cancer may be relevant to the human counterpart and highlights the effect of sulindac treatment on metallohydrolases