Maria Cristina Manfrinato

Cerebrospinal fluid and serum levels and intrathecal production of active matrix metalloproteinase-9 (MMP-9) as markers of disease activity in patients with multiple sclerosis.

In this study, we employed a sensitive activity assay system to measure cerebrospinal fluid (CSF) and serum levels of active matrix metalloproteinase-9 (MMP-9) in 37 relapsing-remitting (RR), 15 secondary progressive (SP) and nine primary progressive (PP) multiple sclerosis (MS) patients, grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also studied, as neurological controls, 48 patients with other inflammatory neurological disorders (OIND) and 48 with non-inflammatory neurological disorders (NIND). To assess active MMP-9/TIMP-1 circuit, CSF and serum levels of MMP-9 tissue inhibitor TIMP-1 were quantified by ELISA in the same patient population. CSF mean levels of active MMP-9, CSF active MMP-9/TIMP-1 ratios and intrathecal active MMP-9 synthesis, as indicated by specific index, were more elevated in MS than in NIND (P <0.05, <0.02 and <0.02, respectively), serum active MMP-9/TIMP-1 ratio was higher in MS (P<0.01) and OIND (P<0.02) than in NIND, and serum TIMP-1 concentrations were lower in MS than in NIND (P<0.05). More importantly, serum active MMP-9 mean levels, serum active MMP-9/TIMP-1 ratio and intrathecal production of active MMP-9 were increased in MS patients with clinical (P<0.001, <0.001 and <0.05, respectively) and MRI (P<0.001, <0.001 and <0.02, respectively) disease activity, whereas CSF mean concentrations of active MMP-9 and CSF active MMP-9/TIMP-1 ratio were enhanced only in MS patients with MRI evidence of disease activity (P<0.02 and <0.01, respectively). Altogether, these findings suggest that a shift in MMP-9/TIMP-1 balance towards proteolytic activity of MMP-9 could be relevant in MS immune dysregulation. In addition, our results indicate that CSF and serum levels of active MMP-9 may represent a potential surrogate biomarker for monitoring MS disease activity. In particular, serum active MMP-9/TIMP-1 ratio seems to be a very appropriate indicator of ongoing MS inflammation, since it is easily measurable.

Matrix metalloproteinase-2 (MMP-2) generates soluble HLA-G1 by cell surface proteolytic shedding

Human leukocyte antigen-G (HLA-G) molecules are non-classical HLA class I antigens with an important role in pregnancy immune regulation and inflammation control. Soluble HLA-G proteins can be generated through two mechanisms: alternative splicing and proteolytic release, which is known to be metalloprotease mediated. Among this class of enzymes, matrix metalloproteinases (MMPs) might be involved in the HLA-G1 membrane cleavage. Of particular interest are MMP-2 and MMP-9, which regulate the inflammatory process by cytokine and chemokine modulation. We evaluated the effect of MMP-9 and MMP-2 on HLA-G1 membrane shedding. In particular, we analyzed the in vitro effect of these two gelatinases on the secretion of HLA-G1 via proteolytic cleavage in 221-G1-transfected cell line, in JEG3 cell line, and in peripheral blood mononuclear cells. The results obtained by both cell lines showed the role of MMP-2 in HLA-G1 shedding. On the contrary, MMP-9 was not involved in this process. In addition, we identified three possible highly specific cleavage sites for MMP-2, whereas none were detected for MMP-9. This study suggests an effective link between MMP-2 and HLA-G1 shedding, increasing our knowledge on the regulatory machinery beyond HLA-G regulation in physiological and pathological conditions.

Influence of Different Strategies of Volume Replacement on the Activity of Matrix Metalloproteinases: An in Vitro and in Vivo Study

Background:Excessive production of matrix metalloproteinase 9 (MMP-9) is linked to tissue damage and anastomotic leakage after large bowel surgery. Hence, the aim of this study was to verify whether different strategies of fluids administration can reduce MMP-9 expression. Methods:In the in vitro experiment, the authors tested the hypothesis of a direct inhibition of MMP-9 by the fluids used perioperatively, i.e., lactated Ringers solution, 3.4% poligeline, and hydroxyethyl starch 130/0.4. In the in vivo experiment, 36 patients undergoing surgery for colon cancer were randomly assigned to three groups to receive lactated Ringers solution, poligeline, or hydroxyethyl starch. MMP-9 and tissue inhibitor of metalloproteinases were measured from venous blood samples; the MMP-9/tissue inhibitor of metalloproteinases ratio was calculated as an index of equilibrium between the action of MMP-9 and its inhibition. Results:In the in vitro experiment, the presence of hydroxyethyl starch 130/0.4 in the MMP-9 assay system showed a strong inhibition of the enzymatic activity compared with lactated Ringers solution. In the in vivo experiment, MMP-9 and tissue inhibitor of metalloproteinases plasma levels did not differ among the three groups at baseline, whereas those levels increased significantly at the end of surgery. At that time, the MMP-9 plasma levels and the MMP-9/tissue inhibitor of metalloproteinases ratio were significantly higher in the lactated Ringers solution and poligeline groups than in the hydroxyethyl starch group. These results were confirmed 72 h after surgery. Conclusions:This study demonstrates that hydroxyethyl starch 130/04 decreases the circulating levels of MMP-9 in patients undergoing abdominal surgery.

Potential relevance of cerebrospinal fluid and serum levels and intrathecal synthesis of active matrix metalloproteinase-2 (MMP-2) as markers of disease remission in patients with multiple sclerosis.

Background Little is known about the involvement of matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor TIMP-2 in multiple sclerosis (MS). Objective To elucidate the actual implication of MMP-2 and TIMP-2 in MS. Methods Cerebrospinal fluid (CSF) and serum levels of active MMP-2 and TIMP-2 were measured by activity assay system and ELISA, respectively, in 67 patients with relapsing–remitting MS (RRMS), categorized according clinical and magnetic resonance imaging (MRI), and in 129 controls. Results Cerebrospinal fluid and serum active MMP-2/TIMP-2 ratio mean values and an intrathecal active MMP-2 production were more increased in RRMS than in non-inflammatory conditions (P < 0.001, P < 0.05, and P < 0.0001, respectively) and in MRI inactive than in MRI active RRMS (P < 0.02, P < 0.01 and P < 0.001, respectively). An intrathecal synthesis of active MMP-2 was more frequent in RRMS than in inflammatory disorders (P < 0.01). Serum active MMP-2/TIMP-2 ratio and MS disease duration were positively correlated (P < 0.02). Conclusion These findings suggest a potential role for MMP-2 activity in the termination of MS neuroinflammation related to remission of the disease and seem to indicate that serum MMP-2/TIMP-2 ratio may represent a useful biomarker for monitoring MS recovery phase.

Matrix metalloproteinase-9 activity detected in body fluids is the result of two different enzyme forms.

In vitro activation of matrix metalloproteinase-9 (MMP-9) (Gelatinase B) with MMP-3 shows the presence of two different forms: an 82 kDa, N-terminal truncated form, and a 65 kDa, N- and C-terminal truncated form. So far the presence of the 65 kDa form has not been reported in vivo. Affinity chromatography was performed to separate MMP-9 from MMP-2 and immunoprecipitation to isolate ∼65 kDa MMP-9 from 82 kDa MMP-9 in sera of healthy donors. The presence of ∼65 kDa active MMP-9 was demonstrated both with gelatin zymography and western blot analysis. The ∼65 kDa MMP-9 lacks the haemopexin domain required for the high-affinity binding of the tissue inhibitor TIMP-1, and can be evaluated by activity assay in the presence of TIMP-1. This opens the possibility to investigate the role of this form of MMP-9 that escapes physiological regulation.

TIMP-1 resistant matrix metalloproteinase-9 is the predominant serum active isoform associated with MRI activity in patients with multiple sclerosis

Background: The activity of matrix metalloproteinase-9 (MMP-9) depends on two isoforms, an 82 kDa active MMP-9 modulated by its specific tissue inhibitor (TIMP-1), and a 65 kDa TIMP-1 resistant active MMP-9. The relevance of these two enzymatic isoforms in multiple sclerosis (MS) is still unknown. Objective: To investigate the contribution of the TIMP-1 modulated and resistant active MMP-9 isoforms to MS pathogenesis. Methods: We measured the serum levels of the 82 kDa and TIMP-1 resistant active MMP-9 isoforms by activity assay systems in 86 relapsing–remitting MS (RRMS) patients, categorized according to clinical and magnetic resonance imaging (MRI) evidence of disease activity, and in 70 inflammatory (OIND) and 69 non-inflammatory (NIND) controls. Results: Serum levels of TIMP-1 resistant MMP-9 were more elevated in MS patients than in OIND and NIND (p < 0.05, p < 0.02, respectively). Conversely, 82 kDa active MMP-9 was higher in NIND than in the OIND and MS patients (p < 0.01 and p < 0.00001, respectively). MRI-active patients had higher levels of TIMP-1 resistant MMP-9 and 82 kDa active MMP-9, than did those with MRI inactive MS (p < 0.01 and p < 0.05, respectively). Conclusion: Our findings suggested that the TIMP-1 resistant MMP-9 seem to be the predominantly active isoform contributing to MS disease activity.

Interplay between Matrix Metalloproteinase-9, Matrix Metalloproteinase-2, and Interleukins in Multiple Sclerosis Patients

Matrix Metalloproteases (MMPs) and cytokines have been involved in the pathogenesis of multiple sclerosis (MS). However, no studies have still explored the possible associations between the two families of molecules. The present study aimed to evaluate the contribution of active MMP-9, active MMP-2, interleukin- (IL-) 17, IL-18, IL-23, and monocyte chemotactic proteins-3 to the pathogenesis of MS and the possible interconnections between MMPs and cytokines. The proteins were determined in the serum and cerebrospinal fluid (CSF) of 89 MS patients and 92 other neurological disorders (OND) controls. Serum active MMP-9 was increased in MS patients and OND controls compared to healthy subjects (p < 0.001 and p < 0.01, resp.), whereas active MMP-2 and ILs did not change. CSF MMP-9, but not MMP-2 or ILs, was selectively elevated in MS compared to OND (p < 0.01). Regarding the MMPs and cytokines intercorrelations, we found a significant association between CSF active MMP-2 and IL-18 (r = 0.3, p < 0.05), while MMP-9 did not show any associations with the cytokines examined. Collectively, our results suggest that active MMP-9, but not ILs, might be a surrogate marker for MS. In addition, interleukins and MMPs might synergistically cooperate in MS, indicating them as potential partners in the disease process.

Thermal stability of the hemagglutinin-neuraminidase from Sendai virus evidences two folding domains

The domain structure of hemagglutinin‐neuraminidase from Sendai virus (cHN) was investigated by studying the thermal stability in the 20–100°C range. Differential scanning calorimetry evidences two conformational transitions. The first transition is apparently a reversible two‐state process, with T m 48.3°C, and is shifted to 50.1°C in the presence of the substrate analogue 2,3‐dehydro‐2‐deoxy‐N‐acetyl neuraminic acid, meaning that the substrate binding domain is involved in the transition. The second transition, with apparent T m 53.2°C, is accompanied by irreversible loss of enzymatic activity of the protein, and the presence of the substrate analogue does not affect the T m. The data indicate that cHN is composed of two independent folding domains, and that only one domain is involved in the binding of the substrate. Our results suggest that the paramyxovirus neuraminidases have the folding properties of a two‐domain protein.

Vaginal Lactoferrin Modulates PGE2, MMP-9, MMP-2, and TIMP-1 Amniotic Fluid Concentrations

Inflammation plays an important role in pregnancy, and cytokine and matrix metalloproteases (MMPs) imbalance has been associated with premature rupture of membranes and increased risk of preterm delivery. Previous studies have demonstrated that lactoferrin (LF), an iron-binding protein with anti-inflammatory properties, is able to decrease amniotic fluid (AF) levels of IL-6. Therefore, we aimed to evaluate the effect of vaginal LF administration on amniotic fluid PGE2 level and MMP-TIMP system in women undergoing genetic amniocentesis. One hundred and eleven women were randomly divided into controls (n = 57) or treated with LF 4 hours before amniocentesis (n = 54). Amniotic fluid PGE2, active MMP-9 and MMP-2, and TIMP-1 and TIMP-2 concentrations were determined by commercially available assays and the values were normalized by AF creatinine concentration. PGE2, active MMP-9, and its inhibitor TIMP-1 were lower in LF-treated group than in controls (p < 0.01, p < 0.005, and p < 0.001, resp.). Conversely, active MMP-2 (p < 0.0001) and MMP-2/TIMP-2 molar ratio (p < 0.001) were increased, whilst TIMP-2 was unchanged. Our data suggest that LF administration is able to modulate the inflammatory response following amniocentesis, which may counteract cytokine and prostanoid imbalance that leads to abortion. This trial is registered with Clinical Trial number NCT02695563.

Influence of 6-aminonicotinamide (6AN) on Leishmania promastigotes evaluated by metabolomics: Beyond the pentose phosphate pathway

6-Aminonicotinamide (6AN) is an antimetabolite used to inhibit the NADPH-producing pentose phosphate pathway (PPP) in many cellular systems, making them more susceptible to oxidative stress. It is converted by a NAD(P)+ glycohydrolase to 6-aminoNAD and 6-aminoNADP, causing the accumulation of PPP intermediates, due to their inability to participate in redox reactions. Some parasites like Plasmodium falciparum and Coccidia are highly sensitive but not all cell types showed a strong responsiveness to 6AN, probably due to the different targeted pathway. For instance, in bacteria the main target is the Preiss-Handler salvage pathway for NAD+ biosynthesis. We were interested in testing 6AN on the kinetoplastid protozoan Leishmania as another model to clarify the mechanisms of action of 6AN, by using metabolomics. Leishmania promastigotes, the life-cycle stage residing in the sandfly, demonstrated a three order of magnitude higher EC50 (mM) compared to P. falciparum and mammalian cells (μM), although pre-treatment with 100 μM 6AN prior to sub-lethal oxidative challenge induced a supra-additive cell kill in L. infantum. By metabolomics, we did not detect 6ANAD/P suggesting that NAD+ glycohydrolases in Leishmania may not be highly efficient in catalysing transglycosidation as happens in other microorganisms. Contrariwise to the reported effect on 6AN-treated cancer cells, we did not detect 6-phosphogluconate (6 PG) accumulation, indicating that 6ANADP cannot bind with high affinity to the PPP enzyme 6 PG dehydrogenase. By contrast, 6AN caused a profound phosphoribosylpyrophosphate (PRPP) decrease and nucleobases accumulation confirming that PPP is somehow affected. More importantly, we found a decrease in nicotinate production, evidencing the interference with the Preiss-Handler salvage pathway for NAD+ biosynthesis, most probably by inhibiting the reaction catalysed by nicotinamidase. Therefore, our combined data from Leishmania strains, though confirming the interference with PPP, also showed that 6AN impairs the Preiss-Handler pathway, underlining the importance to develop compounds targeting this last route.