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.

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.

Balanced and unbalanced solutions modulate the release of Matrix Metalloproteinase-9 (MMP-9) from neutrophils in response to inflammatory stimuli: an in vitro study.

OBJECTIVES AND DESIGN: We investigated the effect of balanced (BS) and unbalanced (UBS) solutions in the absence or presence of hydroxyethyl starch (HES) on neutrophil functionality, evaluating the release of matrix metalloproteinase (MMP)-9, myeloperoxidase (MPO), and MMP-8. MATERIALS AND METHODS: Neutrophils were isolated by gradient centrifugation and dextran sedimentation and incubated in BS or UBS without or with HES, in the absence or presence of Interleukin-8 (IL-8) or Lipopolysaccharide (LPS). MMP-9, MPO, and MMP-8 were assayed by commercially available ELISA kits. RESULTS: There was not any influence of volume replacement solutions on the release of the enzymes from resting neutrophils. After IL-8 stimulation, the release of MMP-9 was higher in BS than in UBS or RPMI-1640, whereas HES enhanced its release regardless of the composition. After LPS stimulation, the release of MMP-9 was higher in both UBS and BS than RPMI-1640, but HES brought its release back to physiological conditions. No difference was found in the release of MPO and MMP-8 after stimulation with IL-8 or LPS. CONCLUSION: Volume replacement solutions might have an impact on the release of MMP-9 depending on the inflammatory milieu, suggesting that the use of balanced or unbalanced solutions is not a neutral choice.

Sex and Gender in Ageing and Longevity: Highlights From an International Course.

Gender medicine is a multidisciplinary science and represents an important perspective for pathophysiological and clinical studies in the third millennium. Here, it is provided an overview of the topics discussed in a recent course on the Role of Sex and Gender in Ageing and Longevity. The paper highlights three themes discussed in the course, i.e., the interaction of gender/sex with, i) the pathophysiology of age-related diseases; ii), the role of genetics and epigenetics in ageing and longevity and, iii) the immune responses of older people to pathogens, vaccines, autoantigens, and allergens. Although largely unexplored, it is clear that sex and gender are modulators of disease biology and treatment outcomes. It is becoming evident that men and women should no longer be considered as subgroups, but as biologically distinct groups of patients deserving consideration for specific therapeutic approaches.

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.

Fast skeletal troponin I, but not the slow isoform, is increased in patients under statin therapy: a pilot study.

INTRODUCTION: Statin therapy is often associated with muscle complaints and increased serum creatine kinase (CK). However, although essential in determining muscle damage, this marker is not specific for skeletal muscle. Recent studies on animal models have shown that slow and fast isoforms of skeletal troponin I (ssTnI and fsTnI, respectively) can be useful markers of skeletal muscle injury. The aim of this study was to evaluate the utility of ssTnI and fsTnI as markers to monitor the statin-induced skeletal muscle damage. MATERIALS AND METHODS: A total of 51 patients (14 using and 37 not using statins) admitted to the intensive care unit of the University of Ferrara Academic Hospital were included in this observational study. Serum activities of CK, aldolase, alanine aminotransferase and myoglobin were determined by spectrophotometric assays or routine laboratory analysis. Isoforms ssTnI and fsTnI were determined by commercially available ELISAs. The creatine kinase MB isoform (CK-MB) and cardiac troponin I (cTnI) were evaluated as biomarkers of cardiac muscle damage by automatic analysers. RESULTS: Among the non-specific markers, only CK was significantly higher in statin users (P = 0.027). Isoform fsTnI, but not ssTnI, was specifically increased in those patients using statins (P = 0.009) evidencing the major susceptibility of fast-twitch fibres towards statins. Sub-clinical increase in fsTnI, but not CK, was more frequent in statin users (P = 0.007). Cardiac markers were not significantly altered by statins confirming the selectivity of the effect on skeletal muscle. CONCLUSIONS: Serum fsTnI could be a good marker for monitoring statin-associated muscular damage outperforming traditional markers.

Effects of anticoagulants on the activity of gelatinases.

OBJECTIVES: To identify the best procedure for preanalytical blood collection in the determination of matrix metalloproteinase (MMP)-2 and -9 by testing the effects of anticoagulants on their activity. DESIGN AND METHODS: Active forms of both gelatinases were measured by specific activity assay systems in serum, plasma EDTA, plasma-heparin and plasma-citrate obtained from 20 healthy volunteers, as well as in a pooled serum sample before and after anticoagulant treatment. RESULTS: : Active MMP-2 and MMP-9 mean concentrations were similar in serum and in plasma-citrate, higher in plasma EDTA than in serum, in plasma-heparin and in plasma-citrate, and lower in plasma-heparin than in serum and plasma-citrate. A similar trend was observed in untreated and treated pooled serum samples. CONCLUSIONS: Our results indicate that MMP-2 and MMP-9 in their active forms are not released by platelets during blood clotting, whereas the use of calcium chelating anticoagulants can profoundly alter the activity of endogenous gelatinases. This suggests that the determination of active forms of MMP-2 and MMP-9 in serum samples represents a suitable procedure.

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.

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 Ringer's 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 Ringer's 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 Ringer's 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 Ringer's 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.