Faecal Matrix Metalloprotease-9 Is a More Sensitive Marker For Diagnosing Pouchitis Than Faecal Calprotectin – Results From a Pilot Study

Background. Potential non-invasive markers of pouchitis would have a great deal of significance within clinical practice. This study is aimed at assessing the diagnostic accuracy of faecal calprotectin and matrix metalloprotease-9 as potential markers in patients both with and without pouchitis. Patients and methods. Stool and blood samples were collected from 33 ileal pouch-anal anastomosis patients before a follow-up pouchoscopy. Biopsy samples were taken for histological purposes. The presence of cuffitis and stenosis was evaluated with an endoscopy. Calprotectin and matrix metalloprotease-9 were quantified with an enzyme-linked immunosorbent assay. Results. Pouchitis was detected in 30.3% of the patients. The levels of faecal calprotectin and matrix metalloprotease-9 increased significantly in patients with pouchitis. The sensitivity and specificity of matrix metalloprotease-9 was higher than that of faecal calprotectin. Only matrix metalloprotease-9 correlated significantly with the severity of pouchitis. Conclusions. Faecal matrix metalloprotease-9 has a high specificity in the diagnosis of pouchitis

plasma matrix metalloprotease 9 correlates with blood lymphocytosis leukemic cell invasiveness and prognosis in b cell chronic lymphocytic leukemia

The complex biology underlying chronic lymphocytic leukemia cell migration and tissue invasiveness is not yet completely understood and might provide novel predictive markers and therapeutic targets. A total of 36 patients out of treatment from at least 3 months were enrolled and followed up for a median period of 44.2 months (range: 4.4-99.2). Matrix metalloprotease 9 and tissue inhibitor of metalloproteases 1 plasma levels and production/release from lymphoid cells were measured by zymography and enzyme-linked immunosorbent assay (ELISA) analysis. Malignant and normal lymphocyte mobility and matrix-degradation capability were studied using a Boyden chamber system, with and without autologous plasma. Free matrix metalloprotease 9 plasma levels were related with blood lymphocytosis, especially in more advanced stages (p = 0.003), and higher concentrations were associated with an increased disease progression risk (hazard ratio = 9.0, 95% confidence interval = 1.5-13.8). Leukemic cells expressed and secreted very little matrix metalloprotease 9. On the contrary, normal lymphocytes derived from the same leukemic patients showed matrix metalloprotease 9 intracellular levels that were lower in subjects with higher blood lymphocytosis (p = 0.024) and more advanced stages (p = 0.03); the released quantities were inversely associated with matrix metalloprotease 9 plasma concentrations (p = 0.035). Leukemic cells had a reduced spontaneous mobility and matrix-degradation capability that were stimulated by autologous plasma (p = 0.001) and normal lymphocytes (p = 0.005), respectively. Matrix metalloprotease 9 affected cell invasiveness depending on concentration and disease stage. In conclusion, chronic lymphocytic leukemia cells have a reduced mobility, matrix-degradation capability, and matrix metalloprotease 9 production compared to their own autologous normal lymphocytes. They are exposed to matrix metalloprotease 9 of prevalently systemic origin whose higher levels are associated with both leukemic and normal lymphocyte accumulation in the peripheral blood and have a negative prognostic value

Reviving the use of inhibitors of matrix metalloproteases in spinal cord injury:a case for specificity

At present, there are no restorative therapies in the clinic for spinal cord injury, with current treatments offering only palliative treatment options. The role of matrix metalloproteases is well established in spinal cord injury, however, translation into the clinical space was plagued by early designs of matrix metalloprotease inhibitors that lacked specificity and fears of musculoskeletal syndrome prevented their further development. Newer, much more specific matrix metalloprotease inhibitors have revived the possibility of using these inhibitors in the clinic since they are much more specific to their target matrix metalloproteases. Here, the evidence for use of matrix metalloproteases after spinal cord injury is reviewed and researchers are urged to overcome their old fears regarding matrix metalloprotease inhibition and possible side effects for the field to progress. Recently published work by us shows that inhibition of specific matrix metalloproteases after spinal cord injury holds promise since four key consequences of spinal cord injury could be alleviated by specific, next-generation matrix metalloprotease inhibitors. For example, specific inhibition of matrix metalloprotease-9 and matrix metalloprotease-12 within 24 hours after injury and for 3 days, alleviates spinal cord injury-induced edema, blood-spinal cord barrier breakdown, neuropathic pain and restores sensory and locomotor function. Attempts are now underway to translate this therapy into the clinic

Immunohistochemical Expression of Matrix Metalloprotease-2 and Matrix Metalloprotease-9 in the Disks of Patients with Temporomandibular Joint Dysfunction

Purpose Matrix metalloproteases (MMPs) are tissue-remodeling enzymes that function during the remodeling process, such as in immune-inflammatory diseases. Metalloprotease-2 (MMP-2) and metalloprotease-9 (MMP-9) are gelatinases that degrade several types of extracellular matrix collagen. It is hypothesized that in temporomandibular joint (TMJ) dysfunction, MMP-2 and MMP-9 expression levels may be elevated. Therefore, the objective of this study is to determine the association of MMP-2 and MMP-9 expression with temporomandibular joint dysfunction using an immunohistochemical approach to evaluate the joint disk. Material and Methods A total of 45 human temporomandibular joint samples were collected, with 36 samples in the test group (patients with anterior disk displacement with reduction (n = 29) and without reduction (n = 7)) and nine samples in the control group. The immunostaining of the TMJ disks was statistically compared between the groups (P \u3c 0.05). Results There was a statistically significant difference for the area of MMP-2 immunostaining between the control group and the displacement disks with reduction group (ADDwR) (P = 0.048) and between the groups with disk displacement and without reduction (ADDwoR) (P = 0.029). The expression of MMP-2 was significantly elevated in the ADDwoR group. Conclusion No statistically significant difference was found between the variable area of MMP-9 expression in the disk with and without disk displacement, as determined by immunohistochemical analysis. However, there was an elevation of MMP-2 expression in the disks of patients with displacement and without reduction (more severe alteration)

Wound-healing effect of Platycodon grandiflorus (Jacq.) extract in rats

Purpose: To evaluate the healing effect of Platycodon grandiflorus (Jacq.). extract (PGE) on experimental burn wounds in rats. Methods: Rats were randomly divided into four groups of eight rats each: control group, silver sulfadiazine (SSD)-treated group, moist exposed burn ointment (MEBO)-treated group and PGE-treated group. PGE, SSD and MEBO were applied topically twice daily for 7 days. SSD and MEBO were used as reference control. External observation of wound area contraction and histological analysis of wound tissues was performed respectively. The effect of PGE on matrix metalloproteases (MMPs), vascular endothelial growth factor (VEGF) and Type-III Collagen proteins of wound tissue in rats were analysed by Western blot. Results: After 10 days of topical treatment with PGE, PGE-treated group showed faster reduction in wound area when compared with control groups (p < 0.01). Matrix metalloprotease-2 (MMP-2), matrix metalloprotease-9 (MMP-9), VEGF and type-III collagen expressions in the wound tissue increased significantly (p < 0.05) when compared with the control burn wounds. Histological results showed an overall early recovery and regeneration in PGE-treated group when compared with control group. Conclusion: PGE possesses a significant wound-healing activity in full-thickness burn wounds in rats. Therefore, it can potentially be developed for the management of burns

Repurposing cancer drugs, batimastat and marimastat, to inhibit the activity of a group I metalloprotease from the venom of the Western Diamondback rattlesnake, Crotalus atrox

Snakebite envenomation causes over 140,000 deaths every year predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with an incredibly complex pathophysiology due to the vast number of unique toxins/proteins found in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a group I metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity was completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 M, while it is partially potentiated by calcium chloride. Molecular docking studies demonstrate that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites

Production and purification of recombinant C-terminal truncated pro-Matrix Metalloprotease-9

MMP-9 consists of N-terminal signal peptide, a pro-domain, catalytic domain, a long flexible hinge region and C- terminal hemopexin domain. In the present study a truncated version of proMMP-9 which lacking hinge region and C-terminal hemopexin domain was produced. To do so Sf9 insect cells and the BaculoDirect Baculovirus expression system were used. Using site directed mutagenesis, single mutation in the beginning of hinge region was introduced that resulted in a stop codon in the MMP-9 DNA. A recombinant Baculovirus was generated containing the Mutated MMP-9 DNA. By transfecting Sf9 cells with the recombinant Baculovirus genome a truncated version of MMP-9(proMMP-9ΔH-HPX) was produced. The proMMP-9ΔH-HPX was purified by Gelatin Sepharose chromatography and Size exclusion chromatography. Gelatin Zymography revealed the activity of proMMP-9ΔH-HPX. Western blot and Mass spectrometry confirmed the identity of proMMP-9ΔH-HPX. The purity of proMMP-9ΔH-HPX was established by SDS-PAGE and Coomassie stainin

MMP-9 cleaves SP-D and abrogates its innate immune functions in vitro

Possession of a properly functioning innate immune system in the lung is vital to prevent infections due to the ongoing exposure of the lung to pathogens. While mechanisms of pulmonary innate immunity have been well studied, our knowledge of how these systems are altered in disease states, leading to increased susceptibility to infections, is limited. One innate immune protein in the lung, the pulmonary collectin SP-D, has been shown to be important in innate immune defense, as well as clearance of allergens and apoptotic cells. MMP-9 is a protease with a wide variety of substrates, and has been found to be dysregulated in a myriad of lung diseases ranging from asthma to cystic fibrosis; in many of these conditions, there are decreased levels of SP-D. Our results indicate that MMP-9 is able to cleave SP-D in vitro and this cleavage leads to loss of its innate immune functions, including its abilities to aggregate bacteria and increase phagocytosis by mouse alveolar macrophages. However, MMP-9-cleaved SP-D was still detected in a solid-phase E. coli LPS-binding assay, while NE-cleaved SP-D was not. In addition, MMP-9 seems to cleave SP-D much more efficiently than NE at physiological levels of calcium. Previous studies have shown that in several diseases, including cystic fibrosis and asthma, patients have increased expression of MMP-9 in the lungs as well as decreased levels of intact SP-D. As patients suffering from many of the diseases in which MMP-9 is over-expressed can be more susceptible to pulmonary infections, it is possible that MMP-9 cleavage of SP-D may contribute to this phenotype

Matrix metalloprotease 9 and remodeling after myocardial infarction

The objective of the work was to study the distribution of metalloprotease 9 (MMP9) in tissues after myocardial infarction. We studied anatomopathological material from 30 patients, the primary victims of acute transmural myocardial infarction. Average age - 63,7 years. There were 18 men and 12 women. 16 patients died in the period up to 3 days, 14 patients - in a period from 3 days to 1 month. Samples were fixed in formalin and immunohistochemical staining for MMP9 was conducted. We used rabbit IgG monoclonal antibody to MMP9 (Epitomics, Clone 1D: EP1254, Cat. N 2551-1, Lot YG 113001P) in the working dilution of 1: 100 - 1: 250. At fatal outcomes that happened within a few hours after the onset of a heart attack a large number of MMP9 was detected in neutrophils in the blood vessels at near infarction zone. In the period from 1 to 2 hours MMP9 was detected in the extracellular matrix in the area of infarction. In the period from 3 to 30 days MMP9 was detected only in fibroclasts at forming cardiosclerosis zone. Thus, the use of staining for MMP9 is convenient for determination of the time elapsed after myocardial infarction in post mortem examination