Análisis de MMPs en fluidos orales en el diagnóstico complementario de las enfermedades periodontales / Oral-fluid MMP analysis in the complementary diagnosis of periodontal diseases

La periodontitis constituye la infección bacteriana más prevalente a nivel mundial y representa un factor de riesgo para diversas patologías sistémicas. El estado de inflamación y destrucción periodontal se manifiestan a través de la presencia de biomarcadores en el suero y fluidos orales, tales como el fluido gingival crevicular (FGC), saliva y enjuague oral. Enzimas como las metaloproteinasas de matriz (MMP) y mieloperoxidasa, constituyen biomarcadores potenciales para ensayos moleculares complementarios a la clínica de uso en el sillón dental. A continuación se presenta una revisión de la literatura respecto de la aplicación potencial del análisis de metaloproteinasas de matriz extracelular (MMPs) en el diagnóstico complementario de las enfermedades periodontales. Se ha demostrado que los niveles de MMP-9, -13 y particularmente de MMP-8, se asocian con el grado de inflamación periodontal, y pueden diferenciar entre sujetos sanos, con gingivitis, periodontitis y peri-implantitis, mientras que la mejoría de los parámetros clínicos en respuesta al tratamiento periodontal se asocia con la reducción de la activación y niveles de estas enzimas en FGC, como así también en el suero. Se concluye que la determinación, particularmente de MMP-8 en fluidos orales presenta un elevado potencial como complemento de los métodos clínicos tradicionales para identificar a los pacientes con periodontitis o en riesgo de desarrollar la enfermedad, monitorear fases del tratamiento y mejoría de signos periodontales e incluso evaluar el estado de inflamación sistémica.

Periodontal disease is the most common bacterial infection worldwide and it can contribute to enhance the risk for the development of several systemic diseases. The status of periodontal inflammation and destruction can be reflected in biomarker measurement in serum and oral fluids, like gingival crevicular fluid (GCF), saliva and mouth-rinse. Some enzymes, such as matrix metalloproteinases (MMPs) and myeloperoxidase are potential candidates for chair-side point-of-care oral fluid assays. This review is focused on the utility of matrix metalloproteinase (MMP) analysis in oral fluid as a complementary diagnostic method to chronic periodontitis. Levels of MMP-9,-13 and specially of MMP-8, reflect oral inflammatory status and discriminate among healthy, gingivitis, periodontitis and periimplantitis individuals, whereas MMP levels and activation in GCF and serum are in line with the improvement of clinical parameters in response to periodontal treatment. As a conclusion, MMP-8 assessment in GCF could represent a helpful adjunctive method to traditional diagnostics to identify periodontitis or patients at risk to develop the disease, monitor treatment phases, improvement of periodontal signs and even screen the systemic inflammation status.

[Investigation of gelatinase- B enzyme role in invasion and metastasis of breast cancer]

Gelatinase B or collagenase type IV is a 92 kDa protein. In case of over-expression of the gene, because of its collagenase activity, it can be involved in metastasis activity of few cancers e.g. bladder, colorectal and gastric carcinoma. Single nucleotide substitution base polymorphism of C to T at -1562 of promotor region can increase gene expression by decreasing transcription inhibitor proteins binding at T alleles. The aim of this case-control study is to investigate the role of this polymorphism in development and invasion of breast cancer in Isfahan women population. At this study 90 breast cancer patients with metastasis and 100 healthy controls were analyzed using polymerase chain reaction-restriction fragment length polymorphism assay [PCR-RFLP]. The mean of follow up period was 2 years. Patients were checked every 3-5 months Data analysis showed a close association between the presence of T allele and invasion of breast cancer [OR = 5.85, 95% CI: 2.64-12.93, p <0.0001]. According to our findings, the major role of this polymorphism is in cancer cell metastasis and invasion of these cells to adjacent tissues

Collagenase specificity in chondrosarcoma metastasis

The treatment of some mesenchymal malignancies has made significant gains over the past few decades with the development of effective systemic therapies. In contrast, the treatment of chondrosarcoma has been limited to surgical resection, with the most significant prognostic indicators being surgical margins and histologic grade. We have reported that MMP-1/TIMP-1 gene expression serves to prognosticate for tumor recurrence in this group of patients. This led to the hypothesis that collagenase activity facilitates cell egression from the cartilaginous matrix. In the current study we examine the specificity of collagenase gene expression in archival human chondrosarcoma samples using semi-quantitative PCR. Messenger RNA was affinity extracted and subject to reverse transcription. The subsequent cDNA was amplified using novel primers and quantitated by densitometry. Ratios of gene expression were constructed and compared to disease-free survival. The data demonstrate that the significance of the MMP-1/TIMP-1 ratio as a predictor of recurrence is confirmed with a larger number of patients. Neutrophil collagenase or MMP-8 was observed in only 5 of 29 samples. Collagenase-3 or MMP-13 was observed in all samples but the level did not correlate with disease-free survival. Since the collagenases have similar activity for fibrillar collagens and cleave the peptide in the same location, post-transcriptional regulatory mechanisms may account for the observed specificity. The determination of the MMP-1/TIMP-1 gene expression ratio not only serves to identify those patients at risk for recurrence but may also serve as a novel therapeutic avenue as an adjunct to surgical resection

Role of tissue inhibitors of metalloproteinases (TIMPs) in colorectal carcinoma

Increased production of matrix metalloproteinases (MMPs) has been associated with increases in invasive and metastatic potential in many types of human carcinoma. Tissue inhibitors of metalloproteinase (TIMP)-1 inhibits most interstitial collagenases and MMP-9. TIMP-2 binds specifically and noncovalently to the pro-form of MMP-2 and inhibits its enzyme activity. In this study, we examined TIMP-1 and TIMP-2 expressions in relation to clinicopathological variables in colorectal carcinoma with in situ hybridization and immunohistochemistry. TIMP-1 and TIMP-2 expressions were localized overwhelmingly to pericancer stromal cells, while malignant and normal mucosal cells were weak or negative. Strong stromal TIMP-1 immunoreactivity correlated with Dukes' stage (p=0.022), status of lymph node metastasis (p=0.044) and poor survival (p= 0.005). The degree of immunohistochemical staining of TIMP-2 did not correlate with all clinicopathological variables. The correlation between enhanced TIMP-1 expression and advanced stage and poor survival suggest a growth promoting activity of TIMP-1 in colorectal carcinoma.

Assay of matrix metalloproteinases in substrate impregnated gels in multiwells.

A zymographic method for the assay of matrix metalloproteinases in substrate impregnated gels in multiwells has been developed for the analysis of a large number of samples at a time. Enzyme was copolymerized with 300 microliters of 10% acrylamide impregnated with gelatin substrate and incubated for 16 hr. The gels were stained with coomassie blue, destained with water and the intensity measured in a densitometer. This method was tested with pure bacterial collagenase and three different gelatinases purified from rat mammary gland. The characteristics of these enzymes such as cation dependence, inhibition and concentration dependence have been examined by this method.

Expresion de MMP-2 (colagenasa tipo IV) en el carcinoma gastrico / MMP-2 expression (type IV collagenase) in gastric cancer

La MMP-2 (colagenosa tipo IV) es una proteína que pertence a la familia de las metaloproteinas, cuya función está relacionada con la degradación de la matriz extracelular. Su capacidad para degradar el colágeno IV de las membranas basales podría transformala en un agente facilitador de la diseminación neoplásica. Para ver su relación con algunas características clínico-patológicas e inmunohistoquímicas del cáncer gástrico se estudiaron 98 casos de adenocarcinoma de estómago determinado por inmunohistoquímica la presencia de MMP-2 en las células neoplásicas. Los resultados mostraron que había correlación entre la presencia de MMP-2 con el nivel de invasión parietal del tumor (p=0.03) y con la presencia de metástasis en ganglios regionales (p=0.05). En cambio no hubo asociación entre la expresión de MMP-2 con la frequencia por sexo, la localización dentro del estómago, el tamaño tumoral, el tipo histológico, el grado histológico, ni la expresión de las proteínas MIB-1, bcl-2, c-erbB-2 y p53. Tampoco se relacionó con la presencia de recidiva de la enfermedad ni con la sobrevida a los 5 años.