Obesity decreases the oxidant stress induced by tobacco smoke in a rat model.

Obesity and emphysema are associated with low-grade systemic inflammation and oxidant stress. Assuming that the oxidant stress induced by emphysema would be decreased by obesity, we analyzed the oxidant/antioxidant state in a rat model combining both diseases simultaneously. Obesity was induced using sucrose, while emphysema by exposure to tobacco smoke. End-points evaluated were: body weight, abdominal fat, plasma dyslipidemia and malondialdehyde (MDA), insulin and glucose AUC, activities of Mn-superoxide dismutase (Mn-SOD), glutathione reductase (GR), glutathione transferase (GST) and glutathione peroxidase (GPx); lung MnSOD and 3-nitrotyrosine (3-NT) immunostaining, and expression of αV and ß6 integrin subunits. In rats with obesity, the body weight, abdominal fat, plasma triglyceride levels, glucose AUC, insulin levels, GST activity, and αV and ß6 integrin expressions were amplified. The rats with emphysema had lower values of body weight, abdominal fat, plasma insulin, triglycerides and glucose AUC but higher values of plasma MDA, GPx activity, and the lung expression of the αV and ß6 integrins. The combination of obesity and emphysema compared to either condition alone led to diminished body weight, abdominal fat, plasma insulin MDA levels, GPx and GST activities, and αV and ß6 integrin expressions; these parameters were all previously increased by obesity. Immunostaining for MnSOD augmented in all experimental groups, but the staining for 3-NT only increased in rats treated with tobacco alone or combined with sucrose. Results showed that obesity reduces oxidant stress and integrin expression, increasing antioxidant enzyme activities; these changes seem to partly contribute to a protective mechanism of obesity against emphysema development.

Oxidative stress and lung injury induced by short-term exposure to wood smoke in guinea pigs.

Oxidative stress and lung injury induced by short-term exposure to wood smoke were evaluated in guinea pigs through cell profile, bronchoalveolar lavage (BAL), conventional histology and immunohistochemistry (4-hydroxynonenal, 3-nitrotyrosine, Mn-superoxide dismutase, heme oxygenase-1); malondialdehyde and 4-hydroxynonenal concentration, Mn-superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase activities in plasma, lung and BAL. Total cells increased in BAL, and the percentage of macrophages, neutrophils and lymphocytes augmented (72-96 h). Histopathological examination of lung tissues showed mild thickening of membranous bronchiole walls, infiltration of foamy macrophages and polymorphonuclear leukocytes in bronchial, bronchiolar and intraalveolar spaces. Goblet cell hyperplasia was also observed in bronchial and bronchiolar epithelia. Plasma malondialdehyde concentration was increased at all times, while 4-hydroxynonenal was increased only in plasma and BAL after 24 h. Plasma glutathione reductase activity increased at 24 and 72 h, BAL glutathione peroxidase activity decreased at 72 and 96 h, whereas catalase activity increased in plasma at 72 h, and decreased in BAL at 24 h. Immunostaining intensity to 4-hydroxynonenal, 3-nitrotyrosine, Mn-superoxide dismutase and heme oxygenase-1 was enhanced mainly in macrophages, bronchial/bronchiolar epithelial cells and type II pneumocytes after 72-96 h of wood smoke exposure. Overall, short-term exposure to wood smoke induces alterations in oxidative/antioxidant state in lung and airway injury, similar to those observed in humans with domestic exposure.

[Factors that affect the dwell time of a central venous catheter]. / Factores que inciden sobre el tiempo de permanencia de un catéter endovenoso central.

OBJECTIVE: To determine the factors that affect the dwell time of a central venous catheter. BACKGROUND: The own immunodeficiency in cancer patient and the collateral effects of their treatment increase the probability of infection and reduce the time of use of a catheter. SUBJECTS: Incipient cohort of 306 patients with cancer, with an average age of 59 +/- 14.5 years. INTERVENTIONS: Time zero like the day of the placement of the catheter was defined. Daily clinical signs of infection by catheter looked for, with taking of simultaneous blood cultures every 7 days. The primary outcome was infection of the catheter and the alternative aim of treatment, obstruction or death of the patient. One compared the dwell time with the infected catheter versus non-infected and its relation with potential risk factors. RESULTS: 306 catheters were placed for a total of 4,043 to days/catheter, became infected 25 (8.2%). The global average survival was of 50 days. One was that to greater dwell time, greater it was the infection incidence. In the fit analysis of survival, the presence of remote infection (OR = 4.71, IC 95% = 1.7-10.1, p = 0.002) was the factor that showed a significant association. CONCLUSIONS: The time of life utility of a catheter is ample, limited by the presence of remote infection like factor of potential risk of infection by catheter.

[Simultaneous haemocultures and diagnosis of catheter-related sepsis]. / Hemocultivos simultáneos y diagnóstico de sepsis relacionada a catéter.

OBJECTIVE: To determine the diagnosis value of simultaneous blood cultures. BACKGROUND: The clinical criteria is not enough to establish catheter-related sepsis diagnosis and remotion or replacement of catheter are not necessary. SUBJECTS: We evaluated 164 catheters used for total parenteral nutrition placement in 127 patients with cancer. Simultaneous blood samples--central and peripheral venous--were taken after seven at ten days of permanency or infection suspicion and compared with tip culture result. INTERVENTIONS: Sensitivity, specificity and predictive values were calculated. The tip culture was the gold standard. RESULTS: A positive central venous blood culture with negative peripheral blood culture result was sensitivity of 87.5% and specificity of 97.9%, an inverse result show a sensitivity of 62.5%. When both blood cultures were positive are correctly identified 83.3% of cases with a sensitivity of 93.9% and diagnostic correlation of 0.87. CONCLUSIONS: The simultaneous blood cultures are high diagnostic value.

Hemocultivos simultáneos y diagnóstico de sepsis relacionada a catéter / Simultaneous haemocultures and diagnosis of catheter-related sepsis

Objetivo: determinar el valor diagnóstico de hemocultivos simultáneos. Antecedentes: El criterio clínico no es suficiente para establecer el diagnóstico de infección relacionada a catéter y en retiro por sospecha o el recambio rutinario no son prácticas recomendadas. Sujetos: Se evalúan 164 catéteres empleados para nutrición endovenosa colocados en 127 pacientes con cáncer, a quienes se les tomó una muestra simultánea desangre a través del catéter y una vena periférica cuando existía sospecha de infección o bien cada 7 a 10 días de permanencia del catéter. Intervenciones: Se calculó la sensibilidad, especificidad y valores predictivos de los hemocultivos como método diagnóstico. El cultivo de punta constituyó el estándar de oro. Resultados: En presencia de un hemocultivo transcatéter positivo con hemocultivo negativo se identificó una sensibilidad de 87,5% y una especificidad de 97,9%, en cambio la presencia de un hemocultivo transcatéter negativo y periférico positivo mostró una sensibilidad de 62,5%. Cuando ambos hemocultivos fueron positivos se identificó correctamente la infección en el 83,3% de los casos con una sensibilidad de 93,7% y una especificidad de 93,9%. La correlación diagnóstica obtenida para ambos hemocultivos positivos fue de 0,87 y de 0,83 cuando el hemocultivo transcatéter fue positivo y el periférico negativo. Conclusiones: La toma de hemocultivos simultáneos tiene un alto valor diagnóstico (AU)

Objective: To determine the diagnosis value of simultaneous blood cultures. Background: The clinical criteria is not enough to establish catheter-related sepsis diagnosis and emotion or replacement of catheter are not necessary. Subjects: We evaluated 164 catheters used for total parenteral nutrition placement in 127 patients with cancer. Simultaneous blood samples —central and peripheral venous— were taken after seven at ten days of permanency or infection suspicion and compared with tip culture result. Interventions: Sensitivity, specificity and predictive values were calculated. The tip culture was the gold standard. Results: A positive central venous blood culture with negative peripheral blood culture result was sensitivity of 87.5% and specificity of 97.9%, an inverse result show a sensitivity of 62.5%. When both blood cultures were positive are correctly identified 83.3% of cases with a sensitivity of 93.9% and diagnostic correlation of 0.87. Conclusions: The simultaneous blood cultures are high diagnostic value (AU)

[Advantages of routine periodic replacement of the central endovenous catheter in total parenteral nutrition]. / Utilidad del recambio periódico rutinario de un catéter endovenoso central para nutrición parenteral total.

OBJECTIVE: To determine the utility of rutinary periodic replacement catheter of total parenteral nutrition (TPN) to prevent the incidence of catheter-related infection. MATERIAL AND METHODS: Cancer patients admitted between Jan. 1, 1996 and Dec. 31, 1997, to receive TNP, were eligible for enrollment in the study. Placed a chateter of poliuretane with double lumen each patient for Nutrition Department. Replacement catheters done over a guidewire after ten days, with or without infection sings. Rates of catheter-related infection were documented for de novo, and guidewire exchanged catheters and analyzed relative risk for infection. RESULTS: Twenty catheter-related infections developed in 140 patients. When the rate of catheter-associated infection was determined for each type of catheterization, the replacement catheter had a high observed rate of infection than either de novo type (19.5% vs 7.8%; p = 0.027). We found that the risk of developing a catheter-related infection in a de novo catheter was significantly less (p < 0.01).

Human leukocyte antigen-associated susceptibility to pulmonary tuberculosis: molecular analysis of class II alleles by DNA amplification and oligonucleotide hybridization in Mexican patients.

BACKGROUND: Pulmonary tuberculosis (PTB) develops by a complex combination of environmental factors with genetic susceptibility. In this context, an association between human leukocyte antigens (HLAs) and tuberculosis has been examined in several populations, but results have been controversial. DESIGN AND MEASUREMENTS: A prospective evaluation of class II HLA genotypes was completed by the polymerase chain reaction (PCR) sequence-specific primer technique and PCR sequence-specific oligonucleotide hybridization in a Mexican population. SETTING: This study was conducted at the Clinical Service of Tuberculosis and the Department of Immunology, National Institute of Respiratory Diseases, Mexico City, Mexico. PATIENTS: Four groups were examined: 95 healthy subjects; 50 nonimmunosuppressed PTB patients; 15 HIV-infected patients (stage IVc in the Centers for Disease Control and Prevention [CDC] classification system for AIDS) with PTB; and 37 HIV-infected patients in the asymptomatic stage (CDC stage II). RESULTS: The frequencies of alleles DQA1*0101 (odds ratio [OR], 6.18; 95% confidence interval [CI], 2.38 to 16.08), DQB1*0501 (OR, 6.16; 95% CI, 2.44 to 17.71), and DRB1*1501 (OR, 7.92; 95% CI, 2.71 to 23.14) were significantly increased in nonimmunosuppressed patients with PTB when compared with healthy subjects. By contrast, frequencies of allele DQB1*0402 and antigens DR4 and DR8 were significantly decreased in patients with PTB. Additionally, a significantly higher frequency of the DRB1*1101 allele was found in HIV-positive subjects (OR, 6.67; 95% CI, 2.13 to 20.83). CONCLUSION: The genetic influence associated with the HLA system appears to have an important role in the development of PTB, although this susceptibility may not be relevant in patients with severe immunodeficiency diseases such as AIDS.

Changes in matrix metalloproteinases during the evolution of interstitial renal fibrosis in a rat experimental model.

The aim of the present study was to analyze the matrix metalloproteinase (MMP) activity during the evolution of interstitial renal fibrosis in a rat experimental model of unilateral ureteral obstruction. The interstitial type I collagenase and the gelatinolytic activities were analyzed by radiolabeled substrate degradation. Interstitial collagenase activity was low at all times while gelatinolytic activity increased on day 6 of evolution, with a decrease in activity from this point. The use of organomercurials revealed the presence of latent enzyme in all cases. Normal kidney samples contained MMP-9 in both active and proenzyme forms as revealed by zymography. On day 3 MMP-9 dimers appeared, and increased activity was observed until day 6. A decrease in the gelatinolytic activity was detected from days 9-15 of evolution. This observation was confirmed by Western blot analysis that revealed the presence of proMMP-9 mainly from days 6-12. Tissue inhibitor of metalloproteinase-1 (TIMP-1) was also detected alone and in combination with proMMP-1 and MMP-1, particularly from days 6-15 of evolution. The presence of MMP-9 and MMP-1 was detected in the cytoplasm of cortical tubular cells by immunohistochemistry, with no difference between the experimental and the normal kidneys. There was also an increase in collagen concentration from day 3 after surgery that increased during the entire evolution of the experimental model. This work reveals that the decrease in the MMP-9 and MMP-1 enzymatic activity, due to their interaction with TIMP-1 and to the lack of activation of the latent forms, may participate in the excessive collagen deposit during the evolution of experimental interstitial renal fibrosis.

72-kD (MMP-2) and 92-kD (MMP-9) type IV collagenase production and activity in different histologic types of lung cancer cells.

In this study we examined the production of gelatinases A and B (MMP-2 and MMP-9), and their natural inhibitors TIMP-1 and TIMP-2 in cell lines derived from different histologic types of lung cancer. Gelatinolytic activity was measured by zymography and radiolabeled gelatin degradation. Immunocytochemistry and Western blot analysis were performed to corroborate the presence of immunoreactive MMP-2, MMP-9, TIMP-1 and TIMP-2 proteins. The highest gelatinolytic activity was identified in the cell extracts from a small-cell carcinoma cell line. MMP-9 was observed in all samples as a proenzyme, while MMP-2 was present as zymogen in the squamous-cell and in the small-cell carcinomas, and in its active form in one squamous-cell carcinoma cell line. TIMPs were also present in the neoplastic lung cell lines. TIMP-1 was observed in the media of all cells as a 21-kD band, and as TIMP-1 polymers with the exception of the small-cell carcinoma samples. TIMP-2 was found as higher-order molecular immunoreactive complexes that may correspond to proMMP-2/TIMP-2 complexes. These results demonstrate that lung neoplastic cells produce both MMP-2 and MMP-9 and their inhibitors, with the small-cell carcinoma cell extracts showing the highest enzymatic activity. This gelatinolytic activity fits well with the clinical metastatic behavior of this type of lung cancer.

Increased matrix metalloproteinase activity and reduced tissue inhibitor of metalloproteinases-1 levels in amniotic fluids from pregnancies complicated by premature rupture of membranes.

OBJECTIVES: It has been suggested that increased matrix metalloproteinases activity promotes the weakening of the amniochorion during normal and premature rupture of membranes. This study was designed to determine whether levels of matrix metalloproteinases and the tissue inhibitor of metalloproteinases-1 in amniotic fluid change in a pattern consistent with this hypothesis. STUDY DESIGN: Gelatinolytic activity, measured by a soluble substrate assay and zymography, and the concentrations of tissue inhibitor of metalloproteinases-1 were estimated in amniotic fluid obtained from (1) normal early gestations, (2) normal term pregnancies with labor, (3) normal term pregnancies without labor, and (4) pregnancies complicated by premature rupture of membranes. The 92 kd type IV collagenase (matrix metalloproteinase-9) was also detected in amniotic fluid by Western blotting. RESULTS: Matrix metalloproteinase activities were higher in amniotic fluid from normal term pregnancies with labor and pregnancies complicated by premature rupture of membranes than from early pregnancies and term gestations without labor. The amniotic fluid from term pregnancies with labor or pregnancies with premature rupture of membranes contained several gelatinases, as revealed by zymography. The major amniotic fluid gelatinolytic activity in premature rupture of membranes and term pregnancies with labor corresponded to matrix metalloproteinase-9. Tissue inhibitor of metalloproteinases-1 concentrations were highest in early-pregnancy amniotic fluid, followed by term gestation with labor, term gestation without labor, and premature rupture of membranes. CONCLUSIONS: Normal labor and premature rupture of membranes are associated with increased levels of matrix metalloproteinases, particularly matrix metalloproteinase-9 in amniotic fluid. Premature rupture of membranes is associated with reduced levels of tissue inhibitor of metalloproteinases-1. The imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 in amniotic fluid may reflect a disorder that promotes premature rupture of membranes.

[The incidence of the refeeding syndrome in cancer patients who receive artificial nutritional treatment]. / Incidencia de síndrome de realimentación en enfermos con cáncer que reciben tratamiento nutricio artificial.

We determine the incidence of the malnutrition syndrome and its relation with probable risk factors, in 106 patients, with a mean age of 53.3 +/- 15.4 years, with a diagnosis of cancer confirmed histologically, and who received artificial nutrition either enterally or endovenously, during an average of 16.8 +/- 2 days, which included a supply of 60 to 100 mmol/day of phosphorus. We considered there to be a renutrition syndrome when there as hypophosphatemia; < 2.5 mmol/l, which took place during the nutritional treatment phase, and previous to which, the patients had normal serum levels of phosphorus. The serum electrolyte concentrations were measured prior to the start of the treatment, and daily during the first week, and later every 3 days until the end. The study variables were: age, sex, type of cancer, degree of malnutrition, degree of hypophosphatemia, day on which in occurred, and clinical manifestations associated to this. The relative risk was calculated for the variables of age, sex, malnutrition and cancer. The incidence of the renutrition syndrome was 24.5%; it was more frequent in the enteral group than in the endovenous group (37.5% vs. 18.9%, p < 0.005); and it took place 72 hours after starting the nutritional support, in 61.5% of the cases, with a mean phosphorus concentrations of 1.9 mmol/l; the most frequent clinical manifestations were the neuromuscular ones (30%), and the most frequent type of cancer was lymphoma (15.4%). The risk factors were age greater than 60 years (RR = 1.7), and moderate or severe malnutrition 8RR = 2.0). We conclude that the prevalence of the renutrition syndrome is high in the cancer patients, despite an intense preventive treatment with phosphorus.

Collagen metabolism in human aqueous humor from primary open-angle glaucoma. Decreased degradation and increased biosynthesis play a role in its pathogenesis.

OBJECTIVE: To explore the influence of aqueous humor from patients with glaucoma on collagen turnover. METHODS: The aqueous humor of patients with primary open-angle glaucoma (POAG), chronic angle-closure glaucoma, congenital glaucoma, neovascular glaucoma, and senile cataract (control group) was analyzed for its capacity to induce fibroblast proliferation, collagen synthesis, collagenolytic activity, and production of tissue inhibitor of metalloproteinase-1. All data were analyzed using the Mann-Whitney U test. RESULTS: Aqueous humor derived from patients with POAG induced a significant decrease of functional collagenase activity (mean +/- SE, 9.12 +/- 1.33 microgram of degraded collagen per milligram of incubated protein vs 20.94 +/- 4.14 micrograms from the control group, P < .05). The enzymatic activity in the other types of glaucoma was similar to that of controls. The POAG samples had a significantly higher concentration of tissue inhibitor of metalloproteinase-1 (mean +/- SE, 3.11 +/- 0.58 vs 0.91 +/- 0.13 micrograms/mL from controls; P < .05). The amount of immunoreactive tissue inhibitor of metalloproteinase-1 found in the other three groups was not significantly different from the control. Aqueous humor stimulated fibroblast proliferation in all cases, but significant differences were not observed between the POAG samples (mean +/- SE, 20,285 +/- 4642 cpm/mg of incubated protein) and the control group samples (26,550 +/- 3688 cpm/mg of incubated protein). Primary open-angle glaucoma fluids increased collagen synthesis significantly compared with the control group (mean +/- SD, 3352 +/- 630 vs 985 +/- 285, P < .05), and the other groups. CONCLUSIONS: An increase of collagen synthesis and a decrease of collagen degradation may contribute to an excessive deposit of collagen with loss of the trabecular cells during the development of POAG, and aqueous humor must be considered as a microenvironment that affects the metabolism or function of the trabecular meshwork or both.

Influence on collagen metabolism of vitreous from eyes with proliferative vitreoretinopathy.

PURPOSE: Proliferative vitreoretinopathy (PVR) is characterized by cell proliferation and membrane formation on the vitreoretinal cavity of the eye. The membranes are composed of extracellular matrix, mainly collagen type I. To explore the possible mechanisms involved in PVR membrane formation, the authors analyzed the role of vitreous humor on collagen turnover. METHODS: The authors studied vitreous samples from ten patients with PVR and from five donor eyes (keratoplasty) as the control group. Human lung fibroblasts were used to study the influence of vitreous on collagen synthesis and cell proliferation. Neutralizing antibodies against transforming growth factor-beta 2 (TGF-beta 2) were used to inhibit the fibroblast collagen synthesis induced by the vitreous samples. Collagenolytic activity was analyzed in vitreous fluid using 3H-labeled collagen. RESULTS: The authors found that samples obtained from patients with PVR significantly increased collagen synthesis (2979 +/- 963.26 versus 800 +/- 232 dpm of 3H-proline incorporated per milligram of vitreous-incubated protein; P < 0.00043), without affecting fibroblast replication. The collagen synthesis induced by the vitreous samples was inhibited by anti-TGF-beta 2 antibodies in both groups (0 and 481 +/- 59 dpm of 3H-proline incorporated per milligram of vitreous-incubated protein for control and PVR samples, respectively). Collagenolytic activity was considerably lower in vitreous derived from PVR samples compared with the control group (19.9 +/- 20.3 versus 234.1 +/- 19.1 micrograms of degraded collagen per milligram of vitreous-incubated protein; P < 0.0032). CONCLUSION: These results suggest that a combined mechanism, including an increase of collagen synthesis mediated at least in part by TGF-beta 2 and a decrease of collagen degradation, may contribute to the exaggerated deposition of collagen observed in PVR membranes, and that vitreous should be considered as a part of the microenvironment that is participating actively in the pathogenesis of this vitreoretinal disorder.

[Transition from the latent to the active enzymatic form as a model of regulation of extracellular matrix degradation in the chorioamnion during human labor]. / Transición de la forma enzimática latente a activa como un modelo de regulación de la degradación de matriz extracelular en el corioamnios durante el trabajo de parto humano.

Matrix metallo proteinases (MMP) are the physiological mediators of collagen degradation and its participation in physiopathogenesis of premature rupture of membranes has been suggested by our group. With the idea of defining if some MMP become active active in a coordinated way with labor in fetal membranes, we analyzed enzymatic activity and immunoreactive protein present in extracts of amnion and chorion. It was possible to identify the presence of MMP-9 in extracts of membranes obtained during cesarean sections, without labor, although its activity/quantity was faintly detectable. Instead, extracts of fetal membranes obtained during active labor showed large activity/quantity of this MMP. With a monoclonal antibody, it was possible to show that the active form of MMP-9 could only be found in samples with labor. MMP-9 and its messenger RNA, were localized by immunohistochemistry and in situ hybridization in amniotic epithelium, in some fibroblasts of the compact layer and in trophoblast-like cells in chorion. It is concluded that: 1. Activity and quantity of MMP-9 increase selectively associated to labor; and 2. That this enzyme is expressed by different cellular populations of fetal membranes.

92-kd type IV collagenase (matrix metalloproteinase-9) activity in human amniochorion increases with labor.

To determine whether specific collagenolytic enzymes are expressed in human fetal membranes with labor, we examined gelatinase activity in extracts of amniochorion by zymography. The 92-kd gelatinase (MMP-9) was barely detectable in extracts of fetal membranes before the onset of labor but was readily demonstrable in extracts prepared from membranes isolated from laboring women or membranes collected immediately after delivery. In contrast, the 72-kd gelatinase (MMP-2) was detectable in extracts from pre- and post-labor membranes. Ethylenediaminetetracetic acid and the tissue inhibitor of metalloproteinases, TIMP-1, inhibited the gelatinase activities detected by zymography, confirming that the enzymes are metalloproteinase. Assay of amniochorion gelatinase activity using a radiolabeled denatured collagen substrate revealed a more than twofold increase in activity comparing pre-labor with post-labor fetal membrane extracts. A function-blocking anti-MMP-9 monoclonal antibody inhibited pre-labor membrane gelatinase activity by approximately 11.5%, which was only slightly greater inhibition than observed with irrelevant monoclonal antibodies. However, post-labor membrane gelatinase activity was reduced by 53% by the function-blocking antibody, indicating that MMP-9 is a major contributor to the increased gelatinase activity extractable from post-labor membranes. Western blot analyses demonstrated increased MMP-9 protein in amniochorion extracts after onset of labor. MMP-9 protein and mRNA were co-localized in amnion epithelium, underlying macrophages and chorion laeve trophoblast and decidual cells after labor. We conclude that 1) MMP-9 activity and protein in human amniochorion increases with labor and 2) MMP-9 is expressed by amnion epithelium, macrophages and chorion laeve trophoblast and decidual cells. The increased expression of MMP-9 may result in degradation of the extracellular matrix of the fetal membranes and facilitate their rupture under both physiological and pathological conditions.

Extraction and partial characterization of a low molecular weight collagenase from normal rat kidney.

A matrix metalloproteinase with selective affinity for collagen was identified and partially purified from normal rat kidney. A two-step purification procedure consisting of gel filtration and affinity chromatography on Sepharose 4B-collagen resulted in an increase in specific activity of more than 3,000 times. The partially purified collagenase cleaved type I collagen and also showed low gelatinolytic activity. The enzyme required Ca2+ and revealed a neutral pH optimum; it was not inhibited by thiol or serineprotease inhibitors. Its activity was fully blocked using recombinant tissue inhibitor of metalloproteinases-1. Using SDS-PAGE and zymography, the estimated Mr of the collagenase was 16.5 x 10(3).

Human amniotic fluid modulation of collagenase production in cultured fibroblasts. A model of fetal membrane rupture.

The participation of a mechanical factor as the only cause of rupture of fetal membranes during normal labor or premature rupture has been criticized, and the involvement of an enzymatic mechanism has been proposed. In this study we analyzed the effect of human amniotic fluids at different gestational ages on the collagenase synthesis of cultured fibroblasts. Our results show that term amniotic fluids are capable of inducing the synthesis of collagenase and other proteases in fibroblasts, as revealed by selective increases in collagenase activity and in immunoreactive collagenase. Nonterm amniotic fluids failed to do the same. This phenomenon is proposed as a model for studying the collagen degradation of fetal membranes during term gestation.

Collagen metabolism in premature rupture of amniotic membranes.

Collagen content, acid-soluble collagen, degradation activity, and collagen biosynthesis were measured in 22 normal and 20 prematurely ruptured membranes from pregnancies near term (37 weeks or more). Although no significant differences were found in the collagen content, a clear correlation was found between this value and the interval from premature rupture to delivery. Collagenolytic activity and collagen solubility were higher and collagen synthesis was lower in amniotic membranes that ruptured prematurely. These studies suggest that premature rupture of membranes is associated with extensive changes in collagen metabolism.

A latent collagenase in human aqueous humor.

It has been suggested that the trabecular meshwork may be involved in the pathogenesis of open-angle glaucoma, and some authors have pointed out that disorders of the extracellular matrix components may play a role; nevertheless, nothing is known about the normal metabolism of connective tissue molecules in this particular tissue. We recently initiated some studies in this field and have focused on the in vitro effects of aqueous humor on collagen metabolism. We report the finding of a latent collagenase of low molecular weight in aqueous humor obtained from cataractous patients; the enzyme was identified through several methods, including its in vitro activity against radiolabelled type I collagen and additionally with a zymogram technique. It was partially characterized by high-performance liquid chromatography.

Experimental diffuse interstitial renal fibrosis. A biochemical approach.

In this paper we describe two simple experimental models of diffuse interstitial renal fibrosis in the rat. One develops 25 days after unilateral renal vein ligation, and the other 15 days after unilateral ureteral double ligation and section. In both models fibrosis is examined morphologically and biochemically, the latter with emphasis on collagen turnover. In addition to a description of the histology, also presented are quantitative biochemical data on four features of tissue collagen turnover, namely total content, concentration, biosynthesis, and degradation. Although the microscopic picture of both models can be adequately described as interstitial diffuse renal fibrosis, their mechanisms are different: unilateral renal vein ligation is an example of collapse fibrosis, whereas unilateral ureteral double ligation and section is a model of absolute fibrosis. In both models however, decreased collagen degradation is the significant metabolic abnormality.