Connective Tissue Breakdown Research Articles

Patients with Hemophilia Have Increased Numbers and Pericyte Coverage of Synovial Vessels.

Abstract 3053Poster Board II-1029In patients with hemophilia, repeated joint bleedings leads to synovitis and bleeding arthropathy is the major cause of morbidity in these patients.The synovitis is characterized by a highly vascular synovial membrane with prominent proliferation of synovial fibroblasts and infiltration by inflammatory cells. This chronic inflammation, as well as a direct toxic effect of blood on chondrocytes ultimately leads to cartilage and bone destruction and a crippling arthropathy. It has recently been shown that. Inflammatory cells isolated from hemophilic joints synthesize pro angiogenic factors (matrix metalloproteas-9, basic fibroblast growth factor and cyclooxygenas-2). Angiogensis in the adult is a complex process involving breakdown of connective tissue, proliferation and migration of endothelial cells but also recruitment of supporting cells, pericytes, important for vessel maturity. The aim of this study was to determine whether repeated joint bleedings in hemophilia patients induce a pro angiogenic reaction [increased micro vascular density (MVD) and expression of vascular endothelial growth factor (VEGF)] in the hemophilic joint and to study if also pericytes are involved in the process. After informed consent, synovial biopsies were collected from patients with severe hemophilia (n=5) when undergoing knee surgery on clinical grounds. As control, synovial biopsies from one patient undergoing diagnostic arthroscopy were used. Biopsies were snap frozen in liquid nitrogen and sectioned by cryotome. After fixation, sections were double stained for CD34 (for detection of endothelial cells) and SMA-a (for detection of pericytes) by immuno fluorescence.Sections were also stained for VEGF by immuno histochemistry. As shown in Table 1 patients with hemophilia had a very high MVD and pericyte coverage as compared to control samples. We also measured levels of VEGF by ELISA in plasma from 23 patients and 4 controls, but in all samples the concentration was below detection level. However, in synovial biopsies from patients with hemophilia, VEGF was clearly expressed by the endothelial cells as well as mononuclear cells present in the section.Table 1Angiogenesis parameters in patients with hemophiliaSynovial biopsiesplasmaMVD (vessels/HPF)Pericyte coverage (%)VEGF+ (cells/HPF)VEGF (pg/mLHemophilia patients17±891±0.122±19<9Control24000Based on these preliminary data we suggest that hemophilic arthropaty is characterized by increased angiogenesis and mobilization of pericytes. Although no increase of VEGF was detected systemically it was shown to be expressed locally, within the inflamed joint. Anti-angiogenic drugs are currently successfully used in different types of cancers and a specificVEGF inhibitor for topical administration has successfully been developed for proliferative diabetic retinopathy. The results presented in this study could be part of a rationale to design studies using these drugs also in hemophilic arthropaty, a condition currently lacking specific treatment. DisclosuresNo relevant conflicts of interest to declare.

Parathyroid hormone administration may modulate periodontal tissue levels of interleukin‐6, matrix metalloproteinase‐2 and matrix metalloproteinase‐9 in experimental periodontitis

Intermittent administration of the parathyroid hormone (1-34) has an anabolic effect on bone and it has been shown to reduce alveolar bone loss in experimental periodontitis models. The aim of the present study was to investigate the effect of parathyroid hormone on tissue degradation-related factors in an experimental periodontitis model in rats. Periodontitis was induced in seventy-six male Wistar rats using ligature around the lower right first molars. The animals were then treated with parathyroid hormone (1-34) (T-group) or vehicle (C-group), three times a week for 15 d (C15, T15) or 30 d (C30, T30). At each experimental time-point, the 19 rats were killed in each group and the gingival tissue around the first lower molar was removed and prepared for the following analyses: mRNA expression of interleukin-1 beta, interleukin-6, matrix metalloproteinase (MMP)-2 and MMP-9, and gelatinolytic activity of MMP-2 and MMP-9. Hemimandibles were decalcified, and serial sections were processed and analyzed for interleukin-6 immohistochemistry. Samples were also histochemically stained by tartrate-resistant acid phosphatase (TRAP) to evaluate the number of osteoclasts present. Parathyroid hormone-treated samples showed decreased of levels of mRNA for interleukin-6 in the T30 group (p < 0.01) and of MMP-2 in the T15 and T30 groups (p < 0.05). Zymography assays demonstrated that treatment with parathyroid hormone led to a decrease in MMP-9 activity (p < 0.01). TRAP staining of alveolar bone revealed that osteoclasts were present in higher numbers (p < 0.05) in the groups not treated with parathyroid hormone. These data suggest that intermittent administration of parathyroid hormone can down-regulate the expression of biomarkers responsible for connective tissue breakdown and bone resorption, and potentially affect alveolar bone resorption activity.

Recent Advances in Molecular Targets and Treatment of Idiopathic Pulmonary Fibrosis: Focus on TGF&amp;#946; Signaling and the Myofibroblast

Idiopathic Pulmonary Fibrosis (IPF) is characterized by injury and loss of lung epithelial cells, accumulation of fibroblasts/myofibroblasts and abnormal remodeling of the lung parenchyma. The prognosis for IPF patients is poor and current therapies are largely ineffective in preventing respiratory failure. Current therapeutic approaches target epithelial cell replacement, manipulation of fibroblasts/myofibroblasts, modulation of procoagulant/fibrinolytic activities, cytokine and growth factor production, angiogenesis, and reduction of oxidative stress. Myofibroblasts are the primary effector cells in fibrosis. These cells may be derived by the activation and proliferation of resident lung fibroblasts, from epithelial-mesenchymal transition (EMT), or through recruitment of circulating fibrocytes. Transforming growth factor beta (TGFbeta) is a profibrotic factor that increases fibroblast proliferation, stimulates the synthesis and deposition of connective tissue, and inhibits connective tissue breakdown. TGFbeta acts through the promoter of the type 1 collagen gene causing increased collagen synthesis. In addition, TGFbeta induces EMT in alveolar epithelial cells (AECs) in vitro and in vivo. AECs exhibit substantial plasticity and may serve as a source of fibroblasts and/or myofibroblasts in lung fibrosis. Therapeutic interventions interfering with the pathways that lead to myofibroblast expansion and AEC apoptosis should be of considerable benefit in the treatment of IPF. This review will focus on the critical role of TGFbeta on AECs EMT and myofibroblasts in the development of fibrosis.

Cytokines That Promote Periodontal Tissue Destruction

Although periodontal diseases are initiated by bacteria that colonize the tooth surface and gingival sulcus, the host response is believed to play an essential role in the breakdown of connective tissue and bone, key features of the disease process. An intermediate mechanism that lies between bacterial stimulation and tissue destruction is the production of cytokines, which stimulates inflammatory events that activate effector mechanisms. These cytokines can be organized as chemokines, innate immune cytokines, and acquired immune cytokines. Although they were historically identified as leukocyte products, many are also produced by a number of cell types, including keratinocytes, resident mesenchymal cells (such as fibroblasts and osteoblasts) or their precursors, dendritic cells, and endothelial cells. Chemokines are chemotactic cytokines that play an important role in leukocyte recruitment and may directly or indirectly modulate osteoclast formation. This article focuses on aspects of osteoimmunology that affect periodontal diseases by examining the role of cytokines, chemokines, and immune cell mediators. It summarizes some of the key findings that attempt to delineate the mechanisms by which immune factors can lead to the loss of connective tissue attachment and alveolar bone. In addition, a discussion is presented on the importance of clarifying the process of uncoupling, a process whereby insufficient bone formation occurs following resorption, which is likely to contribute to net bone loss in periodontal disease.

The use of rodent models to investigate host–bacteria interactions related to periodontal diseases

Even though animal models have limitations, they are often superior to in vitro or clinical studies in addressing mechanistic questions and serve as an essential link between hypotheses and human patients. Periodontal disease can be viewed as a process that involves four major stages: bacterial colonization, invasion, induction of a destructive host response in connective tissue and a repair process that reduces the extent of tissue breakdown. Animal studies should be evaluated in terms of their capacity to test specific hypotheses rather than their fidelity to all aspects of periodontal disease initiation and progression. Thus, each of the models described below can be adapted to test discrete components of these four major steps, but not all of them. This review describes five different animal models that are appropriate for examining components of host-bacteria interactions that can lead to breakdown of hard and soft connective tissue or conditions that limit its repair as follows: the mouse calvarial model, murine oral gavage models with or without adoptive transfer of human lymphocytes, rat ligature model and rat Aggregatibacter actinomycetemcomitans feeding model.

Aerobic Capacity-dependent Changes In Cardiac Gene Expression

Aerobic capacity is a strong predictor of cardiovascular mortality. Rats artificially selected for high aerobic capacity (High Capacity Runners) exhibit an inborn 30 % higher oxygen uptake, increased left ventricular weight and improved myocyte contractility as compared to the Low Capacity Runners with low aerobic capacity. PURPOSE: To determine the cardiac genetic signature and identify patterns of gene expression associated with high and low aerobic capacity. METHODS: RNA was isolated from the left ventricle of the heart and gene expression was analyzed on microarrays. RESULTS: Out of 28.0000 screened genes, 1540 genes were differentially expressed between HCR and LCR. Of those 618 were higher expressed in HCR as compared to LCR, and 922 were higher expressed in LCR versus HCR. The genetic signature of HCR was characterized by a higher expression of genes associated with lipid metabolism, as acyl-CoA dehydrogenase, very low density lipoprotein receptor, carnitine palmitoyltransferase 1 and mitochondrial acyl-CoA thioesterase 1, compared to LCR. These findings suggest a weaker lipid metabolism in LCR, perhaps due to a switch in energy substrate from fatty acids to glucose, an event that occurs in diseased hearts. This is Supported by a higher expression of glycolytic enzymes, like hexokinase 2 and 6-phosphofructo-2 kinase in LCR. Genes encoding proteins involved in cardiac contractility were differentially expressed by the groups. HCR had an over-expression of muscarinic receptor and mitochondrial uncoupling protein 4, whereas LCR over-expressed L type calcium channels and tropomyosin 1.A high expression of genes involved in myocyte growth was found in both groups. In LCR these genes were associated with fetal gene expression, indicating some stage of pathological hypertrophy. In HCR, collagen and matrix metallopepdidase 2 were among the highest expressed genes, suggesting elevated tissue turnover rate in HCR than in LCR, with continuous construction and breakdown of connective tissue. HCR also exhibited a higher expression of insulin-like growth factor 2 and colony stimulating factor 1, which may reflect a low risk of obesity and atherosclerosis respectively. CONCLUSIONS: High versus low aerobic capacity was associated with differences in genes regulating lipid metabolism, contractility and hypertrophy.

Déficit en vitamine C et ulcères de jambe. Résultats d'une enquête cas témoins

Vitamin C is a necessary cofactor for collagen synthesis. A deficiency of vitamin C results in the breakdown of connective tissue in and around the walls of blood vessels. The disease is thus characterized by poor healing of wounds. Chronic leg ulcers are defined as wounds that do not heal. ObjectiveTo investigate whether patients with chronic leg ulcers have vitamin C deficiency. MethodsCase control study; vitamin C was assayed in peripheral blood samples of 42 consecutive patients with chronic leg ulcers and in 37 consecutive patients without chronic leg ulcers. Patients without leg ulcers had peripheral vascular disease, or hypertension, or connective disorders. Patients with diabetes, immunodepression (cancer, HIV infection, corticosteroid therapy) and aged under 65 years were excluded. Reference range for plasma vitamin C was above 26 µmol/l (normal levels, group I), hypovitaminosis C as 6–26 µmol/l (group II) and concentrations<6 µmol/l as scurvy (group III). ResultsMean age was 77.2 years in the ulcers group and 73.8 in the control group (NS), mean weight 73.1 kg in the ulcers group and 67.5 kg in the control group (NS). Smoking was more frequent in the control group (P<0.001). Mean vitamin C levels were lower in the leg ulcers group: 23.9 vs 33.8 µmol/l (P<0.003). Normal levels of vitamin C (group I) were more frequent in the control group: 78.4 vs 50% (P<0.01). Hypovitaminosis C (group II) was more frequent in the leg ulcers group: 23.8 vs 16.2% (P<0.01). Scurvy was more frequent in the leg ulcers group: 26.2 vs 5.4% (P<0.01). C reactive protein levels were higher in the leg ulcers group: 31.8 vs 9.3 mg (P=0.002) and albumin levels were lower in the leg ulcers group: 25 vs 38 g/l (P=0.01) [retrospective data]. ConclusionPatients with chronic leg ulcers have lower levels of vitamin C than patients without leg ulcers, although smoking was more frequent in patients without leg ulcers. The question is whether vitamin C deficiency is a cofactor of impaired healing or is a simple marker of poor healing? It would be interesting to conduct a randomized controlled study about treatment of chronic leg ulcers with vitamin C.

Identification of bactenecin‐1 in cervicovaginal fluid by two‐dimensional electrophoresis in an ovine model of preterm labour

Preterm labour is a major problem in obstetrics. Timely intervention with available treatments is hampered by the lack of a reliable test of imminent preterm delivery. Current methods of diagnosis are based on the detection of breakdown products of foetal membranes or structural changes to the cervix when preterm labour is well established. The aim of this study was to screen the cervicovaginal fluid (CVF) proteome to identify labour-associated proteins that could be used as markers of imminent preterm delivery. Labour was induced in sheep at 135 days of gestation (term 147 days) by foetal infusion of dexamethasone (1 mg/24 h). CVF samples were collected before and 28 h after the start of infusion as well as at delivery (58.7 +/- 1.9 after the start of infusion, n = 5). One protein that was upregulated eight-fold, was bactenecin-1, a member of the cathelicidin family of antimicrobial proteins. This antimicrobial protein warrants further investigation as a marker of preterm labour, particularly during the period after the initiation of labour but before there is marked cervical connective tissue breakdown.

Epidermal Growth Factor Receptor Is a Critical Mediator of Ultraviolet B Irradiation-Induced Signal Transduction in Immortalized Human Keratinocyte HaCaT Cells

Epidermal growth factor receptor (EGFR) is a critical mediator of several types of epithelial cancers. Skin cancer arising from exposure to ultraviolet B irradiation (UVB) from the sun is a prominent form of human cancer. Recent data indicate that in addition to cognate ligands, EGFR is activated by UVB irradiation. We used pharmacological and genetic approaches to investigate the function of EGFR in mediating UVB-induced signal transduction in human skin keratinocyte HaCaT cells. Pharmacological inhibition of EGFR tyrosine kinase significantly inhibited UVB-mediated induction of ERK, p38, and JNK MAP kinases, and their effectors, transcription factors c-Fos and c-Jun. Inhibition of UVB activation of EGFR also suppressed activation of AKT-, PKC-, and PKA-dependent signal transduction pathways. B82 mouse L cells devoid of EGFR were used to further investigate EGFR dependence of UVB-induced signal transduction. UVB failed to induce ERK, and JNK activation was reduced 60% in B82 cells compared to B82K+ cells, which express EGFR. In addition, UVB induced both c-Fos and c-Jun proteins in B82K+ cells, whereas neither were induced in B82 cells. Taken together, these data demonstrate that EGFR is required for UVB-mediated induction of multiple signaling pathways that are known to mediate tumor formation in skin.

A34. Development of novel matrix metalloproteinase inhibitors and their effects on isolated stunned hearts

Matrix metalloproteinases (MMPs) are hydrolytic enzymes involved in the breakdown of myocardial connective tissue with disease. We have undertaken the development of novel non-hydroxamate-based MMP inhibitors (MPIs) intended to enhance the potency and safety profiles of the drugs in light of a troubled clinical history of hydroxamate-based inhibitors. MPI basic design consists of a peptidomimetic backbone attached to a zinc-binding group (ZBG). The drug discovery software LUDI was used to design MPIs with specific inhibition profiles. Interactions between novel MPIs and the enzyme was reproduced using the ZBG portion of the inhibitors bound to a tris(pyrazolyl)borate model complex. This model was then integrated into a known MMP crystal structure. Candidate compounds were initially screened and ranked using a LUDI scoring function. Candidate novel MPIs were subsequently tested at three levels: In vitro using recombinant MMPs, in cell culture using neonatal rat cardiac fibroblasts where cytotoxicity was also monitored, and ex vivo using isolated rat hearts. In both recombinant and cell culture assays new ZBGs were found to be 10–100-fold more potent inhibitors compared to acetohydroxamic acid. Cytotoxicity assays demonstrated that most of these novel compounds were non-toxic at concentrations reaching 100 μM. Two compounds: 200 μM thiomaltol (ZBG) and 10 μM PY2 (PY-2 has nanomolar IC50 vs. MMP-3) were tested ex vivo to assess recovery of cardiac function in stunned myocardium. Stunning was induced by 20 min of no flow ischemia. Rate pressure product results indicated that both compounds conferred a significant increase in recovery of contractile function versus controls. We demonstrate that in silico drug design using a chelator-driven approach can produce potent and selective MPIs.

CCL18/PARC increases pro‐inflammatory cytokines and cell survival in cultured monocytes

CCL18/PARC is a chemokine, which is a chemotactic factor for lymphocytes, immature dendritic cells and for cultured, but not freshly isolated monocytes. Three day old monocytes cultured in the presence of 40 nM CCL18 showed increased survival on day 4 (40–45% surviving cells compared to 15–25% in unstimulated controls). This observation led us to determine differences in gene and cytokine expression in CCL18-stimulated versus control cultures. On the protein level CCL18 caused increased concentrations of IL-6, IL-8, gro-α, ENA-78, MCP-1 and MIP-1α, but down-regulated angiogenin, bFGF and insulin-like growth factor. Interestingly, two inhibitors of membrane metalloproteases (TIMP-1 and TIMP-2) were also down-regulated following CCL18 stimulation. In addition, there was more MMP-9 protein and activity detected in supernatants from CCL18-activated monocytes.. Differential gene expression, which was detected by gene array was verified for several relevant genes by RT-PCR. These genes included IL-15 and stem cell factor, which were both up-regulated and caspase 8, which was down-regulated by CCL18.. In addition, IL-15, - which was shown by FACS to be up-regulated on the cell surface of CCL18-stimulated monocytes, - has an anti-apoptotic function in various leukocytes. Thus CCL18 induces pro-inflammatory effects 1) directly by reducing monocyte apoptosis, 2) indirectly through increased production of cytokines/chemokines, which cause leukocyte influx, and 3) by contributing to connective tissue breakdown due to increased metalloprotease activity.

Fucoidan a sulfated polysaccharide from brown algae is a potent modulator of connective tissue proteolysis

Fucoidans are sulfated fucosylated polymers from brown algae cell wall that exhibit some heparin/heparan sulfate properties. We previously demonstrated that these polysaccharides were able in vitro to stimulate dermal fibroblast proliferation and extracellular matrix deposition. Here, we investigated the action of a 16 kDa fucoidan fraction on parameters involved in connective tissue breakdown. This fucoidan is able to inhibit gelatinase A secretion and stromelysin 1 induction by interleukin-1β on dermal fibroblasts in culture. Furthermore, we observed that fucoidan increases the rate of association of MMPs with their specific inhibitors namely TIMPs. Using tissue sections of human skin in ex vivo experiments, we evidenced that this polysaccharide was able to minimize human leukocyte elastase activity resulting in the protection of human skin elastic fiber network against the enzymatic proteolysis due to this serine proteinase. These results suggested that fucoidan could be used for treating some inflammatory pathologies in which uncontrolled extracellular matrix degradation takes place.

Breakdown of intramuscular connective tissue in cod ( Gadus morhua L.) and spotted wolffish ( Anarhichas minor O.) related to gaping

The purpose of this work was to compare the microstructure of muscle connective tissue in cod and wolffish and to study its degradation during ice-storage to determine which structural alterations are related to the gaping phenomenon. Gaping is very often found in fillets of codfish, but not in wolffish. The results showed that detachments between myofibres occurred prior to the myotomes to myocommata detachments, which occurred more rapidly and to a larger extent in cod than in wolffish. Changes at the ultrastructural level in myocommata proper were mainly related to gaps between collagen fibres and between collagen fibres and cells in the extracellular matrix (ECM). The mean collagen fibre diameter was smaller and the collagen network in myocommata proper was denser in wolffish than in cod. After 7 days of ice-storage the collagen fibres, particular in cod, appeared sparsely packed. The results implied that the increased gaping during ice-storage might be due to degradation of proteoglycans and glycoproteins, important for the spatial organization of the collagen fibres and in anchoring cells in the ECM to the collagen network.

Analysis of superoxide dismutase activity levels in gingiva and gingival crevicular fluid in patients with chronic periodontitis and periodontally healthy controls

Superoxide dismutase (SOD) is an antioxidant enzyme that acts against superoxide, an oxygen radical, released in inflammatory pathways and causes connective tissue breakdown. In this study, SOD activities in gingiva and gingival crevicular fluid (GCF) from patients with chronic periodontitis (CP) and periodontally healthy controls were compared. Twenty-six CP patients and 18 controls were studied. In patients, teeth with moderate-to-severe periodontal breakdown and > or =5 mm pockets that required full-thickness flap surgery in the right or left maxillary quadrant, and in controls, teeth scheduled for extraction for orthodontic reasons were studied. After the clinical measurements (probing depth, clinical attachment level, gingival index, gingival bleeding index, plaque index), GCF samples were collected. Tissue samples were harvested from the same teeth, during flap operation in patients and immediately after tooth extraction in controls. SOD activities were spectrophotometrically assayed. The results were statistically analysed. Gingival SOD activity was significantly higher in the CP group than in controls (p<0.05). No significant difference was found in GCF SOD activity between the groups (p>0.05). Correlations between gingival and GCF SOD activities were not statistically significant in CP and control groups (p>0.05). In CP, SOD activity seems to increase in gingiva, probably as a result of a higher need for SOD activity and protection in gingiva in CP than in periodontal health, while not significantly changing in GCF, suggesting a weak SOD activity in GCF in periodontal disease state. The weak correlation between gingival and GCF SOD activities suggests distinct actions of these SODs.

Adjunctive subantimicrobial dose doxycycline: effect on clinical parameters and gingival crevicular fluid transforming growth factor‐β1 levels in severe, generalized chronic periodontitis

At present there is limited data concerning the efficacy of non-surgical periodontal therapy supplemented with subantimicrobial dose doxycycline (SDD) in the treatment of severe, generalized periodontitis. The purpose of the present study was to evaluate the effect of adjunctive SDD therapy on clinical periodontal parameters and gingival crevicular fluid (GCF) transforming growth factor-beta1 (TGF-beta1) levels in patients with severe, generalized chronic periodontitis over a 6-month period. Thirty-five patients with severe, generalized periodontitis and 11 periodontally healthy subjects were included in the present study. Patients received full-mouth supragingival debridment at baseline and randomized to take either SDD b.i.d. or placebo b.i.d. for 3 months. Patients received root planing and oral hygiene instruction once a week for four consecutive weeks. Clinical measurements including probing depth (PD), clinical attachment level, papilla bleeding index and plaque index and GCF sampling were performed at baseline, 3 and 6 months. The GCF TGF-beta1 levels were analysed by enzyme-linked immunosorbent assay. Thirteen patients in both study groups completed the 6-month trial. Following scaling and root planing (SRP) plus SDD and SRP plus placebo therapy significant improvements in clinical periodontal parameters of both groups were observed (p<0.025). In the SDD group a significantly higher percentage (%73.4) of deep pockets resolved (PD reduction > or =3 mm from baseline) when compared with placebo group (%49.7) at 6 months (p<0.05). At baseline there were no significant differences in GCF TGF-beta1 levels between three groups. Both total amount and concentration of GCF TGF-beta1 in SDD and placebo groups increased when compared with baseline at 3 months. However, only GCF TGF-beta1 levels of SDD group was significantly higher than baseline (p<0.025) and placebo group (p<0.017) at 3 months. At 6 months GCF TGF-beta1 levels of both groups were similar to baseline levels (p<0.025). These data indicate that combination of SDD with non-surgical therapy improves clinical parameters of periodontal disease and increases GCF TGF-beta1 levels together with a decrease in prevalence of residual pockets in patients with severe, generalized chronic periodontitis. Increased GCF TGF-beta1 levels following SDD therapy might suggest a novell pleiotrophic mechanism for tetracyclines to inhibit connective tissue breakdown.

Plasma Gelatinolytic Activity in Leukemias.

Gelatinase A (Matrix Metalloproteinase 2, MMP-2) and B (Matrix Metalloproteinase 9, MMP-9) are secreted by connective tissue, endothelial, epithelial and hematopoietic cells. Elastase and gelatinases produced by granulocytes are released in whole blood in response to varied stimuli. They induce breakdown of connective tissue and play an important role in processes of cell transmigration and tissue remodeling. The aims of this study were to determine and compare the plasma concentration of PMN elastase and the gelatinase activity in patients with chronic myelogenous leukemia (CML, n=16), acute myeloblastic leukemia, AML, n=53 and acute lymphoblastic leukemia, ALL, n=18) and in healthy persons (n=22). We measured activity of MMP-2 and MMP-9 using gelatine zymography and concentration of elastase from polymorphonuclear leukocytes (PMN) in complex with alpha1-proteinase inhibitor using ELISA. The MMP-9 activity in plasma from patients with CML was higher than that from AML, ALL and reference groups. The MMP-9 activity in AML and ALL group was lower than in healthy persons. There was no significant difference in the MMP-2 levels between the plasma samples from patients with CML or ALL and those from healthy subjects, but MMP-2 activity was lower in the reference group than in AML. PMN elastase concentration in plasma from the healthy reference group was lower than that from CML and AML patients. Furthermore, PMN elastase concentration in plasma from CML patients was higher than that from AML and ALL. We found significant correlation between MMP-9 activity and white blood cells count in the reference group (r=0,56), CML group (r=0,70) and AML group (r=0,44). The PMN elastase concentration increased proportionally to the white blood-cell count (r=0,51) in plasma from patients with CML only. There was significant relationship between the count of granulocytes in different stages of maturation with PMN elastase and MMP-9 activity: promyelocytes (r=0,73 for elastase and r=0,75 for MMP-9), myelocytes (r=0,63 and r=0,79), metamyelocytes (r=0,56 and r=0,83), band neutrophils (r=0,64 and r=0,74), segmented neutrophils (r=0,73 for MMP-9) and eosinophils (r=0,64 for MMP-9) in blood from CML patients. Increased MMP-9 activity was correlated with increased MMP-2 activity in the reference group (r=0,58) and AML group (r=0,50), however it was correlated with decreased MMP-2 activity in the CML group (r=−0,59). In the CML group it was observed that decreased MMP-2 activity correlated with an increased count of: blasts (r=−0,64), promyelocytes (r=−0,77), myelocytes (r=−0,63), metamyelocytes (r=−0,64) and band neutrophils (r=−0,60). Gelatin zymography can be employed to display differences of plasma MMP-2 and MMP-9 activity between patients with chronic myelogenous leukemia, acute leukemia and healthy persons.

Lack of Effect of Oral 4‐Phenylbutyrate on Serum Alpha‐1‐Antitrypsin in Patients with α‐1‐Antitrypsin Deficiency: A Preliminary Study

ABSTRACTObjective:In homozygotes with ZZ genotype alpha‐1‐antitrypsin (α1AT) deficiency, mutant α1ATZ protein (α1ATZ) accumulates in hepatocytes, rather than being secreted into the blood. Homozygous individuals experience emphysema as a result of reduced levels of circulating α1AT in the lung with which to inhibit connective tissue breakdown. Homozygotes may also experience liver disease from the accumulation of α1ATZ within hepatocytes, which causes liver damage. A previous study indicated that the compound 4‐phenylbutyrate (4‐PBA) mediated a significant increase in release of α1ATZ from cells in tissue culture and in a mouse model of α1AT deficiency. The authors hypothesized that 4‐PBA could be used to treat both the liver and lung disease of humans with α1AT deficiency.Methods:In this preliminary, open label study the authors evaluated the effect of 14 days of oral 4‐PBA therapy on α1AT blood levels in 10 patients with α1AT deficiency.Results:There was no significant increase in α1AT blood level associated with 4‐PBA administration. Symptomatic and metabolic side effects were significant.Conclusion:4‐PBA did not increase α1AT blood levels in humans with α1AT deficiency in this preliminary trial.

Lack of effect of oral 4-phenylbutyrate on serum alpha-1-antitrypsin in patients with alpha-1-antitrypsin deficiency: a preliminary study.

In homozygotes with ZZ genotype alpha-1-antitrypsin (alpha1AT) deficiency, mutant alpha1ATZ protein (alpha1ATZ) accumulates in hepatocytes, rather than being secreted into the blood. Homozygous individuals experience emphysema as a result of reduced levels of circulating alpha1AT in the lung with which to inhibit connective tissue breakdown. Homozygotes may also experience liver disease from the accumulation of alpha1ATZ within hepatocytes, which causes liver damage. A previous study indicated that the compound 4-phenylbutyrate (4-PBA) mediated a significant increase in release of alpha1ATZ from cells in tissue culture and in a mouse model of alpha1AT deficiency. The authors hypothesized that 4-PBA could be used to treat both the liver and lung disease of humans with alpha1AT deficiency. In this preliminary, open label study the authors evaluated the effect of 14 days of oral 4-PBA therapy on alpha1AT blood levels in 10 patients with alpha1AT deficiency. There was no significant increase in alpha1AT blood level associated with 4-PBA administration. Symptomatic and metabolic side effects were significant. 4-PBA did not increase alpha1AT blood levels in humans with alpha1AT deficiency in this preliminary trial.

Interleukin-1beta induces matrix metalloproteinase-1 expression in cultured human gingival fibroblasts: role of cyclooxygenase-2 and prostaglandin E2.

Matrix metalloproteinases (MMPs) degrade extracellular matrices and are responsible for excessive connective tissue breakdown in inflammatory disorders. We investigated the mechanism of MMP-1 expression in human gingival fibroblasts in response to the stimulation with interleukin-1beta (IL-1beta), and the role of inducible-type cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in the regulation of MMP-1 expression. We stimulated cultured human gingival fibroblasts with r(h)IL-1beta, and examined the expression of MMP-1 mRNA and protein by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of indomethacin, dexamethasone, or cycloheximide (CHX) on the IL-1beta-induced expression of MMP-1 was examined. The expression of MMP-1 in gingival fibroblasts stimulated with PGE2 was also examined. IL-1beta stimulated the expressions of mRNA and protein for MMP-1, in cultured fibroblasts, in time- and concentration-dependent manners. Pretreatment of the cells with indomethacin or dexamethasone inhibited the IL-1beta-induced MMP-1 expression. CHX, a protein synthesis inhibitor, also suppressed the MMP-1 expression. IL-1beta also induced COX-2 expression in gingival fibroblasts, and PGE2, a major COX-2 product, was found to enhance MMP-1 expression. The IL-1beta-induced MMP-1 expression in gingival fibroblasts may be mediated, at least in part, by COX-2 and its product PGE2.

Imaging microvascular structure with contrast enhanced MRI

Over the past 10 years there has been increasing interest in the development of novel MRI techniques that enable researchers and clinicians to study the development and structure of vasculature in pathological tissues. Although the majority of this work has been performed in studies of human tumours the techniques are potentially widely applicable in other pathological states [1–4]. All tissues depend on their vasculature for an adequate supply of nutrients and removal of waste metabolic materials. As tissues develop, an adequate and appropriately structured vascular supply must develop at the same time. This process, known as angiogenesis, is also an essential component of the behaviour of many pathological tissues including tumours and inflammatory disease [1, 5]. More importantly, the angiogenic process appears to be largely independent of the developing tissue. This has given rise to the concept of antiangiogenic therapies which could be effective in a wide range of tumours independent of the tissue type. The angiogenic process is complex and can be stimulated by any one of several mechanisms. Typically, growth of tissue which has outstripped its local blood supply results in regional hypoxia and hypoglycaemia which stimulates the release of local chemical messengers from the cells of the tissue itself. The best known of these messengers is the cytokine, vascular endothelial growth factor (VEGF) [6–9]. VEGF is a common and potent angiogenic stimulator, which is found in many pathological tissues. It is released in response to local hypoglycaemia and/or hypoxia and has several effects each of which will improve metabolic supply. In the short term VEGF will act directly on local capillaries to increase endothelial permeability resulting in an immediate increase in the supply of nutrients [10]. This increase in permeability is also believed to form an important part of the metastatic mechanism allowing passage of tumour cells into the circulation. In the medium to long term, VEGF will stimulate mitosis in endothelial cells from local blood vessels so that they divide and develop a new vascular infrastructure to supply the tumour. The angiogenic mechanism also is responsible for breakdown of local connective tissues, which allows in-growth of new blood vessels. Where the angiogenic process fails tissue development cannot occur and novel antiangiogenic therapies are currently being developed which use this mechanism for the treatment of a wide range of cancers and other pathologies [11, 12]. The increasing understanding of the role of the angiogenic process in disease progression has led to interest in methods for documenting the presence and activity of angiogenesis [12–14]. In particular there has been considerable interest in the development of reliable quantitative methods which can provide independent indicators of the status of, and changes in, microvascular structure [13, 14]. In pathological tissues the angiogenic process is often abnormal, leading to the development of distorted vascular beds characterized by an excessive proportion of blood vessels and blood vessels with abnormal morphology and flow characteristics [15]. Central areas of a rapidly growing tumour will commonly exhibit inadequate blood flow due to reduce local perfusion pressure resulting from a combination of inadequate vascularization and increased interstitial tumour pressure. Finally, the angiogenic neovasculature will exhibit increase endothelial permeability to medium and large sized molecules [16, 17]. Initial studies of microvascular structure in tumours were undertaken using histological techniques, however these are essentially unsatisfactory for a number of reasons [18, 19]. Pathological assessment relies on the acquisition of tissue samples which is clearly invasive and which can be repeated only infrequently. Furthermore, many tumours and other pathological tissues demonstrate considerable heterogeneity in microvascular structure so that isolated regional biopsies may give misleading information. These limitations have led to the development of imaging based methods for quantification of microvascular structure. These methods are commonly based on dynamic contrast enhanced imaging techniques using MRI or CT data collection combined with analytic algorithms to calculate descriptive parameters related to microvascular structure. The availability of quantitative imaging based methods to describe the microvasculature in biological tissues is potentially of considerable importance. Such methods can provide important surrogate markers of therapeutic response in trials of novel antiangiogenic therapy and the majority of applications to date have been in this area. However, these techniques can also be seen to have directly clinical relevance providing information about diagnosis, tumour grade, therapeutic response, tumour recurrence and prognosis. At the present time the majority of technical development of these techniques has been centred around the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). This article will describe the development and range of DCE-MRI techniques and the range of image analysis approaches available. The article aims to provide insight into the benefits and disadvantages of specific approaches rather than to detail the actual analysis mechanisms in detail.