A fine-tuned defense at the pea root caps: Involvement of border cells and arabinogalactan proteins against soilborne diseases.

Plants have to cope with a myriad of soilborne pathogens that affect crop production and food security. The complex interactions between the root system and microorganisms are determinant for the whole plant health. However, the knowledge regarding root defense responses is limited as compared to the aerial parts of the plant. Immune responses in roots appear to be tissue-specific suggesting a compartmentalization of defense mechanisms in these organs. The root cap releases cells termed root "associated cap-derived cells" (AC-DCs) or "border cells" embedded in a thick mucilage layer forming the root extracellular trap (RET) dedicated to root protection against soilborne pathogens. Pea (Pisum sativum) is the plant model used to characterize the composition of the RET and to unravel its function in root defense. The objective of this paper is to review modes of action of the RET from pea against diverse pathogens with a special focus on root rot disease caused by Aphanomyces euteiches, one of the most widely occurring and large-scale pea crop diseases. The RET, at the interface between the soil and the root, is enriched in antimicrobial compounds including defense-related proteins, secondary metabolites, and glycan-containing molecules. More especially arabinogalactan proteins (AGPs), a family of plant extracellular proteoglycans belonging to the hydroxyproline-rich glycoproteins were found to be particularly present in pea border cells and mucilage. Herein, we discuss the role of RET and AGPs in the interaction between roots and microorganisms and future potential developments for pea crop protection.

Deep Learning-Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard-to-Transfect Cells.

Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human-induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.

Deep Learning and Computer Vision Strategies for Automated Gene Editing with a Single-Cell Electroporation Platform.

Single-cell delivery platforms like microinjection and nanoprobe electroporation enable unparalleled control over cell manipulation tasks but are generally limited in throughput. Here, we present an automated single-cell electroporation system capable of automatically detecting cells with artificial intelligence (AI) software and delivering exogenous cargoes of different sizes with uniform dosage. We implemented a fully convolutional network (FCN) architecture to precisely locate the nuclei and cytosol of six cell types with various shapes and sizes, using phase contrast microscopy. Nuclear staining or reporter fluorescence was used along with phase contrast images of cells within the same field of view to facilitate the manual annotation process. Furthermore, we leveraged the near-human inference capabilities of the FCN network in detecting stained nuclei to automatically generate ground-truth labels of thousands of cells within seconds, and observed no statistically significant difference in performance compared to training with manual annotations. The average detection sensitivity and precision of the FCN network were 95±1.7% and 90±1.8%, respectively, outperforming a traditional image-processing algorithm (72±7.2% and 72±5.5%) used for comparison. To test the platform, we delivered fluorescent-labeled proteins into adhered cells and measured a delivery efficiency of 90%. As a demonstration, we used the automated single-cell electroporation platform to deliver Cas9-guide RNA (gRNA) complexes into an induced pluripotent stem cell (iPSC) line to knock out a green fluorescent protein-encoding gene in a population of ~200 cells. The results demonstrate that automated single-cell delivery is a useful cell manipulation tool for applications that demand throughput, control, and precision.

Nanofountain Probe Electroporation Enables Versatile Single-Cell Intracellular Delivery and Investigation of Postpulse Electropore Dynamics.

Introducing exogenous molecules into cells with high efficiency and dosage control is a crucial step in basic research as well as clinical applications. Here, the capability of the nanofountain probe electroporation (NFP-E) system to deliver proteins and plasmids in a variety of continuous and primary cell types with appropriate dosage control is reported. It is shown that the NFP-E can achieve fine control over the relative expression of two cotransfected plasmids. Finally, the dynamics of electropore closure after the pulsing ends with the NFP-E is investigated. Localized electroporation has recently been utilized to demonstrate the converse process of delivery (sampling), in which a small volume of the cytosol is retrieved during electroporation without causing cell lysis. Single-cell temporal sampling confers the benefit of monitoring the same cell over time and can provide valuable insights into the mechanisms underlying processes such as stem cell differentiation and disease progression. NFP-E parameters that maximize the membrane resealing time, which is essential for increasing the sampled volume and in meeting the challenge of monitoring low copy number biomarkers, are identified. Its application in CRISPR/Cas9 gene editing, stem cell reprogramming, and single-cell sampling studies is envisioned.

A critical role for ABC transporters in persistent lung inflammation in the development of emphysema after smoke exposure.

Macrophage infiltration is common to both emphysema and atherosclerosis, and cigarette smoke down-regulates the macrophage cholesterol efflux transporter ATP binding cassette (ABC)A1. This decreased cholesterol efflux results in lipid-laden macrophages. We hypothesize that cigarette smoke adversely affects cholesterol transport via an ABCA1-dependent mechanism in macrophages, enhancing TLR4/myeloid differentiation primary response gene 88 (Myd88) signaling and resulting in matrix metalloproteinase (MMP) up-regulation and exacerbation of pulmonary inflammation. ABCA1 is significantly down-regulated in the lung upon smoke exposure conditions. Macrophages exposed to cigarette smoke in vivo and in vitro exhibit impaired cholesterol efflux correlating with significantly decreased ABCA1 expression, up-regulation of the TLR4/Myd88 pathway, and downstream MMP-9 and MMP-13 expression. Treatment with liver X receptor (LXR) agonist restores ABCA1 expression after short-term smoke exposure and attenuates the inflammatory response; after long-term smoke exposure, there is also attenuated physiologic and morphologic changes of emphysema. In vitro, treatment with LXR agonist decreases macrophage inflammatory activation in wild-type but not ABCA1 knockout mice, suggesting an ABCA1-dependent mechanism of action. These studies demonstrate an important association between cigarette smoke exposure and cholesterol-mediated pathways in the macrophage inflammatory response. Modulation of these pathways through manipulation of ABCA1 activity effectively blocks cigarette smoke-induced inflammation and provides a potential novel therapeutic approach for the treatment of chronic obstructive pulmonary disease.-Sonett, J., Goldklang, M., Sklepkiewicz, P., Gerber, A., Trischler, J., Zelonina, T., Westerterp, M., Lemaître, V., Okada, V., D'Armiento, J. A critical role for ABC transporters in persistent lung inflammation in the development of emphysema after smoke exposure.

Monoclonal Cell Line Generation and CRISPR/Cas9 Manipulation via Single-Cell Electroporation.

Stably transfected cell lines are widely used in drug discovery and biological research to produce recombinant proteins. Generation of these cell lines requires the isolation of multiple clones, using time-consuming dilution methods, to evaluate the expression levels of the gene of interest. A new and efficient method is described for the generation of monoclonal cell lines, without the need for dilution cloning. In this new method, arrays of patterned cell colonies and single cell transfection are employed to deliver a plasmid coding for a reporter gene and conferring resistance to an antibiotic. Using a nanofountain probe electroporation system, probe positioning is achieved through a micromanipulator with sub-micron resolution and resistance-based feedback control. The array of patterned cell colonies allows for rapid selection of numerous stably transfected clonal cell lines located on the same culture well, conferring a significant advantage over slower and labor-intensive traditional methods. In addition to plasmid integration, this methodology can be seamlessly combined with CRISPR/Cas9 gene editing, paving the way for advanced cell engineering.

Laminar shear stress upregulates endothelial Ca²âº-activated K⁺ channels KCa2.3 and KCa3.1 via a Ca²âº/calmodulin-dependent protein kinase kinase/Akt/p300 cascade.

In endothelial cells (ECs), Ca²âº-activated K⁺ channels KCa2.3 and KCa3.1 play a crucial role in the regulation of arterial tone via producing NO and endothelium-derived hyperpolarizing factors. Since a rise in intracellular Ca²âº levels and activation of p300 histone acetyltransferase are early EC responses to laminar shear stress (LS) for the transcriptional activation of genes, we examined the role of Ca²âº/calmodulin-dependent kinase kinase (CaMKK), the most upstream element of a Ca²âº/calmodulin-kinase cascade, and p300 in LS-dependent regulation of KCa2.3 and KCa3.1 in ECs. Exposure to LS (15 dyn/cm²) for 24 h markedly increased KCa2.3 and KCa3.1 mRNA expression in cultured human coronary artery ECs (3.2 ± 0.4 and 45 ± 10 fold increase, respectively; P < 0.05 vs. static condition; n = 8-30), whereas oscillatory shear (OS; ± 5 dyn/cm² × 1 Hz) moderately increased KCa3.1 but did not affect KCa2.3. Expression of KCa2.1 and KCa2.2 was suppressed under both LS and OS conditions, whereas KCa1.1 was slightly elevated in LS and unchanged in OS. Inhibition of CaMKK attenuated LS-induced increases in the expression and channel activity of KCa2.3 and KCa3.1, and in phosphorylation of Akt (Ser473) and p300 (Ser1834). Inhibition of Akt abolished the upregulation of these channels by diminishing p300 phosphorylation. Consistently, disruption of the interaction of p300 with transcription factors eliminated the induction of these channels. Thus a CaMKK/Akt/p300 cascade plays an important role in LS-dependent induction of KCa2.3 and KCa3.1 expression, thereby regulating EC function and adaptation to hemodynamic changes.

The intermediate conductance calcium-activated potassium channel KCa3.1 regulates vascular smooth muscle cell proliferation via controlling calcium-dependent signaling.

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF ß-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

Ecto-5'-nucleotidase, CD73, is an endothelium-derived hyperpolarizing factor synthase.

OBJECTIVE: Adenosine dilates human coronary arteries by activating potassium channels in an endothelial cell-independent manner. Cell surface ecto-5'-nucleotidase (CD73) rapidly dephosphorylates extracellular adenosine 5'-monophosphate to adenosine. We tested the hypothesis that coronary vasodilation to adenine nucleotides is mediated by an endothelial CD73-dependent, extracellular production of adenosine that acts as an endothelium-derived hyperpolarizing factor. METHODS AND RESULTS: Videomicroscopy showed that adenine nucleotides, but not inosine, potently dilated and hyperpolarized human coronary arteries independent of nitric oxide, prostacyclin, and classical endothelium-derived hyperpolarizing factors, whereas endothelial denudation, adenosine receptor antagonism, adenosine deaminase, or CD73 blockers reduced vasodilations. Liquid chromatography-electrospray ionization-mass spectrometry revealed adenosine accumulation in perfusates from arteries in the presence of adenosine 5'-diphosphate. CD73 was localized on the cell surface of endothelial cells, but not of vascular smooth muscle cells, and its deficiency suppressed vasodilation of mouse coronary arteries to adenine nucleotides and augmented vasodilation to adenosine. Adenosine dose-dependently dilated and hyperpolarized human coronary arteries to a similar extent as adenosine 5'-diphosphate. CONCLUSIONS: Coronary vasodilation to adenine nucleotides is associated with endothelial CD73-dependent production of extracellular adenosine that acts as an endothelium-derived hyperpolarizing factor by relaxing and hyperpolarizing underlying vascular smooth muscle cells via activating adenosine receptors. Thus, CD73 is a novel endothelium-derived hyperpolarizing factor synthase in human and mouse coronary arteries.

Vitamin D2 versus vitamin D3 supplementation in hemodialysis patients: a comparative pilot study.

BACKGROUND: In patients with chronic kidney disease, vitamin D insufficiency is highly prevalent. It can be corrected by supplementation with either vitamin D(2) or vitamin D3. Recent studies in patients without impaired kidney function suggest that vitamin D(3) is more efficient than vitamin D(2) in correcting vitamin D insufficiency. However, no direct comparison has been made in hemodialysis (HD) patients. METHODS: Thirty-nine HD patients with serum 25-hydroxyvitamin D (25(OH)D) levels =20 ng/mL were enrolled in this comparative, prospective pilot study. They were divided into 3 groups and treated over a 3-month period. Each patient received oral doses of 200,000 international units (IU) vitamin D per month according to the following treatment schedule: (i) vitamin D(2) in small fractionated doses at each HD session, 3 times per week (group D2S); (ii) vitamin D(2) once a month (group D2M); or (iii) vitamin D(3) once a month (group D3M). Changes in serum 25(OH)D levels were measured at the end of the study. RESULTS: Posttreatment serum 25(OH)D levels increased significantly in all groups. The mean ± SD serum 25(OH)D value for group D3M patients (40 ± 13 ng/mL) was significantly higher than that for groups D2M (25 ± 9 ng/mL, p<0.01) and D2S patients (25 ± 9 ng/mL, p<0.01). Serum 25(OH)D increased to levels >30 ng/mL in 84% of group D3M patients, but in only 15% and 27% of group D2M and D2S subjects, respectively. CONCLUSION: Vitamin D(3) is more effective than vitamin D(2) in providing adequate 25(OH)D serum levels in HD patients.

Activation of the TLR4 signaling pathway and abnormal cholesterol efflux lead to emphysema in ApoE-deficient mice.

Smokers with airflow obstruction have an increased risk of atherosclerosis, but the relationship between the pathogenesis of these diseases is not well understood. To determine whether hypercholesterolemia alters lung inflammation and emphysema formation, we examined the lung phenotype of two hypercholesterolemic murine models of atherosclerosis at baseline and on a high-fat diet. Airspace enlargement developed in the lungs of apolipoprotein E-deficient (Apoe(-/-)) mice exposed to a Western-type diet for 10 wk. An elevated number of macrophages and lymphocytes accompanied by an increase in matrix metalloproteinase-9 (MMP-9) activity and MMP-12 expression was observed in the lungs of Apoe(-/-) mice on a Western-type diet. In contrast, low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice did not exhibit lung destruction or inflammatory changes. Most importantly, we revealed augmented expression of the downstream targets of the Toll-like receptor (TLR) pathway, interleukin-1 receptor-associated kinase 1, and granulocyte colony-stimulating factor, in the lungs of Apoe(-/-) mice fed with a Western-type diet. In addition, we demonstrated overexpression of MMP-9 in Apoe(-/-) macrophages treated with TLR4 ligand, augmented with the addition of oxidized LDL, suggesting that emphysema in these mice results from the activation of the TLR pathway secondary to known abnormal cholesterol efflux. Our findings indicate that, in Apoe(-/-) mice fed with an atherogenic diet, abnormal cholesterol efflux leads to increased systemic inflammation with subsequent lung damage and emphysema formation.

[Disconnection of arteriovenous fistula: standardize the coverage of the hemorragic risk]. / Débranchement de fistule artérioveineuse : rationaliser la prise en charge du risque hémorragique.

During the disconnection of the arteriovenous fistulas, the nursing staff manages a hemorrhagic risk function of various factors requiring the use of an adapted haemostatic treatment. In front of numerous available references, it seemed interesting to us to standardize the coverage of this risk within the service of hemodialysis. We realized an audit to analyze the practices of disconnections and estimate the context of use of the haemostatic agents. A review of the consumptions as well as a medical economic study were realized on the haemostatic agents used in dialysis. A revaluation of the practices after 6 months was programmed to validate the institution of the protocol. Fifty observed disconnections brought to light numerous critical points. A decision tree was established to direct the choice of the haemostatic method. The importance of the direct compression in first intention was reminded and data sheets were realized. Two absorbable bandages were deleted by the allocation and an alginate of class III was subjected to name specific prescription. The assessment in 6 months proves the support of the teams to this new protocol by the impact on the consumptions and the follow-up of the decision tree during the disconnections. This study allowed us to harmonize a common act and to put at the disposal of the nursing only bandage haemostatics indicated in the disconnection of the arteriovenous fistulas. The spread good use was a major axis of improvement of the practices as well as a help for the integration of the new procedure.

Medical practice patterns and socio-economic factors may explain geographical variation of end-stage renal disease incidence.

BACKGROUND: Incidence rates of renal replacement therapy (RRT) for end-stage renal disease (ESRD) vary geographically not only between but within countries. This study uses data from the French REIN registry to quantify the extent to which socio-economic environment, health care supply and medical practice patterns such as early dialysis initiation or greater propensity to accept frail or elderly patients for dialysis, may explain spatial patterns of ESRD incidence in 85 French districts. METHODS: The association between age- and sex-adjusted incidence rates of RRT in 2008-09 and 17 indicators was explored at the district level with geographically appropriate methods, before and after controlling for the effects of diabetes and the other significant indicators. Rate ratios (RR) and credible intervals (CI) were estimated for a 1-SD increase of each covariate. RESULTS: Crude RRT incidence by district ranged from 85.8 to 225.5 per million inhabitants. The age- and sex-adjusted RRT incidence increased with the proportion of people unemployed (RR: 1.05, 95% CI 1.01-1.09), the population density (RR: 1.07, 95% CI 1.02-1.12) and the prevalence of diabetes (RR: 1.08, 95% CI 1.03-1.12). It also increased with the proportions of incident ESRD patients >85 years (RR: 1.02, 95% CI 0.99-1.06), of deaths within the first 3 months of RRT (RR: 1.03, 95% CI 1.0-1.06) and of nephrologists in private practice (RR: 1.05, 95% CI 1.01-1.08) and with the median estimated glomerular filtration rate (eGFR) at dialysis initiation (RR: 1.06, 95% CI 1.02-1.09). CONCLUSION: This study confirms that socio-economic factors and diabetes explain substantial between-area variations in RRT incidence and highlights the variability of practice patterns, especially decisions about RRT and their potential impact on incidence.

Cigarette smoke components induce matrix metalloproteinase-1 in aortic endothelial cells through inhibition of mTOR signaling.

Smoking is a major risk factor for heart disease, but the molecular effects of cigarette smoke on vascular cells are poorly understood. In this study, we demonstrate that matrix metalloproteinase-1 (MMP-1), a collagenase expressed in atherosclerosis and aneurysms but not in the normal vessel wall, is induced in the aortic endothelium of rabbits exposed to cigarette smoke. In vitro cigarette smoke extract (CSE) and one of its components, acrolein, inhibit the mammalian target of rapamycin (mTOR)/p70S6K pathway in human endothelial cells, and chemical inhibition of this pathway by rapamycin resulted in elevated MMP-1. Moreover, the tissue inhibitor of metalloproteases-3 (TIMP-3), a major regulator of angiogenesis, is significantly downregulated in aortic endothelial cells treated with CSE, acrolein, or rapamycin. These data indicate that inhibition of mTOR by cigarette smoke components is a key event in the modulation of endothelial MMP-1 and TIMP-3 expression. Our study suggests that circulating smoke components, including acrolein, contribute to vascular diseases through enhanced MMP-1 and decreased TIMP-3 secretion in the endothelium, potentially leading to impaired angiogenesis, matrix disruption, and vessel injury.

Anemia normalization in patients with type 2 diabetes and chronic kidney disease: results of the NEPHRODIAB2 randomized trial.

STATEMENTS OF THE PROBLEM: Correction of anemia in type 2 diabetes (T2DM) patients with chronic kidney disease stages 3-4 may slow the decline of kidney function but may increase cardiovascular risk through higher hematocrit. The NEPHRODIAB2 study was designed to assess efficacy and safety of complete hemoglobin (Hb) normalization in these patients. METHODS: We randomly assigned 89 T2DM patients with an estimated glomerular filtration rate (eGFR; abbreviated 175 Modification of Diet in Renal Disease formula) of 25 to 60 ml/min per 1.73 m(2) and moderate anemia (Hb, 100-129 g/l) to a target Hb value in subnormal range (110-129g/l, group 1, n=43) or normal range (130-149 g/l, group 2, n=46). The primary end point was eGFR decline after 2 years of follow-up. Secondary end points included iron and erythropoietin dosage, quality of life (Medical Outcomes Study 36-item Short-Form Health Survey scores) and adverse events. RESULTS: Six months after randomization, the mean Hb levels were <120 g/l in group 1 and >130 g/l in group 2 (P<.05 at 6, 12, 18 and 24 months). Blood pressure, 24-h proteinuria and HbA1c did not differ during follow-up (P>.05). Two-year declines in eGFR were -8.7±12.2 in group 1 and -5.1±7.8 ml/min per 1.73 m(2) in group 2 (P=.29). Mean weekly use of erythropoietin was 7.8±11.6 µg in group 1 and 30.1±33.6 µg in group 2 (P<.0001). There was no significant difference regarding Medical Outcomes Study 36-item Short-Form Health Survey score change or adverse event occurrence. CONCLUSIONS: In this trial, normalization of Hb level in T2DM patients with chronic kidney disease was safe but did not significantly slow renal function decline and increased treatment cost due to erythropoietin use.

[Renal malakoplakia: an underestimate cause of renal failure]. / Malacoplakie renale: une cause méconnue d'insuffisance rénale.

Malakoplakia is an inflammatory granulomatous disease induced by defective phagocytic activity of macrophage. Malakoplakia is histologically characterized by the presence of Michaelis-Gutmann bodies in macrophages. Although not uncommon in the genito-urinary tract, isolated malakoplakia of the kidney is rarely found. Its main clinical presentation associates acute renal failure and acute pyelonephritis. The clue for diagnosis of renal malakoplakia is based on renal biopsy showing Michaelis-Gutmann bodies. Establishing the diagnosis of renal malakoplakia is essential as it determines the choice of antibiotics and duration of treatment. Prognosis remains poor, leading frequently to chronic renal failure. In this paper, we report four cases of renal malakoplakia and discuss clinical presentation, biological and pathological features, treatment and prognosis of this disease.

Matrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs in non-neoplastic diseases.

Cellular functions within tissues are strictly regulated by the tissue microenvironment which comprises extracellular matrix and extracellular matrix-deposited factors such as growth factors, cytokines and chemokines. These molecules are metabolized by matrix metalloproteinases (MMP), a disintegrin and metalloproteinases (ADAM) and ADAM with thrombospondin motifs (ADAMTS), which are members of the metzincin superfamily. They function in various pathological conditions of both neoplastic and non-neoplastic diseases by digesting different substrates under the control of tissue inhibitors of metalloproteinases (TIMP) and reversion-inducing, cysteine-rich protein with Kazal motifs (RECK). In neoplastic diseases MMP play a central role in cancer cell invasion and metastases, and ADAM are also important to cancer cell proliferation and progression through the metabolism of growth factors and their receptors. Numerous papers have described the involvement of these metalloproteinases in non-neoplastic diseases in nearly every organ. In contrast to the numerous review articles on their roles in cancer cell proliferation and progression, there are very few articles discussing non-neoplastic diseases. This review therefore will focus on the properties of MMP, ADAM and ADAMTS and their implications for non-neoplastic diseases of the cardiovascular system, respiratory system, central nervous system, digestive system, renal system, wound healing and infection, and joints and muscular system.

Altered lymphocyte trafficking and diminished airway reactivity in transgenic mice expressing human MMP-9 in a mouse model of asthma.

Matrix metalloproteinase-9 (MMP-9) is hypothesized to facilitate leukocyte extravasation and extracellular remodeling in asthmatic airways. Careful descriptive studies have shown that MMP-9 levels are higher in the sputum of asthmatics; however, the consequence of increased MMP-9 activity has not been determined in this disease. We induced asthma in transgenic mice that express human MMP-9 in the murine lung tissue macrophage to determine the direct effect of human MMP-9 expression on airway inflammation. Transgenic (TG) and wild-type (WT) mice were immunized and challenged with ovalbumin. Forty-eight hours after the ovalbumin challenge, airway hyperresponsiveness (AHR) was measured, and inflammatory cell infiltration was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue. Baseline levels of inflammation were similar in the TG and WT groups of mice, and pulmonary eosinophilia was established in both groups by sensitization and challenge with ovalbumin. There was a significant reduction in AHR in sensitized and challenged trangenics compared with WT controls. Although total BALF cell counts were similar in both groups, the lymphocyte number in the lavage of the TG group was significantly diminished compared with the WT group (0.25 +/- 0.08 vs. 0.89 +/- 0.53; P = 0.0032). In addition, the draining lymphocytes were found to be larger in the TG animals compared with the WT mice. Equal numbers of macrophages, eosinophils, and neutrophils were seen in both groups. IL-13 levels were found to be lower in the sensitized TG compared with the WT mice. These results demonstrate an inverse relationship between human MMP-9 and AHR and suggest that MMP-9 expression alters leukocyte extravasation by reducing lymphocyte accumulation in the walls of asthmatic airways.

Role for cathepsin K in emphysema in smoke-exposed guinea pigs.

The protease-antiprotease imbalance in the lung plays an important role in the pathogenesis of smoke-induced emphysema. The aim of this study was to characterize the proteolytic responses leading to emphysema formation in the guinea pig smoke exposure model. Guinea pigs were exposed to cigarette smoke for 1, 2, 4, 8, and 12 weeks. Age-matched guinea pigs exposed to room air served as controls. Cigarette smoke induced inflammation after 4 weeks and generated emphysematous changes in the guinea pigs after 12 weeks of smoke exposure. Increased phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases was demonstrated post cigarette smoke exposure. A decrease in elastin and collagen and the loss of type III collagen were observed in the alveolar wall of smoke-exposed guinea pigs. Interestingly, no change was seen in the expression of collagenolytic matrix metalloproteinases. Furthermore, the authors observed a 3-fold increase in cathepsin K activity in the lungs of smoke-exposed guinea pigs. The significance of this finding was supported by human studies that demonstrate increased expression of cathepsin K in the lungs of patients with emphysema. Elevation of cathepsin K in guinea pig lungs after smoke exposure likely constitutes a critical event leading to the disruption of lung extracellular matrix in this model.

Higher parathyroid hormone (PTH) concentrations with the Architect PTH assay than with the Elecsys assay in hemodialysis patients, and a simple way to standardize these two methods.

BACKGROUND: The Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines recommend maintaining serum parathyroid hormone (PTH) concentration between 150 and 300 pg/mL in patients with chronic kidney disease (CKD) stage 5. However, a marked inter-method variability in PTH measurement has been reported recently. The aim of this study was to evaluate whether harmonization of the results measured with two commercial kits known to produce significantly different serum PTH concentrations could be reasonably achieved by a simple procedure. METHODS: The study comprised a total of 216 hemodialyzed patients in whom blood was collected immediately before a dialysis session. The patients were from three dialysis centers, which defined three groups (119, 34, and 63 patients for groups 1, 2, and 3, respectively). PTH was measured by two automated assays, the Elecsys (Roche Diagnostics) and Architect (Abbott Diagnostics) assays, in three different laboratories and with different lots of reagents. We arbitrarily chose the Roche assay as the reference method, because several studies had previously shown that the concentrations measured with this assay were very close to the Allegro assay used in the studies that defined the K/DOQI thresholds. Data are median (interquartile range). RESULTS: The median PTH concentrations were higher (p<0.001) in the Architect assay [238 (140-434) pg/mL] when compared to the Elecsys assay [182 (109-338) pg/mL]. Bland-Altman plots in the three groups showed a similar proportional bias between both kits. The Architect PTH/Elecsys PTH ratios were similar in the three groups [1.30 (1.25-1.35), 1.30 (1.19-1.39), and 1.31 (1.25-1.35)], and the ratio was 1.30 (1.25-1.35) in the cohort (pooling the three groups). In the whole population, 53 patients (24.5%) were classified differently by the two kits according to the K/DOQI cut-off values. We divided the Architect values by 1.3 to obtain "corrected" values. These corrected Architect values were not different to the measured Elecsys values, and the Bland-Altman plot comparing the Elecsys and the corrected Artchitect values did not show any systematic proportional bias. Only six patients (2.8%) were still classified differently by the Elecsys and the corrected Architect concentrations. CONCLUSIONS: We propose to divide the PTH values measured with the Architect PTH assay by 1.3 so that the corrected values are almost identical to those measured with the Elecsys assay.