Secretory Protease Research Articles

Characterization of The Surgical Leakage Collected After Breast Cancer Surgery and Studying Their Effect on Breast Cancer Cell Line

Globally, breast cancer is the most common cancer in women and the second leading cause of death. The choice of breast cancer treatment is based on its clinical staging. Although there is a development in the adjuvant of systemic treatments, surgery is a primary choice for treatment. One of those complications is surgical leakage which is suggested to have many cell growth factors, cytokines, and chemokines in order to tissue repair process. Many studies reported that surgical leakage may have a role in tumor progression and local recurrence by inducing the lasting cancer cells. Invasion and metastasis are one of several cellular mechanisms in cancer development and progression through secretions of many inflammatory mediators. For example, TNF-α is one of the inflammatory mediators that up-regulates the expression and secretion of proteases (such as matrix metalloproteinases MMPs family) which are involved in carcinoma cell motility, invasion, and metastasis. Indeed the role of TNF-α in cancer progression and metastasis is depending on its ability to produce and stimulate (MMP) which have a role in tumor angiogenesis, invasion and metastasis process. Also one of TNF-α function is to induce epithelial-mesenchymal transition (EMT) process which known to occur through embryogenesis, metastasis. Previous studies by the authors showed that TNF- α is highly expressed and secreted by tumor microenvironment by Egyptian cancer patients. Using different concentrations of recombinant TNF-α, we test the morphological changes of cancer cells from a round shape to spindle shape (migratory cells), in addition, we studied how different concentrations of TNF- α induce invasion and migration on MDA-MB-231 breast cancer cell line using Matrigel Invasion chambers.

Loss of control of the culturable bacteria in the hindgut of Bombyx mori after Cry1Ab ingestion

Bt protein, produced by Bacillus thuringiensis, can bind receptors to destroy the physiological functions of the insect midgut. It is unknown whether Bt can also target the hindgut and influence its defense against fecal bacteria. Here we show that Crystal protein 1Ab (Cry1Ab), a Bt protein, was detected in the larval hindgut contents of Bombyx mori after ingestion of this toxin protein. The number of fecal bacteria that can be inhibited by the hindgut prophenoloxidase-induced melanization was significantly enhanced after oral ingestion of Cry1Ab. Although the hindgut contents became brown, the activity of hindgut phenoloxidase was decreased. LC-MS/MS analysis of the hindgut lumen contents revealed that many new proteins including several proteases were newly secreted. The enhanced secretion of proteases cleaved prophenoloxidase to decrease its activity, including the corresponding activity to inhibit the fecal bacteria. In addition, after ingestion of Cry1Ab, the pylorus (between the midgut and hindgut) could not autonomously contract due to the physical detachment of the acellular cuticle-like membrane from the epidermal cells, which prevented the movement of food from the midgut to the hindgut. Some cells in the cryptonephry of the hindgut became swollen and degraded, possibly due to the presence of Cry1Ab in the hindgut. These findings demonstrate that the inhibition of feces bacteria by the hindgut prophenoloxidase-induced melanization is out of control after Cry1Ab ingestion.

Out-Smarting the Host: Bacteria Maneuvering the Immune Response to Favor Their Survival.

Bacteria adapt themselves to various environmental conditions in nature, which can lead to bacterial adaptation and persistence in the host as commensals or pathogens. In healthy individuals, host defense mechanisms prevent the opportunistic bacteria/commensals from becoming a pathological infection. However, certain pathological conditions can impair normal defense barriers leading to bacterial survival and persistence. Under pathological conditions such as chronic lung inflammation, bacteria employ various mechanisms from structural changes to protease secretion to manipulate and evade the host immune response and create a niche permitting commensal bacteria to thrive into infections. Therefore, understanding the mechanisms by which pathogenic bacteria survive in the host tissues and organs may offer new strategies to overcome persistent bacterial infections. In this review, we will discuss and highlight the complex interactions between airway pathogenic bacteria and immune responses in several major chronic inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD).

Antimicrobial peptide (AMP) from Zingiber zerumbet rhizomes with inhibitory effect on Pythium myriotylum secretory proteases and zoospore viability

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50-80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75kDa and 25kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000MW cut-off) filter, yielded a prominent band of 25kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4µg protein and an IC50 value of 0.59µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.

Practical context of enzymatic treatment for wound healing: A secreted protease approach (Review).

Skin wounds have been extensively studied as their healing represents a critical step towards achieving homeostasis following a traumatic event. Dependent on the severity of the damage, wounds are categorized as either acute or chronic. To date, chronic wounds have the highest economic impact as long term increases wound care costs. Chronic wounds affect 6.5 million patients in the United States with an annual estimated expense of $25 billion for the health care system. Among wound treatment categories, active wound care represents the fastest-growing category due to its specific actions and lower costs. Within this category, proteases from various sources have been used as successful agents in debridement wound care. The wound healing process is predominantly mediated by matrix metalloproteinases (MMPs) that, when dysregulated, result in defective wound healing. Therapeutic activity has been described for animal secretions including fish epithelial mucus, maggot secretory products and snake venom, which contain secreted proteases (SPs). No further alternatives for use, sources or types of proteases used for wound healing have been found in the literature to date. Through the present review, the context of enzymatic wound care alternatives will be discussed. In addition, substrate homology of SPs and human MMPs will be compared and contrasted. The purpose of these discussions is to identify and propose the stages of wound healing in which SPs may be used as therapeutic agents to improve the wound healing process.

The feather degradation mechanisms of a new Streptomyces sp. isolate SCUT-3

Feather waste is the highest protein-containing resource in nature and is poorly reused. Bioconversion is widely accepted as a low-cost and environmentally benign process, but limited by the availability of safe and highly efficient feather degrading bacteria (FDB) for its industrial-scale fermentation. Excessive focuses on keratinase and limited knowledge of other factors have hindered complete understanding of the mechanisms employed by FDB to utilize feathers and feather cycling in the biosphere. Streptomyces sp. SCUT-3 can efficiently degrade feather to products with high amino acid content, useful as a nutrition source for animals, plants and microorganisms. Using multiple omics and other techniques, we reveal how SCUT-3 turns on its feather utilization machinery, including its colonization, reducing agent and protease secretion, peptide/amino acid importation and metabolism, oxygen consumption and iron uptake, spore formation and resuscitation, and so on. This study would shed light on the feather utilization mechanisms of FDBs.

Oral feeding with probiotic Lactobacillus rhamnosus attenuates cigarette smoke-induced COPD in C57Bl/6 mice: Relevance to inflammatory markers in human bronchial epithelial cells.

COPD is a prevalent lung disease with significant impacts on public health. Affected airways exhibit pulmonary neutrophilia and consequent secretion of pro-inflammatory cytokines and proteases, which result in lung emphysema. Probiotics act as nonspecific modulators of the innate immune system that improve several inflammatory responses. To investigate the effect of Lactobacillus rhamnosus (Lr) on cigarette smoke (CS)-induced COPD C57Bl/6 mice were treated with Lr during the week before COPD induction and three times/week until euthanasia. For in vitro assays, murine bronchial epithelial cells as well as human bronchial epithelial cells exposed to cigarette smoke extract during 24 hours were treated with Lr 1 hour before CSE addition. Lr treatment attenuated the inflammatory response both in the airways and lung parenchyma, reducing inflammatory cells infiltration and the production of pro-inflammatory cytokines and chemokines. Also, Lr-treated mice presented with lower metalloproteases in lung tissue and lung remodeling. In parallel to the reduction in the expression of TLR2, TLR4, TLR9, STAT3, and NF-κB in lung tissue, Lr increased the levels of IL-10 as well as SOCS3 and TIMP1/2, indicating the induction of an anti-inflammatory environment. Similarly, murine bronchial epithelial cells as well as human bronchial epithelial cells (BEAS) exposed to CSE produced pro-inflammatory cytokines and chemokines, which were inhibited by Lr treatment in association with the production of anti-inflammatory molecules. Moreover, the presence of Lr also modulated the expression of COPD-associated transcription found into BALF of COPD mice group, i.e., Lr downregulated expression of NF-κB and STAT3, and inversely upregulated increased expression of SOCS3. Thus, our findings indicate that Lr modulates the balance between pro- and anti-inflammatory cytokines in human bronchial epithelial cells upon CS exposure and it can be a useful tool to improve the lung inflammatory response associated with COPD.

Effect of Salt and pH on the Growth and Production of Alkaline Proteases From Haloalkaliphilic Bacteria Isolated From Saline Desert

Saline desert of Kutch is yet unexplored for its microbial diversity and biotechnological potential. Proteases are significant with respect to their applications and cellular roles. A total of 15 bacteria isolated from the Little Rann of Kutch were isolated and screened for their growth and protease secretion at different pH and salt concentrations. Effect of NaCl concentrations and pH was assessed on growth and production of the proteases. Overall, 50 % of the isolates grew in the range of 0-20 % (w/v) NaCl, while nearly 45 % of them optimally produced extracellular proteases at 10 % (w/v) NaCl and 20 % isolates produced protease optimally at 15% (w/v) NaCl concentration. Majority of the isolates secreted protease in pH range 7-11 and nearly 70 % the isolates produced enzyme in the broad range of pH, 7-11 and nearly 6 % isolates produced protease in the range of pH 9-11. Further, around 40% of the isolates produced proteases optimally at pH 9-11. Therefore, the isolates demonstrated a broad range of pH and salt for growth and protease production. The study suggests wide prospective of the applications of these organisms and their enzymes.

Rubus idaeus reduces inflammation and prevents chondrocyte catabolism and cartilage loss

Purpose: Osteoarthritis is characterized by cartilage degradation resulting from several mechanisms controlling the function of chondrocytes. The activation of chondrocyte results in an increased secretion of matrix proteases, contributing to aggrecan degradation and cleavage of type II collagen network. Moreover, cartilage loss is also associated with a synovial inflammation with immune cell infiltration and cytokines secretion. It has been suggested that natural products possess anti-inflammatory properties that may inhibit the release of key osteoarthritis-related cytokines.

Exploring the Potential for Fungal Antagonism and Cell Wall Attack by Bacillus subtilis natto.

To develop more ecologically sustainable agricultural practices requires that we reduce our reliance on synthetic chemical pesticides for crop protection. This will likely involve optimized biocontrol approaches – the use of beneficial soil microbes to attack potential plant pathogens to protect plants from diseases. Many bacterial species, including strains of Bacillus subtilis, have been explored for their biocontrol properties, as they can control the growth of harmful fungi, often by disrupting the fungal cell wall. A strain that is not often considered for this particular application is Bacillus subtilis natto, primarily known for fermenting soybeans via cell wall degradation in the Japanese probiotic dish “natto.” Because deconstruction of the fungal cell wall is considered an important biocontrol trait, we were motivated to explore the possible anti-fungal properties of the B. subtilis natto strain. We show that B. subtilis natto can use complex fungal material as a carbon source for growth, and can effectively deconstruct fungal cell walls. We found degradation of fungal cell wall proteins, and showed that growth on a mix of peptides was very strong. We also found that intact fungal cell walls can induce the secretion of chitinases and proteases. Surprisingly, we could show that chitin, the bulk component of the fungal cell wall, does not permit successful growth of the natto strain or induce the secretion of chitinolytic enzymes, although these were produced during exposure to proteins or to complex fungal material. We have further shown that protease secretion is likely a constitutively enabled mechanism for nutrient scavenging by B. subtilis natto, as well as a potent tool for the degradation of fungal cell walls. Overall, our data highlight B. subtilis natto as a promising candidate for biocontrol products, with relevant behaviors that can be optimized by altering growth conditions. Whereas it is common for bacterial biocontrol products to be supplied with chitin or chitosan as a priming polysaccharide, our data indicate that this is not a useful approach with this particular bacterium, which should instead be supplied with either glucose or attenuated fungal material.

A computational bio-chemo-mechanical model of in vivo tissue-engineered vascular graft development.

Stenosis is the primary complication of current tissue-engineered vascular grafts used in pediatric congenital cardiac surgery. Murine models provide considerable insight into the possible mechanisms underlying this situation, but they are not efficient for identifying optimal changes in scaffold design or therapeutic strategies to prevent narrowing. In contrast, computational modeling promises to enable time- and cost-efficient examinations of factors leading to narrowing. Whereas past models have been limited by their phenomenological basis, we present a new mechanistic model that integrates molecular- and cellular-driven immuno- and mechano-mediated contributions to in vivo neotissue development within implanted polymeric scaffolds. Model parameters are inferred directly from in vivo measurements for an inferior vena cava interposition graft model in the mouse that are augmented by data from the literature. By complementing Bayesian estimation with identifiability analysis and simplex optimization, we found optimal parameter values that match model outputs with experimental targets and quantify variability due to measurement uncertainty. Utility is illustrated by parametrically exploring possible graft narrowing as a function of scaffold pore size, macrophage activity, and the immunomodulatory cytokine transforming growth factor beta 1 (TGF-β1). The model captures salient temporal profiles of infiltrating immune and synthetic cells and associated secretion of cytokines, proteases, and matrix constituents throughout neovessel evolution, and parametric studies suggest that modulating scaffold immunogenicity with early immunomodulatory therapies may reduce graft narrowing without compromising compliance.

Molecular and Cellular Pathways Contributing to Joint Damage in Rheumatoid Arthritis.

Rheumatoid arthritis is a chronic autoimmune syndrome associated with several genetic, epigenetic, and environmental factors affecting the articular joints contributing to cartilage and bone damage. Although etiology of this disease is not clear, several immune pathways, involving immune (T cells, B cells, dendritic cells, macrophages, and neutrophils) and nonimmune (fibroblasts and chondrocytes) cells, participate in the secretion of many proinflammatory cytokines, chemokines, proteases (MMPs, ADAMTS), and other matrix lysing enzymes that could disturb the immune balance leading to cartilage and bone damage. The presence of autoantibodies preceding the clinical onset of arthritis and the induction of bone erosion early in the disease course clearly suggest that initiation events damaging the cartilage and bone start very early during the autoimmune phase of the arthritis development. During this process, several signaling molecules (RANKL-RANK, NF-κB, MAPK, NFATc1, and Src kinase) are activated in the osteoclasts, cells responsible for bone resorption. Hence, comprehensive knowledge on pathogenesis is a prerequisite for prevention and development of targeted clinical treatment for RA patients that can restore the immune balance improving clinical therapy.

Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles.

Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases in humans. The wide distribution of Acanthamoeba, the characteristic of being amphizoic and the severity of the disease motivate researchers to focus on the isolation of these organisms, but also in demonstrating direct and indirect factors that could indicate a possible pathogenic potential. Here, we performed the characterization of the pathogenic potential of an Acanthamoeba T5 isolate collected from a water source in a hospital. Osmo- and thermotolerance, the secretion of proteases and the effect of trophozoites over cell monolayers were analyzed by different methodologies. Additionally, we confirm the secretion of extracellular vesicles (EVs) of this isolate incubated at two different temperatures, and the presence of serine and cysteine proteases in these vesicles. Finally, using atomic force microscopy, we determined some nanomechanical properties of the secreted vesicles and found a higher value of adhesion in the EVs obtained at 37 °C, which could have implications in the parasite´s survival and damaging potential in two different biological environments.

Auto-Regulation of the Thyroid Gland Beyond Classical Pathways.

This mini-review asks how self-regulation of the thyroid gland is realized at the cellular and molecular levels by canonical and non-canonical means. Canonical pathways of thyroid regulation comprise thyroid stimulating hormone-triggered receptor signaling. As part of non-canonical regulation, we hypothesized an interplay between protease-mediated thyroglobulin processing and thyroid hormone release into the circulation by means of thyroid hormone transporters like Mct8. We proposed a sensing mechanism by different thyroid hormone transporters, present in specific subcellular locations of thyroid epithelial cells, selectively monitoring individual steps of thyroglobulin processing, and thus, the cellular thyroid hormone status. Indeed, we found that proteases and thyroid hormone transporters are functionally inter-connected, however, in a counter-intuitive manner fostering self-thyrotoxicity in particular in Mct8- and/or Mct10-deficient mice. Furthermore, the possible role of the G protein-coupled receptor Taar1 is discussed, because we detected Taar1 at cilia of the apical plasma membrane of thyrocytes in vitro and in situ. Eventually, through pheno-typing Taar1-deficient mice, we identified a co-regulatory role of Taar1 and the thyroid stimulating hormone receptors. Recently, we showed that inhibition of thyroglobulin-processing enzymes results in disappearance of cilia from the apical pole of thyrocytes, while Taar1 is re-located to the endoplasmic reticulum. This pathway features a connection between thyrotropin-stimulated secretion of proteases into the thyroid follicle lumen and substrate-mediated self-assisted control of initially peri-cellular thyroglobulin processing, before its reinternalization by endocytosis, followed by extensive endo-lysosomal liberation of thyroid hormones, which are then released from thyroid follicles by means of thyroid hormone transporters.

Capillary-assisted microfluidic biosensing platform captures single cell secretion dynamics in nanoliter compartments

Cancer cells continuously secrete inflammatory biomolecules which play significant roles in disease progression and tumor metastasis toward secondary sites. Despite recent efforts to capture cancer cells' intercellular secretion heterogeneity using microfluidics, the challenges in operation of these systems as well as the complexity of designing a biosensing assay for long-term and real-time measurement of single cell secretions have become grand research barriers. Here, we present a new capillary-based microfluidic biosensing approach to easily and reliably capture ~500 single cells inside isolated dead-end nanoliter compartments using simple pipette injection, and quantify their individual secretion dynamics at the single cell resolution over a long period of culture (~16 h). We first present a detailed investigation of the fluid mechanics underlying the formation of nanoliter compartments in the microfluidic system. Based on the measurement of single cell capture efficiency, we employ a one-step FRET-based biosensor which monitors the single cancer cells' protease activity. The sensor reports the fluorescent signal as a product of amino acid chain cleavage and reduction in its quenching capability. Using the single cell protease secretion data, we identified modes of cell secretion dynamics in our cell sample. While most of the cells had low secretion levels, two other smaller and more aggressive secretion dynamics were cells with secretion modes that include sharp spikes or slow but progressive trend. The method presented here overcomes the difficulties associated with performing single cell secretion assays, enabling a feasible and reliable technique for high throughput measurement of metabolic activities in cancer cells.

Collagen-Immobilized Extracellular FRET Reporter for Visualizing Protease Activity Secreted by Living Cells.

Despite the diverse roles of cell-secreted proteases in the extracellular matrix (ECM), classical methods to analyze protease activity have not been explored at the cell culture site. Here, we report a stable, matrix-sticky, and protease-sensitive extracellular reporter that comprises a collagen-binding protein and a Förster resonance energy transfer (FRET) coupler of an enhanced green fluorescent protein and a small dye molecule. The extracellular FRET reporter via split intein-mediated protein trans-splicing is able to adhere to collagen matrices, leading to fluorescence changes by matrix metalloproteinase-2 (MMP2) activity during living cell culture without impeding cell viability. When a proMMP2 mutant (Y581A) with altered protease secretion and activity was transfected into cancer cells, the reporter revealed a dramatic reduction in MMP2 activity in both two- and three-dimensional culture systems, compared with cells transfected with wild-type proMMP2. Our reporter is immediately amenable to monitor protease activity in diverse ECM-resident cells as well as to study protease-related extracellular signaling and tissue remodeling.

The motif EXEXXXL in the cytosolic tail of the secretory human proprotein convertase PC7 regulates its trafficking and cleavage activity

Many secretory proteins are activated by cleavage at specific sites. The proprotein convertases (PCs) form a family of nine secretory subtilisin-like serine proteases, seven of which cleave at specific basic residues within the trans-Golgi network, granules, or at the cell surface/endosomes. The seventh member, PC7, is a type-I transmembrane (TM) protein with a 97-residue-long cytosolic tail (CT). PC7 sheds human transferrin receptor 1 (hTfR1) into soluble shTfR1 in endosomes. To better understand the physiological roles of PC7, here we focused on the relationship between the CT-regulated trafficking of PC7 and its ability to shed hTfR1. Deletion of the TMCT resulted in soluble PC7 and loss of its hTfR1 shedding activity. Extensive CT deletions and mutagenesis analyses helped us zoom in on three residues in the CT, namely Glu-719, Glu-721, and Leu-725, that are part of a novel motif, EXEXXXL725, critical for PC7 activity on hTfR1. NMR studies of two 14-mer peptides mimicking this region of the CT and its Ala variants revealed that the three exposed residues are on the same side of the molecule. This led to the identification of adaptor protein 2 (AP-2) as a protein that recognizes the EXEXXXL725 motif, thus representing a potentially new regulator of PC7 trafficking and cleavage activity. Immunocytochemistry of the subcellular localization of PC7 and its Ala variants of Leu-725 and Glu-719 and Glu-721 revealed that Leu-725 enhances PC7 localization to early endosomes and that, together with Glu-719 and Glu-721, it increases the endosomal activity of PC7 on hTfR1.

Killer Immunoglobulin-Like Receptor 2DL4 (CD158d) Regulates Human Mast Cells both Positively and Negatively: Possible Roles in Pregnancy and Cancer Metastasis.

Killer immunoglobulin-like receptor (KIR) 2DL4 (CD158d) was previously thought to be a human NK cell-specific protein. Mast cells are involved in allergic reactions via their KIT-mediated and FcɛRI-mediated responses. We recently detected the expression of KIR2DL4 in human cultured mast cells established from peripheral blood of healthy volunteers (PB-mast), in the human mast cell line LAD2, and in human tissue mast cells. Agonistic antibodies against KIR2DL4 negatively regulate the KIT-mediated and FcɛRI-mediated responses of PB-mast and LAD2 cells. In addition, agonistic antibodies and human leukocyte antigen (HLA)-G, a natural ligand for KIR2DL4, induce the secretion of leukemia inhibitory factor and serine proteases from human mast cells, which have been implicated in pregnancy establishment and cancer metastasis. Therefore, KIR2DL4 stimulation with agonistic antibodies and recombinant HLA-G protein may enhance both processes, in addition to suppressing mast-cell-mediated allergic reactions.

Hypermutation as an Evolutionary Mechanism for Achromobacter xylosoxidans in Cystic Fibrosis Lung Infection.

Achromobacter xylosoxidans can cause chronic infections in the lungs of patients with cystic fibrosis (CF) by adapting to the specific environment. The study of longitudinal isolates allows to investigate its within-host evolution to unravel the adaptive mechanisms contributing to successful colonization. In this study, four clinical isolates longitudinally collected from two chronically infected patients underwent whole genome sequencing, de novo assembly and sequence analysis. Phenotypic assays were also performed. The isolates coming from one of the patients (patient A) presented a greater number of genetic variants, diverse integrative and conjugative elements, and different protease secretion. In the first of these isolates (strain A1), we also found a large deletion in the mutS gene, involved in DNA mismatch repair (MMR). In contrast, isolates from patient B showed a lower number of variants, only one integrative and mobilizable element, no phenotypic changes, and no mutations in the MMR system. These results suggest that in the two patients the establishment of a chronic infection was mediated by different adaptive mechanisms. While the strains isolated from patient B showed a longitudinal microevolution, strain A1 can be clearly classified as a hypermutator, confirming the occurrence and importance of this adaptive mechanism in A. xylosoxidans infection.

Role of Cyclosporine in Gingival Hyperplasia: An In Vitro Study on Gingival Fibroblasts.

Background: Gingival hyperplasia could occur after the administration of cyclosporine A. Up to 90% of the patients submitted to immunosuppressant drugs have been reported to suffer from this side effect. The role of fibroblasts in gingival hyperplasia has been widely discussed by literature, showing contrasting results. In order to demonstrate the effect of cyclosporine A on the extracellular matrix component of fibroblasts, we investigated the gene expression profile of human fibroblasts after cyclosporine A administration. Materials and methods: Primary gingival fibroblasts were stimulated with 1000 ng/mL cyclosporine A solution for 16 h. Gene expression levels of 57 genes belonging to the “Extracellular Matrix and Adhesion Molecules” pathway were analyzed using real-time PCR in treated cells, compared to untreated cells used as control. Results: Expression levels of different genes were significantly de-regulated. The gene CDH1, which codes for the cell adhesion protein E-cadherin, showed up-regulation. Almost all the extracellular matrix metalloproteases showed down-regulation (MMP8, MMP11, MMP15, MMP16, MMP24, MMP26). The administration of cyclosporine A was followed by down-regulation of other genes: COL7A1, the transmembrane receptors ITGB2 and ITGB4, and the basement membrane constituents LAMA2 and LAMB1. Conclusion: Data collected demonstrate that cyclosporine inhibits the secretion of matrix proteases, contributing to the accumulation of extracellular matrix components in the gingival connective tissue, causing gingival overgrowth. Patients affected by gingival overgrowth caused by cyclosporine A need to be further investigated in order to determine the role of this drug on fibroblasts.