Characterization of Biofilm Formation by Mycobacterium chimaera on Medical Device Materials.

Non-tuberculous mycobacteria (NTM) are widespread in the environment and are a public health concern due to their resistance to antimicrobial agents. The colonization of surgical heater-cooler devices (HCDs) by the slow-growing NTM species Mycobacterium chimaera has recently been linked to multiple invasive infections in patients worldwide. The resistance of M. chimaera to antimicrobials may be aided by a protective biofilm matrix of extracellular polymeric substances (EPS). This study explored the hypothesis that M. chimaera can form biofilms on medically relevant materials. Several M. chimaera strains, including two HCD isolates, were used to inoculate a panel of medical device materials. M. chimaera colonization of the surfaces was monitored for 6 weeks. M. chimaera formed a robust biofilm at the air-liquid interface of borosilicate glass tubes, which increased in mass over time. M. chimaera was observed by 3D Laser Scanning Microscopy to have motility during colonization, and form biofilms on stainless steel, titanium, silicone and polystyrene surfaces during the first week of inoculation. Scanning electron microscopy (SEM) of M. chimaera biofilms after 4 weeks of inoculation showed that M. chimaera cells were enclosed entirely in extracellular material, while cryo-preserved SEM samples further revealed that an ultrastructural component of the EPS matrix was a tangled mesh of 3D fiber-like projections connecting cells. Considering that slow-growing M. chimaera typically has culture times on the order of weeks, the microscopically observed ability to rapidly colonize stainless steel and titanium surfaces in as little as 24 h after inoculation is uncharacteristic. The insights that this study provides into M. chimaera colonization and biofilm formation of medical device materials are a significant advance in our fundamental understanding of M. chimaera surface interactions and have important implications for research into novel antimicrobial materials, designs and other approaches to help reduce the risk of infection.

Impact of contact lens materials on multipurpose contact lens solution disinfection activity against Fusarium solani.

OBJECTIVE: To investigate the effects of eight different soft contact lenses on disinfection efficacy of a multipurpose solution (MPS) containing polyhexamethylene biguanide (PHMB) against Fusarium solani. METHODS: Six silicone hydrogel lenses (galyfilcon A, senofilcon A, comfilcon A, enfilcon A, balafilcon A, and lotrifilcon B) and two conventional hydrogel lenses (polymacon and etafilcon A) were placed in polypropylene lens cases filled with MPS containing 0.0001% PHMB and soaked for 6, 12, 24, 72, and 168 hours. After each interval, depleted MPS from lens cases were removed and assayed for activity against F. solani according to International Organization for Standardization (ISO) 14729 stand-alone procedure. A portion was aliquoted for chemical analysis. RESULTS: Soaking etafilcon A, balafilcon A, and polymacon lenses for 6 hours reduced the concentration of PHMB in MPS by more than half the stated labeled concentration, with concentrations below the limit of detection for etafilcon A-depleted and balafilcon A-depleted solutions after 12 and 72 hours of soaking, respectively. Except for comfilcon A-depleted solutions, all others failed to consistently obtain one log reduction of F. solani. The solutions soaked with etafilcon A, balafilcon A, and polymacon lenses for 24 hours or more lost all or almost all fungicidal activity against F. solani. CONCLUSIONS: Over time, the disinfectant uptake by some lenses can significantly reduce the PHMB concentration and the fungicidal activity of the MPS against F. solani. Current ISO methodology does not address the reduction in microbiocidal efficacy when lenses are soaked in MPS. The ISO committee should consider adding "soaking experiments" to quantify the effect that contact lens materials have on the performance of MPSs.

Down-regulation of myeloid cell leukemia 1 by epigallocatechin-3-gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor alpha-induced apoptosis.

OBJECTIVE: Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor alpha (TNFalpha)-induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin-3-gallate (EGCG) in down-regulating Mcl-1 expression and its mechanism of RA synovial fibroblast sensitization to TNFalpha-induced apoptosis. METHODS: EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP-ribose) polymerase (PARP), Mcl-1, Bcl-2, Akt, and nuclear translocation of NF-kappaB. RESULTS: In RA synovial fibroblasts, EGCG (5-50 microM) inhibited constitutive and TNFalpha-induced Mcl-1 protein expression in a concentration- and time-dependent manner (P<0.05). Importantly, EGCG specifically abrogated Mcl-1 expression in RA synovial fibroblasts and affected Mcl-1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl-1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down-regulation of the TNFalpha-induced Akt and NF-kappaB pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF-kappaB inhibitors. Interestingly, Mcl-1 degradation by EGCG sensitized RA synovial fibroblasts to TNFalpha-induced PARP cleavage and apoptotic cell death. CONCLUSION: Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNFalpha-induced apoptosis by specifically blocking Mcl-1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA.

The role of vascular cell adhesion molecule 1/ very late activation antigen 4 in endothelial progenitor cell recruitment to rheumatoid arthritis synovium.

OBJECTIVE: Marrow-derived endothelial progenitor cells (EPCs) are important in the neovascularization that occurs in diverse conditions such as cardiovascular disorders, inflammatory diseases, and neoplasms. In rheumatoid arthritis (RA), synovial neovascularization propels disease by nourishing the inflamed and hyperproliferative synovium. This study was undertaken to investigate the hypothesis that EPCs selectively home to inflamed joint tissue and may perpetuate synovial neovascularization. METHODS: In a collagen-induced arthritis (CIA) model, neovascularization and EPC accumulation in mouse ankle synovium was measured. In an antibody-induced arthritis model, EPC recruitment to inflamed synovium was evaluated. In a chimeric SCID mouse/human synovial tissue (ST) model, mice were engrafted subcutaneously with human ST, and EPC homing to grafts was assessed 2 days later. EPC adhesion to RA fibroblasts and RA ST was evaluated in vitro. RESULTS: In mice with CIA, cells bearing EPC markers were significantly increased in peripheral blood and accumulated in inflamed synovial pannus. EPCs were 4-fold more numerous in inflamed synovium from mice with anti-type II collagen antibody-induced arthritis versus controls. In SCID mice, EPC homing to RA ST was 3-fold greater than to normal synovium. Antibody neutralization of vascular cell adhesion molecule 1 (VCAM-1) and its ligand component alpha4 integrin potently inhibited EPC adhesion to RA fibroblasts and RA ST cryosections. CONCLUSION: These data demonstrate the selective recruitment of EPCs to inflamed joint tissue. The VCAM-1/very late activation antigen 4 adhesive system critically mediates EPC adhesion to cultured RA fibroblasts and to RA ST cryosections. These findings provide evidence of a possible role of EPCs in the synovial neovascularization that is critical to RA pathogenesis.

Differential E-selectin expression by iris versus retina microvascular endothelial cells cultured from the same individuals.

The microvasculature of the eye plays a critical role in many ophthalmic diseases, including diabetic retinopathy and macular degeneration. Transcriptional profiling by gene array allows characterization of endothelial cells (EC) and can test for inherent EC diversity relative to the tissue source of the EC. Here, we established highly purified microvascular EC cultures from donor-matched human irises and retinae (4 donor pairs). We used nylon-based gene array kits to compare gene expression in paired confluent EC monolayers, under both quiescent and inflammatory agent (bacterial lipopolysaccharide, LPS; or tumor necrosis-factor alpha, TNFalpha)-activated conditions. In the absence of an inflammatory agent, iris and retinal ECs from the same donor were remarkably similar in overall gene expression profiles, except for possible differences in the expression of platelet-derived growth factor-A and a DNA mismatch repair protein (mutL homologue). Several detectable transcripts had never previously been reported in the eye. After inflammatory stimulation, significantly greater expression of the adhesion molecule E-selectin mRNA was consistently detected in retinal versus iris EC, and this difference was maintained at the protein level both in cell-surface-expressed and secreted soluble E-selectin protein in paired cultures. Thus, cultured EC derived from adjacent microvascular beds are biologically distinct. Such endothelial diversity could play a role in the pathogenesis of tissue-specific inflammation, infection, neovascularization, and malignancy.

Constitutive and inflammatory mediator-regulated fractalkine expression in human ocular tissues and cultured cells.

PURPOSE: Fractalkine (FKN) is a dual-adhesion molecule-chemokine that plays a role in inflammation but has not been explored in the eye. In the current study, constitutive expression of FKN was identified in human iris and retina, and its regulation by various cytokines in endothelial cells (ECs) and stromal cells from human iris, retina, and choroid was investigated. METHODS: Human iris and retina explants were evaluated for FKN mRNA and protein expression using RT-PCR and immunohistochemistry, respectively. Cultured ocular ECs and stromal cells were stimulated with various inflammatory mediators (endotoxin; TNFalpha; interferon-gamma; interleukin (IL)-1alpha, -4, -10, -13, -17, and -18; and/or CD40 ligand, or combinations thereof), with FKN mRNA being subsequently evaluated by cDNA array and/or RT-PCR and FKN protein by enzyme-linked immunoculture assay (ELICA) and/or by Western blot analysis. RESULTS: Iris and retina explants constitutively expressed FKN protein in microvascular ECs and also in several stromal cell types. Iris and retina both express FKN mRNA. TNFalpha upregulated FKN in iris explants. All ocular microvascular ECs and stromal cultures expressed low FKN mRNA and/or protein levels, which were variably upregulated by endotoxin, TNFalpha, interferon-gamma, IL-1alpha, and/or CD40 ligand, but not by IL-18. In ECs, the Th2 cytokines IL-4 and -13, but not IL-10, reduced TNFalpha-induced FKN protein. IL-17, usually considered proinflammatory, reduced TNFalpha-induced FKN protein in ocular ECs. CONCLUSIONS: FKN is expressed in various ocular tissues and cells. Inflammatory mediator modulation of ocular FKN expression suggests that this adhesive chemokine may play important roles in regulating leukocyte efflux in inflammatory eye diseases, such as anterior uveitis and retinochoroiditis.

Homocysteine upregulates vascular cell adhesion molecule-1 expression in cultured human aortic endothelial cells and enhances monocyte adhesion.

Elevated plasma homocysteine is an independent risk factor for atherosclerosis. We hypothesized that homocysteine enhances monocyte/human aortic endothelial cell (HAEC) interactions, a pivotal early event in atherogenesis, by upregulating endothelial adhesion molecules. After incubation of cultured HAECs with reduced DL-homocysteine for up to 24 hours, adhesion of human monocytes to homocysteine-stimulated HAECs was significantly upregulated in a time- and dose-dependent fashion. Pretreatment of HAECs with 100 micromol/L homocysteine caused a 4.5-fold increase in the adhesion of normal human monocytes (P<0.001). Similarly, adhesion of monocytic U937 cells was maximally elevated by 3.5-fold at 100 micromol/L homocysteine (P<0.001). In support of our hypothesis, vascular cell adhesion molecule (VCAM)-1 mRNA expression increased 5-fold in HAECs after 3 hours of treatment with 100 micromol/L homocysteine, as assessed by quantitative reverse transcription- polymerase chain reaction. Neutralizing antibody studies confirmed the involvement of VCAM-1 in mediating monocyte adhesion to homocysteine-stimulated HAECs. Coincubation of HAECs with homocysteine and tumor necrosis factor-alpha synergistically elevated monocyte adhesion as well as VCAM-1 protein expression, with the latter evaluated by flow cytometry. Preincubation of HAECs with cyclooxygenase inhibitors completely abrogated homocysteine-induced monocyte adhesion, whereas scavenging reactive oxygen species and the elevation of NO caused partial inhibition only. These data support the notion that the proinflammatory effects of homocysteine may have important implications in atherogenesis.