Removal Of Non-adherent Cells Research Articles

Is there an optimal method to detach Candida albicans biofilm from dental materials?

Introduction. Candida albicans can produce a complex, dynamic and resistant biofilm on the surface of dental materials, especially denture base acrylic resins and temporary soft liners. This biofilm is the main aetiological factor for denture stomatitis, an oral inflammatory condition characterized by chronic and diffuse erythema and oedema of the denture bearing mucosa.Gap Statement. There is no consensus in the literature regarding the best method to detach biofilms from dental materials. In order to assess the antifungal efficacy of new materials and treatments, the biofilm needs to be properly detached and quantified.Aim. This study compared different methods of detaching C. albicans biofilm from denture base acrylic resin (Vipi Cril) and temporary soft liner (Softone) specimens.Methodology. Specimens of each material were immersed in an inoculum of C. albicans SC5314 and remained for 90 min in orbital agitation at 75 r.p.m. and 37 °C. After the removal of non-adherent cells, the specimens were immersed in RPMI-1640 medium for 48 h. Biofilm formation was evaluated with confocal laser scanning microscopy (n=5). Then, other specimens (n=7) were fabricated, contaminated and immersed in 3 ml of sterile phosphate-buffered saline (PBS) and vortexed or sonicated for 1, 2, 5, or 10 min to detach the biofilm. The quantification of detached biofilm was performed by colony-forming unit (c.f.u.) ml-1 count. Results were submitted to one-way analysis of variance (ANOVA)/Tukey HSD test (α=0.05).Results. A mature and viable biofilm was observed on the surfaces of both materials. For both materials, there was no significant difference (P>0.05) among detachment methods.Conclusion. Any of the tested methods could be used to detach C. albicans biofilm from hard and soft acrylic materials.

Antibiofilm activity and mode of action of DMSO alone and its combination with afatinib against Gram-negative pathogens.

Biofilms are complex microbial communities that tend to attach to either biotic or abiotic surface. Enclosed in a self-produced extracellular polymeric substance (EPS) matrix, the biofilms often cause persistent infections. The objective of this study was to investigate the antibiofilm activity of dimethyl sulfoxide (DMSO) and afatinib against Gram-negative pathogens. Test microorganisms used in this study were Escherichia coli ATCC 1299, Pseudomonas aeruginosa ATCC 10145, and Salmonella typhimurium ATCC 14028. Biofilms were developed in 96-well microplate at 37°C for 24h. Following removal of non-adherent cells, analysis of biofilm viability, biofilm biomass, and extracellular polymeric substances (EPS) matrix were performed using resazurin assay, crystal violet assay, and attenuated total reflectance fourier transform infrared (ATR-FTIR) spectroscopy, respectively. Bradford protein assay was conducted to determine the total amount of EPS proteins. The results demonstrated that both 32% DMSO alone and its combination with 3.2μg/mL afatinib were effective in killing biofilm cells and reducing biofilm biomass. IR spectral variations of EPS matrix of biofilms in the range between 1700 and 900cm-1 were also observed. Reduction in EPS proteins verified the chemical modifications of EPS matrix. In conclusion, 32% DMSO alone and its combination with 3.2μg/mL afatinib showed remarkable antibiofilm activities against Gram-negative pathogens. It was suggested that the biofilm inhibition was mediated by the chemical modification of EPS matrix.

Development of an in vitro model on cellular adhesion on granular natural bone mineral under dynamic seeding conditions--a pilot study.

Adhesion of osteogenic cells on biomaterials can be studied with static in vitro models, whereas models representing dynamic seeding conditions are rare. Herein, we present an in vitro model to study cell adhesion on granular biomaterials under dynamic seeding conditions. Radiolabeled osteogenic MC3T3-E1 cells were allowed to adhere to granules of natural bovine bone mineral (NBM) under constant rotation. Adhesion of MC3T3-E1 cells was determined by liquid scintillation counting, and cell morphology was visualized by scanning electron microscopy. Cell viability was determined by MTT assay under static and dynamic conditions, at room and body temperature, and in the presence or absence of serum. We show here that MC3T3-E1 cells rapidly adhere to NBM, reaching a peak 3 h after seeding. Attached cells display characteristic signs of spreading. Five to ten percent of total radioactivity remained on NBM after the removal of nonadherent cells. Viability is maintained at room temperature and under rotation for upto 3 h. This data suggests that the dynamic in vitro model presented here provides a tool to study cell adhesion on granular biomaterials.

Proteasomes Control Caspase-1 Activation in Anthrax Lethal Toxin-mediated Cell Killing

Activation of caspase-1 through the inflammasome protein Nalp1b controls anthrax lethal toxin (LT)-induced necrosis in murine macrophages. In this study we analyzed physiological changes controlled by caspase-1 in LT-treated murine macrophages. The caspase-1 inhibitor Boc-D-cmk blocked caspase-1 activity and membrane impairment in LT-treated cells. To determine the relationship between caspase-1 activation and membrane integrity, we added Boc-D-cmk to J774A.1 macrophages at different time points following LT exposure. Remarkably, Boc-D-cmk rescued LT-treated macrophages, even when added at the peak of caspase-1 activation. Late addition of the caspase-1 inhibitor reversed the losses of plasma membrane integrity and metabolic activity in these cells. Similar results were obtained with the proteasome inhibitor MG132, one of the most potent inhibitors of LT toxicity. LT-treated macrophages displaying evidence of membrane impairment recovered upon the addition of MG132, mirroring the Boc-D-cmk response. Strikingly, late addition of proteasome inhibitors also abrogated caspase-1 activity in LT-treated macrophages. Proteasomal control of caspase-1 activity and membrane impairment, however, was restricted to LT-induced cytolysis, because proteasome inhibitors did not block caspase-1 activation and cell death triggered by lipopolysaccharide and nigericin. Our findings indicate that proteasome inhibitors do not target caspase-1 directly but instead control an upstream event in LT-treated macrophages leading to caspase-1 activation. Taken together, caspase-1-mediated necrosis appears to be tightly controlled and differentially regulated by proteasomes depending on the source of caspase-1 induction.

Sensitization by blood transfusion results in antibody production that correlates with C4d deposition and rejection

vascular homeostasis and in multiple vascular pathologies. We analyzed the effects of the calcineurin inhibitors cyclosporin A (CyA) and tacrolimus (T), and the mTOR inhibitor sirolimus (S) on EPC survival. Methods: Peripheral blood mononuclear cells were isolated from healthy subjects by density gradient centrifugation, resuspended in endothelial growth medium and plated on to fibronectin-coated wells at a density of 0.5x10 cells/cm. At day 4 after plating, non-adherent cells were removed and cells were cultured to day 7. Cells were cultured with CyA (0.01 to 10000 ng/ml), T (0.001 to 1000 ng/ml), S (0.001 to 1000 ng/ml) or vehicle control (DMSO 0.1%). Immunosuppressants (IS) were added either at day 0 and maintained for 7 days or at day 4 after removal of non-adherent cells. At day 7, cell number and viability was quantified by XTT labeling. Human aortic endothelial cells (HAEC) were exposed to IS for the same times. Apoptosis was measured by annexin-FITC labeling. Results: S extensively reduced EPC viability. Clinically relevant concentrations of S reduced EPC viability by 90% compared with controls after 7 days of exposure. This effect was quite marked even at the sub-clinical doses. For CyA, there was a reduced viability with supra-clinical concentrations. For T, no effect was noticed at any dose. When HAEC were analyzed, S reduced viability, but to a much lower extent than with EPC. At clinical concentrations, S reduced EPC viability by 25%, whereas treatment with CyA or T did not significantly affect viability. EPC stained with annexin-V demonstrated a marked increase in apoptosis after S exposure. Conclusions: Sirolimus induces EPC apoptosis and greatly reduces EPC survival, even at low, sub-clinical concentrations. This effect may partially explain the findings that sirolimus inhibits angiogenesis, reduces postangioplasty restenosis and transplant vasculopathy.

Role of Osteoclasts in Regulation of Human Hematopoietic Stem Cell Niche and Fate.

Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (>95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (>90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after >6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

Formation of short-lived multinucleated giant cells (MGCS) from cultured gilthead seabream macrophages.

Monocytes/macrophages obtained from the head kidney and peritoneal cavity of gilthead seabream (Sparus aurata) were cultured using plates from three different manufacturers, and were maintained under different conditions. The effects on the morphology and fusion of monocytes/macrophages of initial cell loading, removal of non-adherent cells at different times after plating, and addition of serum and antibiotics were evaluated by light microscopy, and transmission (TEM) and scanning (SEM) electron microscopy. Despite variations in adherence, the behaviour and the morphological changes in kidney monocytes/macrophages were similar in all three types of plates. When foetal calf serum (FCS) was added to the incubation medium, most of the cells resembling monocytes/macrophages were connected by cytoplasmic extensions that formed bridges after 24 hr in culture. After 30 hr, the monocytes/macrophages started to fuse, forming multinucleated giant cells (MGCs) which gradually increased in size until the culture was 4-5 days old. After 5 days the MGCs started to die, and after a week most had disappeared from the cultures. Cells incubated with medium without serum showed changes similar to those fed with FCS, but some cells survived for 3 weeks. The addition of fish serum to the medium appeared to accelerate all processes: the monocytes/macrophages and MGCs died after 3 days in culture. Antibiotics had no apparent effect on the cultures. Removal of non-adherent cells at different times after plating did not appear to affect cell fusion. Coating the wells with extracellular matrix proteins reduced adherence but did not inhibit cell fusion. Curiously, not all macrophages fused with MGCs, and, unlike MGCs, these macrophages phagocytosed sheep red blood cells (SRBCs). Peritoneal macrophages also fused and formed MGCs in culture, similarly to kidney cells.

Focussing Human B Cell Specificity by Immunoselection via Antigen-Presenting Cells in vitro

We present a protocol for in vitro immunization of B cells using monocyte-derived accessory cells (MoAC). MoAC are developed from human peripheral blood monocytes in culture and represent functionally competent inducers of antigen-specific immune responses. Using MoAC, we attempted to immunoselect TT-specific lymphocytes by rosetting. Adherent human MoAC were pulsed with tetanus toxoid (TT) and allowed to form clusters with autologous lymphocytes, followed by removal of non-adherent cells. After one week of culture, a specific anti-TT antibody response emerged on a low background of unspecific Ig. In comparison, cultures which had not been selected for adherent cells produced a high polyclonal background. Our results demonstrate that from peripheral blood cells, previously not a favourable source for in vitro immunization, in a majority of tests antigen-specific B cells could efficiently be immunoselected via adherence to autologous antigen-presenting cells, leading to a high-titre in vitro immunization.

Macrophage tropism: fact or fiction?

Primary HIV-1 isolates can be distinguished by phenotypic qualities such as the ability to productively infect cells of established CD4-positive lines and to induce syncytia in MT-2 cells. Such viral phenotypes have also been reported to confer host cell specificity. It is perceived that primary isolates with the syncytium-inducing phenotype (SI or rapid/high) are T cell tropic and are therefore unable to infect primary cells of the monocyte/macrophage lineage. However, we have consistently found that these isolates are as capable of establishing infection in monocyte-derived macrophages (MDM) as the monocytotropic, non-syncytium-inducing variants (NSI or slow/low). It is known that differentiation, activation, and proliferation of human monocytes are affected by both isolation methods and culture conditions. Therefore, to test whether our inability to discriminate macrophage tropic HIV-1 isolates could be explained by differences in culturing techniques, we isolated monocytes by elutriation or short-term adherence and allowed the cells to mature and differentiate in either the presence or absence of autologous lymphocytes. After removal of nonadherent cells, MDM were infected with a panel of SI and NSI primary HIV-1 isolates. MDM were susceptible to infection by the SI as well as the NSI isolates, regardless of whether or not the cells were allowed to mature in the presence of autologous lymphocytes. However, MDM matured in the presence of autologous lymphocytes replicated HIV-1 isolates (both NSI and SI) to a higher titre than MDM matured in the absence of lymphocytes. In light of these findings and recently published reports on HIV-1 phenotype and chemokine receptor usage we believe that the term macrophage-tropic strains of HIV-1 is no longer appropriate.

Monocyte adherence to the subendothelial basement membrane increases Interleukin-8 gene expression and antigen release

The emigration of peripheral blood monocytes into the interstitium allows for contact with a variety of surfaces which may provide signals important for monocyte function in both normal and inflammatory states. In the present study, we examined the effect of adherence to an endothelial cell-derived basement membrane and to collagen I, the major collagen of the interstitium, on monocyte release and gene expression of the potent chemotactic cytokine Interleukin-8 (IL-8). We further evaluated neutrophil chemotactic activity of the conditioned media containing antigenic IL-8 from monocytes adherent to these same surfaces. Elutriation-purified monocytes were adhered for 1 hour to plastic tissue culture wells either uncoated (PL) or coated with bovine serum albumin (BSA), collagen type I (C-I), or endothelial cell-derived basement membrane (BM). Following removal of nonadherent cells, monocytes were further incubated in a serum-free media for 18 hours in the presence or absence of lipopolysaccharide (IPS). Following 18 hrs of incubation there were significantly less monocytes remaining adherent to BM when compared to other surfaces tested. In the absence of LPS, adherent monocytes released significant amounts of IL-8 that was not surface specific. In the presence of LPS, monocytes adherent to BM released significantly more IL-8, when corrected for adherent cell number, than monocytes adherent to PL, BSA, or C-I. Conditioned media from adherent monocytes expressed IL-8 dependent neutrophil chemotactic activity that was not influenced by the surfaces tested. Northern blot analysis indicated greater induction for IL-8 mRNA by monocytes adhered to BM after 18 hrs in the presence of LPS. These results suggest that monocyte adherence to the subendothelial basement membrane provides a priming signal for the induction and secretion of the chemotactic cytokine IL-8 in response to inflammatory stimuli.

An adhesion assay using minimal shear force to remove nonadherent cells

A new 96-well microtiter plate based adhesion assay was developed to measure weak cell adhesion. This assay is distinct from other adhesion assays by the procedure in which the nonadherent cells are removed. In most conventional adhesion assays, nonadherent cells are removed by aspiration followed by repeated washes. However, the shear force generated by such washing also detaches weakly adherent cells. In the minimal shear force adhesion assay (MSFA) described here, the removal of nonadherent cells is carried out by applying a gentle shear force in a fluid environment. In this procedure, adherent cells are not subjected to harsh and variable washing forces and are not exposed to surface tension caused by the removal of washing fluid between successive washes. Using the lymphoid cell lines XC1.5/51 and MPC11, the number of adherent cells determined by this new adhesion assay is three times higher than the conventional adhesion assay. This MSFA assay is simple, consistent, and easy to perform. With modifications for applying a defined shear force, this assay can be adopted to compare cell adhesion strength to various substrata.

Neuroimmunomodulatory effects of acupuncture in mice

The purpose of this study was to assess the effect of acupuncture on the immunological response. The induction of anti-sheep red blood cells (SRBC) plaque-forming cells (PFC) was used as a measurement of the immune response to treatment. In normal non-immunized mice, enhancement of PFC was seen after a single acupuncture treatment when spleen cells from stimulated mice were cultured with SRBC in vitro. After 3 acupuncture treatments, spleen cells from mice did not show PFC enhancement after treatment with anti-Thy-1.2 antibody and complement, nor after the removal of non-adherent cells. Serum obtained from mice 1 h after acupuncture stimulation enhanced the PFC of normal spleen cells in vitro, but the enhancement was abolished by the addition of propranolol. These results suggest that acupuncture, by activation of the autonomic nervous system, modulates the immune response.

In Vitro and In Vivo Comparison of the Activity of Human Lymphokine-Activated Killer (LAK) Cells and Adherent LAK Cells

The effectiveness of "standard" lymphokine-activated killer (LAK) cells, recovered after 6 days, and of "standard" adherent LAK (A-LAK) cells, recovered after 14 days of culture in the presence of recombinant interleukin-2 (rIL-2) from peripheral blood lymphocytes of 21 healthy donors, was assessed through comparison of their proliferation, surface markers, cytotoxic activity, lymphokine production, and antitumor activity. In the presence of rIL-2, plastic adherent precursors of A-LAK cells proliferated much better than those of "standard LAK" cells and expanded even more than 300-fold. However, the final cell recovery of A-LAK was always lower because of their very few precursors, and the total lytic units (LUs) generated in A-LAK cultures were always lower for the same reason. On the other hand, the lytic activity of each A-LAK cell was always higher than that of a LAK cell. This was particularly evident on day 6 of culture. Removal of nonadherent cells after the first 24 h culture resulted in a significant enrichment in CD3-CD56+ and CD8+CD56+ cells in A-LAK cells, with a marginal number of CD4+ cells. A significant direct correlation between LUs and A-LAK CD3-CD56+ percentage was found. In the presence of rIL-2, A-LAK cells produced higher amounts of tumor necrosis factor-alpha and interferon-gamma than LAK cells, while only A-LAK cells produced IL-1 beta and small amounts of IL-4. Neither LAK nor A-LAK produced IL-2. In the absence of injections of IL-2, LAK and A-LAK cells were equally able to inhibit the growth of a human T-cell lymphoma in immunosuppressed nude mice.

The enhancement of the immune response by pain stimulation in mice. I. The enhancement effect on PFC production via sympathetic nervous system in vivo and in vitro.

Effects of catecholamines and osmotical and physical stimuli on the induction of anti-sheep red blood cells (SRBC) plaque-forming cells (PFC) were investigated in (C57BL/6 X BALB/c)F1 mice in vivo and in vitro. The anti-SRBC PFC from mice immunized with 5 X 10(7) SRBC was markedly increased by daily s.c. injections of epinephrine. The enhancement of PFC by epinephrine was completely blocked by preadministration with propranolol and hexamethonium, but not with phentolamine. The PFC was increased by osmotic and physical stimuli given once a day for 4 days after immunization with SRBC. The enhancement of PFC by these stimuli was completely blocked by preadministration with propranolol and hexamethonium. The enhancement of PFC by physical stimuli was observed in nonimmunized mice when spleen cells from stimulated mice were cultured with SRBC in vitro. In normal mice, the enhancement of PFC was observed 2 hr after one physical stimulation. However, spleen cells from mice given two physical stimuli did not show the enhancement of PFC after treatment with anti-Thy-1.2 antibody and complement, nor after removal of nonadherent cells. Next, the serum obtained from mice 30 to 60 min after a physical stimulation enhanced PFC of normal mice spleen cells in vitro, but the enhancement was abolished by the addition of propranolol. The enhancement of anti-SRBC PFC by s.c. injection of epinephrine suggested that the autonomic nervous system, especially the sympathetic nervous system, was activated by a local stimulus effect of the injection. This enhancement of anti-SRBC PFC appear to be due to the activation of antigen non-specific helper T lymphocytes by the beta-actin of endogenous catecholamines from the adrenal gland.

Binding of antigen-specific, H-2-restricted T cell hybridomas to antigen-pulsed adherent cell monolayers.

Two methods were investigated for studying the binding of radiolabeled hybridoma T cells to antigen (Ag) and H-2 products for which they bore receptors. In both cases hybridoma T cells were labeled with tritiated thymidine. In one method labeled cells were added to adherent splenic cells prepulsed with antigen, and the mixture was incubated overnight at 37 degrees C before nonadherent cells were gently washed away. The percent of adherent hybridoma T cells was then estimated by harvesting the adherent monolayers and measuring tritium counts bound. In a second method radiolabeled hybridoma T cells were added to adherent antigen-pulsed B cell lymphomas or hybridomas for between 15 min and 1 hr at 37 degrees C before removal of nonadherent cells and harvesting of the adherent monolayers. In both cases binding was both antigen- and I-region specific. In the second case binding was also rapid; significant binding could be measured after 15 min incubation. These techniques were used to study subclones of one of our T cell hybridomas that were thought by a functional assay (interleukin 2 release) to have lost receptors for Ag/H-2. It was found that subclones of the hybridoma that no longer secreted interleukin 2 in response to Ag/H-2, even though they continued to secrete interleukin 2 in response to concanavalin A, also no longer bound specifically to Ag-pulsed monolayers of the appropriate H-2 type. This confirmed the idea that these subclones had lost the ability to synthesize receptors for Ag/H-2. It is hoped that assays of this type will be useful in the future for the study of Ag/H-2 receptors on T cells.

Improved method for the isolation of purified mouse peritoneal macrophages

Mouse peritoneal macrophages were cultured for 45 min in medium supplemented with fetal calf serum (FCS) in petri dishes coated overnight with heat-inactivated FCS. After removal of non-adherent cells by washing, adherent cells were detached by a brief incubation in the presence of sub-toxic levels of ethylenediamine tetraacetate (EDTA). Overall peritoneal macrophage recoveries of 90% can be routinely achieved with this method, and full cell viability is maintained.