Functionalization of hybrid surface microparticles for in vitro cellular antigen classification.

Hybrid material surfaces on microparticles are emerging as vehicles for many biomedical multiplexing applications. Functionalization of these hybrid surface microparticles to biomolecules presents unique challenges related to optimization of surface chemistries including uniformity, repeatability, and sample sparring. Hybrid interfaces between microlevel surfaces and individual biomolecules will provide different microenvironments impacting the surface functionalization optimization and efficiency. Here, we propose and validate the first demonstration of streptavidin adsorption-based antibody functionalization on unmodified, hybrid surface microparticles for in vitro analysis. We test this analytical technique and fabricate hybrid surface microparticles with a polystyrene core and aluminum oxide semi-coating. Additionally, we optimize the streptavidin-biotin functionalization chemistry in both assay implementation and sample sparring via analytical mass balances for these microparticles and subsequently conjugate anti-human CD11b antibodies. Result confirmation and characterization occurs from ultraviolet protein absorbance and ImageJ processing of fluorescence microscopy images. Additionally, we design and implement the multi-sectional imaging (MSI) approach to support functionalization uniformity on the hybrid surface microparticles. Finally, as a proof-of-concept performance, we validate anti-CD11b antibodies functionalization by visualizing hybrid surface microparticles conjugate to human neutrophils isolated from blood samples collected from potentially septic patients. Our study introduces and defines a category of functionalization for hybrid surface microparticles with the intent of minuscule sample volumes, low cost, and low environmental impact to be used for many cellular or proteomic in vitro multiplexing applications in the future. Graphical abstract.

Salivary immunity in elderly individuals presented with Candida-related denture stomatitis.

OBJECTIVES: Elderly individuals with Candida-related denture stomatitis (DS) present with a reduced defence against Candida albicans. This study evaluated levels of antimicrobial mediators in the elderly DS saliva and salivary neutrophils' activation characteristics compared with elderly and young without DS. METHODS: Salivary peroxidases (SPO) and elastase activities (ELA), nitric oxide (NO), transforming growth factor beta (TGF-ß), IL-6 and CCL3 production were determined in saliva from elderly with or without DS, and young control individuals. TLR4, CXCR1, CD11b, CD16 and CD32 expression on salivary neutrophils were evaluated. Correlations between number and apoptosis rate of salivary neutrophils, enzymatic activities and cytokine levels were determined. RESULTS: Elderly DS individuals exhibited the lowest SPO and ELA activities. Also, the activity of both enzymes was low in elderly without DS. Although both elderly groups showed higher salivary NO and TGF-ß levels compared to young control groups, elderly DS presented the highest salivary NO, TGF-ß, IL-6 and CCL3 levels. Decreased percentages of salivary TLR4(+) and CD16(+) neutrophils were detected in both elderly groups. Although these damages could influence the establishment and persistence of DS, the highest levels of salivary IL-6 and CCL3 in elderly DS could be preventing more serious complications.

Interleukin-34 is expressed by giant cell tumours of bone and plays a key role in RANKL-induced osteoclastogenesis.

Interleukin-34 (IL-34) is a newly discovered regulator of myeloid lineage differentiation, proliferation, and survival, acting via the macrophage-colony stimulating factor receptor (M-CSF receptor, c-fms). M-CSF, the main ligand for c-fms, is required for osteoclastogenesis and has been already identified as a critical contributor of the pathogenesis of giant cell tumours of bone (GCTs), tumours rich in osteoclasts. According to the key role of M-CSF in osteoclastogenesis and GCTs, the expression of IL-34 in human GCTs was first assessed. Quantitative analysis of IL-34 mRNA expression in 14 human GCTs revealed expression of this cytokine in GCTs as well as M-CSF and c-fms. Immunohistochemistry demonstrated that osteoclast-like cells exhibited a huge immunostaining for IL-34 and that mononuclear stromal cells were slightly positive for this protein. In contrast to osteoblasts, bone-resorbing osteoclasts showed very strong staining for IL-34, suggesting its potential role in the pathogenesis of GCTs by facilitating osteoclast formation. The role of IL-34 in osteoclastogenesis was then studied in murine and human models. IL-34 was able to support RANKL-induced osteoclastogenesis in the absence of M-CSF in all models. Multinucleated cells generated in the presence of IL-34 and RANKL expressed specific osteoclastic markers and resorbed dentine. IL-34 induced phosphorylation of ERK 1/2 and Akt through the activation of c-fms, as revealed by the inhibition of signalling by a specific c-fms tyrosine kinase inhibitor. Furthermore, IL-34 stimulated RANKL-induced osteoclastogenesis by promoting the adhesion and proliferation of osteoclast progenitors, and had no effect on osteoclast survival. Overall, these data reveal that IL-34 can be entirely substituted for M-CSF in RANKL-induced osteoclastogenesis, thus identifying a new biological activity for this cytokine and a contribution to the pathogenesis of GCTs.

Periodontal inflammation and bone loss in aged mice.

BACKGROUND AND OBJECTIVE: Young mice do not develop measurable periodontal bone loss, unless heavily infected with human periodontal pathogens. However, mice with a genetically altered immune system are unable to control their own oral flora and develop periodontitis early in life. Based on the potential of the indigenous oral microbiota to cause periodontitis, we hypothesized that normal mice may ultimately develop inflammatory periodontal bone loss, i.e. as a function of age. If confirmed, this could serve as an aging model of chronic periodontitis. MATERIAL AND METHODS: Periodontal bone levels were measured as the distance from the cementoenamel junction to the alveolar bone crest in young mice (8-10 wk of age), old mice (>or= 18 mo of age) and mice of intermediate ages. Differential expression of inflammatory mediators in the gingivae of young and old mice was determined by quantitative real-time PCR. RESULTS: In comparison with young mice, old mice displayed significantly (p < 0.05) increased periodontal bone loss, accompanied by elevated expression of proinflammatory cytokines (interleukin-1 beta, tumor necrosis factor alpha and interleukin-17A) and innate immune receptors involved in the induction or amplification of inflammation (Toll-like receptor 2, CD14, CD11b, CD18, complement C5a receptor and triggering receptor expressed on myeloid cells 3). CONCLUSION: Mice develop naturally induced periodontal bone loss as a function of age. This aging model of periodontitis represents a genuinely chronic model to study mechanisms of periodontal tissue destruction.

Interactions of neutrophils with silver-coated vascular polyester grafts.

BACKGROUND: In reconstructive vascular surgery, infection is one of the most feared complications because of the high mortality. While the antimicrobial effect of a silver-coated endoprosthesis has been proven in experimental trials, there are no reports on its interactions with granulocytes, the first effector cells in general inflammation and in infection. MATERIALS AND METHODS: Therefore, we investigated whether silver coating of vascular polyester grafts affects receptor expression, mediator release, and functions of human neutrophils relevant for microbicidal activity and the wound-healing process. Naïve neutrophils were analyzed for their cellular receptors such as cluster of differentiation (CD)62L, CD11b, CXCR2, and fMLP-R, the mediators interleukin 8, granulocyte elastase (human neutrophil elastase), and leukotriene B4 (LTB4) as well as for microbicidal capacity (oxidative burst) in vitro. In addition, the role of plasma coating for receptor expression was addressed. RESULTS: There was both a decrease of CD62L and CXCR2 expression and an increase of CD11b, fMLP-R expression, elastase release, and LTB4 generation, which were statistically significant (p = 0.04; p = 0.01; p = 0.0; p = 0.0; p = 0.01; p = 0.02, respectively) in the presence of the silver-coated graft compared with non-silver-coated vascular grafts. In addition, microbicidal activity was significantly (p = 0.0) impaired by the silver-coated graft. Coating of the vascular grafts with plasma did not alter the former observations significantly. CONCLUSION: The results may indicate that silver-coated vascular polyester grafts activate neutrophils chronically which may favor tissue destruction and impaired antimicrobial effects.

A new hydrophilic polysulfone hemodialysis membrane can prevent platelet-neutrophil interactions and successive neutrophil activation.

PURPOSE:: Microaggregates have often been observed during hemodialysis and are clearly associated with complications of hemodialysis therapy. In this study, we aimed to clarify the effects of two polysulfone membranes, with different abilities to activate blood cells, on the formation of these microaggregates; we also investigated their molecular mechanisms. METHODS:: Human whole blood was circulated through a mini-module dialyzer using the membranes in vitro; platelet-neutrophil complexes in blood were determined by flow cytometry. Isolated human neutrophils were incubated with the membranes in plasma, in the presence or absence of platelets, followed by flow cytometric analysis of intracellular reactive oxygen species and cell-surface activated CD11b on neutrophils. RESULTS:: CX-U, a conventional polysulfone membrane with remarkable cell activation, induced the formation of platelet-neutrophil complexes; however, NV-U, a new hydrophilic polysulfone membrane with slight or no cell activation, did not cause complex formation. Moreover, CX-U-induced reactive oxygen species production and the increase in activated CD11b expression on neutrophils were enhanced by platelets. On the other hand, NV-U hardly affected neutrophil activation, regardless of whether platelets were present or not. The enhancement of CX-U-induced neutrophil activations by platelets was greatly inhibited by anti-CD62P antibody. CONCLUSION:: The ability of polysulfone membranes to activate blood cells is closely related to platelet-neutrophil interaction. Therefore, a biocompatible membrane, like NV-U, can be expected to prevent microaggregate formation during hemodialysis and avoid subsequent cell activation.

Effective induction of anti-tumor immune responses with oligomannose-coated liposome targeting to intraperitoneal phagocytic cells.

We recently established a novel drug delivery system (DDS) using oligomannose-coated liposomes (OMLs) which are probably taken up by macrophages (Mvarphi) to carry anti-cancer drugs to milky spots known as preferential metastatic sites of gastric cancers [Y. Ikehara, T. Niwa, L. Biao, S.K. Ikehara, N. Ohashi, T. Kobayashi, Y. Shimizu, N. Kojima, H. Nakanishi, A carbohydrate recognition-based drug delivery and controlled release system using intraperitoneal macrophages as a cellular vehicle, Cancer Res. 66 (2006) 8740-8748]. In the present study, we applied this intraperitoneal DDS for systemic cancer immunotherapy employing ovalbumin (OVA) as a model antigen. The cells taking up the OMLs containing FITC-OVA injected into the peritoneal cavity were predominantly Mvarphi, as they showed adhesive characteristics and expressed F4/80 and CD11b almost exclusively. The phagocytic cells also took up bare OVA directly to the same extent as OML-enclosed OVA (OML-OVA), as it is a highly mannosilated protein. The phagocytic cells taking up OML-OVA, however, could activate OVA-specific CD8+ (from OT-I: H-2Kb/OVA257-264-specific) and CD4+ (from OT-II: H-2Ab/OVA323-339-specific) T cells much more effectively in vitro than those taking up bare OVA. Furthermore, only the mice pre-immunized with OML-OVA rejected E.G7-OVA (OVA-transfected EL4) but not EL4. These results indicate that the OMLs can also be used as an effective antigen delivery system for cancer immunotherapy activating both CTL and Th subsets.

Periodontal manifestation of leukocyte adhesion deficiency type I.

BACKGROUND: Leukocyte adhesion deficiency type I (LAD-I) is an autosomal recessive immunodeficiency disorder characterized by defects in the integrin receptors of white blood cells that lead to impaired adhesion and chemotaxis. Affected patients are susceptible to recurrent bacterial and fungal infections, impaired pus formation, delayed wound healing, and periodontitis. METHODS: A case of generalized advanced periodontal destruction of the permanent and deciduous dentition in a young Jordanian girl with a severe phenotype of LAD-I is presented in this report. The medical diagnosis was made based on the characteristic clinical and laboratory findings, in particular the total absence of CD18, CD11b, and CD11c as determined by flow cytometry sorting. RESULTS: Periodontal findings in this patient include the early onset of the disease, which affected the primary teeth and permanent dentitions, the intense redness and inflammation of the gingiva, and the rapid periodontal destruction that seems refractory to conventional non-surgical periodontal therapy. CONCLUSION: This report emphasizes the importance of the differential diagnosis of severe immunodeficiency disorders in children and adolescents and mandates the importance of combined care by medical and dental practitioners to prevent tooth loss and control oral infection.

Systemic Injection of Substance P Promotes Murine Calvarial Repair Through Mobilizing Endogenous Mesenchymal Stem Cells.

Craniofacial defect is a critical problem in dental clinic, which has a tremendous impact on patients' quality of life. Mesenchymal stem cell-based therapy has emerged as a promising approach for tissue defect repair. However, reduced survival after mesenchymal stem cells (MSCs) transplantation remains as a major problem in this area, which hampers the outcome of regeneration. Recently, the mechanism to mobilize endogenous MSCs for tissue regeneration has received increasing attentions, as it does not require exogenous cell transplantation. The primary goal of this study was to confirm the role of intravenous substance P in mobilizing endogenous CD45-CD11b-CD29+ MSCs in critical-sized bone defect animals and to investigate the effects of substance P on calvarial bone repair. Flow cytometry analyses revealed that intravenous substance P promoted the mobilization of endogenous CD45-CD11b-CD29+ MSCs after bone defect. In addition, Micro-CT showed that intravenous substance P improved the outcomes of calvarial bone repair. Furthermore, we discovered that systemic injection of substance P attenuated inflammation and enhanced the survival of the local-transplanted GFP+ MSCs. Our findings suggested that substance P together with its mobilized CD45-CD11b-CD29+ MSCs helped improve calvarial defect repair through regulating inflammatory conditions and promoting the survival of local-transplanted cells.

In vitro blood reactivity to hydroxylated and non-hydroxylated polymer surfaces.

Complement activation on hydroxyl-group-bearing surfaces is regarded as the main reason for granulocyte activation in applications of blood-contacting medical devices such as extracorporeal blood purification. However, the factors inducing the cell adhesion so far remained ambiguous. For a dedicated research, whole blood was incubated with a set of structurally similar polymer coatings on glass with either hydroxy or ether functionalities. By co-incubation of an activating with a non-activating surface, the reaction of granulocytes activated by complement fragments on non-activating surfaces could be evaluated. As expected, hydroxyl-terminated polymer layers induced much higher levels of complement activation than those with ether functionalities. Leukocyte activation, as measured by the expression of CD11b, correlated closely with the presence of free complement fragment C5a. However, adhesion of leukocytes was rather associated with the adsorption of activated fragments of C3 than with the activation level of the cells. Moreover, it was found that adsorbed quantities of fibrin and fibrinogen had little influence on leukocyte adhesion. It is concluded that the activation of leukocytes is triggered by soluble complement factors such as C5a while their adhesion on hydroxy-bearing surfaces is mainly triggered by the presence of surface-bound complement fragment C3b.

Effect of vascular endothelial growth factor on RANK gene expression in osteoclast precursors and on osteoclastogenesis.

OBJECTIVE: The aim of this study was to determine if vascular endothelial growth factor (VEGF) can upregulate the gene expression of receptor activator of nuclear factor kappa B (RANK) in osteoclast precursors, as does CSF-1. A secondary aim was to determine if VEGF can promote osteoclastogenesis in vitro comparable to CSF-1. DESIGN: Osteoclast precursors (mononuclear cells) were incubated with different concentrations of VEGF, CSF-1, or a combination of the two, and the gene expression of RANK was determined by RT-PCR. A TRAP assay also was conducted to determine their effect on osteoclastogenesis. An Alamar blue assay was done to analyse the effect of the molecules on proliferation of the osteoclast precursors. RESULTS: VEGF upregulated RANK expression in osteoclast precursors as effectively as CSF-1. VEGF did not promote osteoclastogenesis, as did CSF-1. A combination of the two did. CSF-1 enhanced proliferation of the osteoclast precursors but VEGF did not. However, VEGF in combination with CSF-1 did increase proliferation. CONCLUSIONS: At the time of the secondary burst of osteoclastogenesis prior to tooth eruption, VEGF expression in the dental follicle is high but the expression of CSF-1 is low. This study demonstrates that VEGF can fully substitute for CSF-1 to upregulate the RANK expression in osteoclast precursors that is needed for osteoclastogenesis. However, VEGF alone neither can promote osteoclastogenesis nor stimulate proliferation of the osteoclast precursors in vitro. For proliferation and osteoclastogenesis, a low dose of CSF-1 in combination with VEGF is needed.

Effect of complement inhibition and heparin coating on artificial surface-induced leukocyte and platelet activation.

BACKGROUND: Exposure of blood to artificial surfaces, as in cardiopulmonary bypass, induces an inflammatory response involving complement, leukocyte and platelet activation. To elucidate the specific role of complement in this process, studies were performed on blood circulated in polyvinyl chloride tubing in the absence and presence of complement inhibitors. Parallel experiments were performed with heparin-coated polyvinyl chloride tubing, which is known to prevent complement and cell activation. METHODS: A novel experimental model was used, based on human whole blood anticoagulated with lepirudin. Complement activation products, myeloperoxidase, lactoferrin, and thrombospondin were quantified in enzyme immunoassays. Leukocyte CD11b expression and leukocyte-platelet conjugates were detected by flow cytometry. RESULTS: Increased levels of C3 activation products, alternative pathway convertase, and the terminal SC5b-9 complex, combined with unchanged levels of C1rs-C1-inhibitor complexes and marginal changes in C4 activation demonstrated that complement was activated through the alternative pathway. Granulocyte and monocyte CD11b expression and granulocyte-platelet conjugate formation were efficiently attenuated by blocking either factor D, C3, C5, or C5a receptor. In contrast, monocyte-platelet conjugate formation and release of myeloperoxidase, lactoferrin, and thrombospondin were not reduced by complement inhibition. Heparin-coated polyvinyl chloride tubing efficiently reduced all inflammatory markers studied, except for C1rs-C1-inhibitor complexes, which increased, consistent with the enhancing effect of heparin on C1-inhibitor function. This effect did not, however, reduce fluid-phase classic pathway activation induced by heat-aggregated immunoglobulin G. CONCLUSIONS: Leukocyte and platelet activation in response to artificial materials occur by mechanisms that vary in their dependence on complement. Heparin coating precludes both the complement-dependent and complement-independent reactions.

Clinical and immunological findings of two siblings in a family with generalized aggressive periodontitis.

PURPOSE: The clinical and immunological data from 2 siblings with generalized aggressive periodontitis in a family are presented. METHODS: Peripheral blood lymphocyte subpopulations were analyzed by dual color flow cytometry by using monoclonal antibodies for the lymphocyte receptors CD2, CD3, CD4, CD5, CD8, CD16, CD19, CD11b, CD11c, and HLA-DR. RESULTS: Both patients showed a higher increase in CD11b/CD18 expression. CONCLUSIONS: The evaluation of CD11b and CD11c receptors may be important in explaining leukocyte abnormalities in the pathogenesis of the disease. One of the patients showed decreased counts of natural killer cells while the other was normal. In both patients, the authors observed a slight elevation in Ts lymphocytes.

Dental pulp dendritic cells migrate to regional lymph nodes.

Dendritic cell (DC) migration to regional lymph nodes (RLNs) is an essential step in adaptive immunity, and cell-surface antigens on migrating DCs greatly affect the quality and quantity of subsequent immune responses. Although MHC class II(+) DC-like cells exist in the dental pulp, the lineage and function of these cells remain unknown. Here, we identified migratory DCs from the dental pulp after cusp trimming and acid etching in KikGR mice, in which the photoconvertible fluorescent protein changed from green to red upon violet light exposure. Two major cell fractions from the dental pulp had migrated to the RLNs at 16 hrs after cusp treatment, which showed the following lineage markers in the main and second fractions: CD11c(high)CD11b(++)Ly6C(low) Ly6G(low) F4/80(+) and CD11c(med)CD11b(+++)Ly6C(++)Ly6G(+++)F4/80(-), respectively. These lineage markers indicate that the former cells were DCs that had migrated through afferent lymphoid vessels, and the latter were granulocytes recruited via blood circulation. Migratory dental pulp DCs were mature, expressing the highest levels of CD273 (B7-DC) and CD86 co-stimulators and MHC class II. Our results suggest that cariogenic-bacteria-exposed dental pulp DCs migrate to RLNs and there trigger adaptive immune responses.

Standardization and safety of alveolar bone-derived stem cell isolation.

Cell therapies utilizing mesenchymal stem cells (MSCs) could overcome limitations of traditional treatments for reconstructing craniofacial tissues. This large-scale study explored a standardized methodology for the isolation and clinical-scale expansion of alveolar bone marrow-derived MSCs (aBMSCs). We harvested 103 alveolar bone marrow samples from 45 patients using 1 of 3 standardized methodologies. Following aBMSC isolation, cells were characterized through cell-surface marker expression and lineage-specific differentiation. Long-term cultures (> 50 population doublings [PDs]) were evaluated for transformational changes through senescence, gene expression, and karyotyping. Finally, aBMSC bone-forming potential was determined in vivo. More than 0.5 cc of bone marrow was needed to predictably isolate aBMSCs, and, regardless of methodology for harvest, cell-surface marker expression of CD73, CD90, CD105, and Stro-1 was similar for aBMSCs, being 89.8%, 98.8%, 93.8%, and 3.2%, respectively; all cells were negative for CD11b, CD19, and CD45. aBMSCs exhibited multipotency, and karyotypes were normal up to 30 PDs, with significant cell senescence beginning following 35 PDs. Additionally, aBMSCs induced ectopic bone formation following subcutaneous transplantation into mice. These findings demonstrate a predictable approach for the isolation and safe clinical-scale expansion of aBMSCs, and thus, their clinical use could be considered for craniofacial regenerative therapies.

Irreversible inflammation is associated with decreased levels of the alpha1-, beta1-, and alpha2-subunits of sGC in human odontoblasts.

The nitric oxide (NO) receptor enzyme soluble guanylate cyclase (sGC) contains one prosthetic heme group as an αß heterodimer, and two heterodimer isoforms (α(1)ß(1), α(2)ß(1)) were characterized to have enzyme activity. To test the irreversible inflammation-dependent regulation of sGC in odontoblasts, we incubated decalcified frozen sections of healthy and inflamed human third molars with antibodies against ß-actin, nitrotyrosine, inducible nitric oxide synthase (iNOS), α(1)-, ß(1)-, and α(2)-subunits of sGC and analyzed them at protein levels by quantitative immunohistochemistry. The irreversible inflammation induced an increase in the signal intensities for nitrotyrosine and iNOS and a decrease for the α(1)-, ß(1)-, and α(2)-subunits of sGC in odontoblasts. Inflammatory mediators, reactive oxygen, and nitrogen species may impair the expression of the α(1)-, ß(1)-, and α(2)-subunits in odontoblasts. The decrease of sGC at the protein level in inflamed odontoblasts is compatible with a critical role for sGC to mediate biological effects of NO in health.

Intraperitoneal immunization with oligomannose-coated liposome-entrapped soluble leishmanial antigen induces antigen-specific T-helper type immune response in BALB/c mice through uptake by peritoneal macrophages.

The present study demonstrates that the intraperitoneal administration of soluble leishmanial antigen (SLA) entrapped in liposomes coated with neoglycolipids containing oligomannose residues (mannopentaose or mannotriose) strongly induces an antigen-specific T-helper type 1 (Th1) immune response in BALB/c mice. In response to in vitro stimulation with SLA, spleen cells from mice that had received oligomannose-coated liposomes encasing SLA (SLA-OML) displayed greater interferon (IFN)-gamma and interleukin (IL)-2 production and lower IL-4 and IL-5 production than spleen cells from mice that had received SLA alone, indicating that the SLA-specific Th1 immune response had predominantly been induced in the mice that had received SLA-OML. After subsequent infection with Leishmania major, mice that had received SLA-OML were effectively protected against the disease, with a predominant production of IFN-gamma. OML were preferentially and rapidly incorporated into peritoneal macrophages, and the transplantation of macrophages containing SLA-OML into the peritoneal cavity also induced protection against L. major infection. Thus, SLA-OML were shown to successfully induce a specific Th1 immune response capable of controlling L. major infection in BALB/c mice through the effective uptake of OML by peritoneal macrophages.

The effect of haemodilution on blood-biomaterial contact-mediated CD11b expression on neutrophils: ex vivo studies.

Modern cardiopulmonary bypass (CPB) systems are getting smaller, both in terms of the exposed surface area of biomaterials and the priming volume. In a series of studies utilizing a rat recirculation model, we demonstrated that the magnitude of the inflammatory response seen under these conditions is proportional to the surface area of exposed material, a finding that supports the use of miniature systems in terms of moderating the inflammatory response. However, the second impact of miniature perfusion systems, the reduced priming volume with concomitant reduction in haemodilution, was not investigated with reference to inflammation. The present study was designed to determine whether this change in CPB haematocrit profile has any effect on the inflammatory response. In common with previous studies by this group, we employed the expression of the integrin CD11b on neutrophils as a marker of neutrophil activation, and hence the inflammatory response, in a rat recirculation biomaterial testing model, containing di-(2-ethyl-hexyl)-phthalate plasticized polyvinyl chloride of the type commonly employed in CPB circuits. The results demonstrated that neutrophil activation is influenced by haemodilution. We studied five groups of animals, each with different mean induced haematocrit: Group 1 (41.3 +/- 1.27%); Group 2 (30.93 +/- 2.85%); Group 3 (24.83 +/- 1.36%); Group 4 (20.60 +/- 3.47%); Group 5 (20.48 +/- 1.31%). Groups 1 and 5 animals were controls, neither of which underwent the period of recirculation. Rather, these controls were employed to isolate the noncontact effect of haemodilution on CD11b expression. We found that there were differences in per cent change in CD11b expression from start to end of the recirculation period between Group 1 (109.54 +/- 49.53%), Group 2 (189.1 +/- 18.68%), Group 3 (224.28 +/- 43.97), Group 4 (368.97 +/- 24.28%) and Group 5 (127 +/- 57.8%). There were intergroup statistically significant differences (p < 0.05). These results confirm that there is a relationship between haematocrit level and biomaterial contact-mediated activation of neutrophils. Furthermore, these studies confirm that haemodilution alone has no effect on neutrophil activation. One possible explanation for this outcome is that with higher levels of haemodilution, neutrophils have a greater opportunity to contact surface 'receptor' sites on the biomaterial, resulting in more neutrophil activation. Whatever the mechanism, these data tend to support the modern trend towards lower circuit surface area and higher haematocrit.

Impact of hemodialysis membrane and permeability on neutrophil transmigration in vitro.

BACKGROUND/AIM: The impact of dialysis membrane permeability on neutrophil transmigration properties in vitro was examined in the present study. This issue has not been fully scrutinized before. METHODS: We studied the capacity of neutrophils collected from a group of dialysis patients randomly treated with cuprophan, low- or high-flux polysulfone, to transmigrate in vitro through a membrane covered with fibronectin (a main constituent of the endothelial basement membrane). The hemodialysis-induced quantitative changes in expression of adhesion molecules were examined in parallel. RESULTS: At the end of dialysis, neutrophils collected from patients treated with high-flux polysulfone dialyzers had a significantly higher transmigration index than neutrophils from patients treated with low-flux polysulfone membrane (p < 0.01) or cuprophan membrane (p < 0.01), and approached the level of transmigration observed in neutrophils collected from healthy controls. In the groups treated with low-flux polysulfone and cuprophan dialyzers, the transmigration capacity was significantly lower (p < 0.02) compared to neutrophils from healthy subjects. We also noted that differences between low- and high-flux polysulfone dialysis, in the context of transmigration properties, were not mirrored by changes in adhesion phenotype, which strengthens the view that there is no strict relationship between these two features. CONCLUSION: The study demonstrates that high-flux polysulfone dialysis, as opposed to low-flux polysulfone and cuprophan treatment, improves the transmigration properties of circulating neutrophils, despite similar effects on adhesion molecule phenotypes. A plausible mechanism is that potentially toxic middle range molecules that inhibit neutrophil migration are more efficiently eliminated during high-flux polysulfone dialysis, but this explanation requires further support.

[Experimental investigations onto the biocompatibility of coated vascular grafts as a function of the primary disease in various patients groups]. / Experimentelle Untersuchungen zur Biokompatibilität beschichteter Gefässprothesen in Abhängigkeit von der Grundkrankheit verschiedener Patientenkollektive.

INTRODUCTION: This work presents initial experimental results obtained with co-incubation of human neutrophilic granulocytes from patients with various diseases (5 patients each group) and collagen- or silver-coated polyester grafts (25 patients each graft). METHODS: After two-hour co-cultivation of the graft with human granulocytes of different patients (normal controls; peripheral occlusive vascular disease; diabetes mellitus; carcinoma/chemotherapy; sepsis), the following parameters were determined by flow cytometry: CD11b, CD62L, fMLP, CXCR2. RESULTS: The change in the receptor expression was taken as a measure of responsiveness, with statistically significant differences seen within and between the groups. For the silver-coated graft, such differences were mainly noted for the fMLP receptor (p=0.01). For the two-hour incubation with collagen coating, the receptors CD62L and CXCR2 were indicative of differences between the various diseases (CD62L p=0.01; CXCR2 p=0.01). Comparison between the grafts revealed statistically significant differences for the CXCR2 and the fMLP receptors (CXCR2 p=0.00; fMLP p=0.03). CONCLUSIONS: The differences noted between the grafts and between the patients groups are suggestive of an altered responsiveness of the granulocytes to the grafts. This enables new aspects with respect to the genesis of accompanying clinical symptoms.