Profiling of immune-related gene expression in children with familial hypercholesterolaemia.

BACKGROUND: Innate and adaptive immune responses are pivotal in atherosclerosis, but their association with early-stage atherosclerosis in humans is incompletely understood. In this regard, untreated children with familial hypercholesterolaemia may serve as a human model to investigate the effect of elevated low-density lipoprotein (LDL)-cholesterol. OBJECTIVES: We aimed to study the immunological and inflammatory pathways involved in early atherosclerosis by examining mRNA molecules in peripheral blood mononuclear cells (PBMCs) from children with FH. METHODS: We analysed the level of 587 immune-related mRNA molecules using state-of-the-art Nanostring technology in PBMCs from children with (n = 30) and without (n = 21) FH, and from FH children before and after statin therapy (n = 10). RESULTS: 176 genes (30%) were differentially expressed between the FH and healthy children at P < 0.05. Compared to healthy children, the dysregulated pathways in FH children included the following: T cells (18/19); B cells (5/6); tumour necrosis factor super family (TNFSF) (6/8); cell growth, proliferation and differentiation (5/7); interleukins (5/9); toll-like receptors (2/5); apoptosis (3/7) and antigen presentation (1/7), where the ratio denotes higher expressed genes to total number of genes. Statin therapy reversed expression of thirteen of these mRNAs in FH children. CONCLUSION: FH children display higher PBMC expression of immune-related genes mapped to several pathways, including T and B cells, and TNFSF than healthy children. Our results suggest that LDL-C plays an important role in modulating expression of different immune-related genes, and novel data on the involvement of these pathways in the early atherosclerosis may represent future therapeutic targets for prevention of atherosclerotic progression.

Phagocytosis of live and dead Escherichia coli and Staphylococcus aureus in human whole blood is markedly reduced by combined inhibition of C5aR1 and CD14.

BACKGROUND: Sepsis is a dysregulated host response to infection. The aim of this study was to investigate the effects of complement- and CD14 inhibition on phagocytosis of live and dead Gram-negative and Gram-positive bacteria in human whole blood. METHODS: Lepirudin-anticoagulated blood was incubated with live or dead E. coli or S. aureus at 37 °C for 120 min with or without the C5aR1 antagonist PMX53 and/or anti-CD14. Granulocyte and monocyte phagocytosis were measured by flow cytometry, and five plasma cytokines by multiplex, yielding a total of 28 mediators of inflammation tested for. RESULTS: 16/28 conditions were reduced by PMX53, 7/28 by anti-CD14, and 24/28 by combined PMX53 and CD14 inhibition. The effect of complement inhibition was quantitatively more pronounced, in particular for the responses to S. aureus. The effect of anti-CD14 was modest, except for a marked reduction in INF-ß. The responses to live and dead S. aureus were equally inhibited, whereas the responses to live E. coli were inhibited less than those to dead E. coli. CONCLUSION: C5aR1 inhibited phagocytosis-induced inflammation by live and dead E. coli and S. aureus. CD14 blockade potentiated the effect of C5aR1 blockade, thus attenuating inflammation.

Complement component 5 does not interfere with physiological hemostasis but is essential for Escherichia coli-induced coagulation accompanied by Toll-like receptor 4.

There is a close cross-talk between complement, Toll-like receptors (TLRs) and coagulation. The role of the central complement component 5 (C5) in physiological and pathophysiological hemostasis has not, however, been fully elucidated. This study examined the effects of C5 in normal hemostasis and in Escherichia coli-induced coagulation and tissue factor (TF) up-regulation. Fresh whole blood obtained from six healthy donors and one C5-deficient individual (C5D) was anti-coagulated with the thrombin inhibitor lepirudin. Blood was incubated with or without E. coli in the presence of the C5 inhibitor eculizumab, a blocking anti-CD14 monoclonal antibody (anti-CD14) or the TLR-4 inhibitor eritoran. C5D blood was reconstituted with purified human C5. TF mRNA was measured by quantitative polymerase chain reaction (qPCR) and monocyte TF and CD11b surface expression by flow cytometry. Prothrombin fragment 1+2 (PTF1·2) in plasma and microparticles exposing TF (TF-MP) was measured by enzyme-linked immunosorbent assay (ELISA). Coagulation kinetics were analyzed by rotational thromboelastometry and platelet function by PFA-200. Normal blood with eculizumab as well as C5D blood with or without reconstitution with C5 displayed completely normal biochemical hemostatic patterns. In contrast, E. coli-induced TF mRNA and TF-MP were significantly reduced by C5 inhibition. C5 inhibition combined with anti-CD14 or eritoran completely inhibited the E. coli-induced monocyte TF, TF-MP and plasma PTF1·2. Addition of C5a alone did not induce TF expression on monocytes. In conclusion, C5 showed no impact on physiological hemostasis, but substantially contributed to E. coli-induced procoagulant events, which were abolished by the combined inhibition of C5 and CD14 or TLR-4.

Staphylococcus aureus-induced complement activation promotes tissue factor-mediated coagulation.

Essentials Complement, Toll-like receptors and coagulation cross-talk in the process of thromboinflammation. This is explored in a unique human whole-blood model of S. aureus bacteremia. Coagulation is here shown as a downstream event of C5a-induced tissue factor (TF) production. Combined inhibition of C5 and CD14 efficiently attenuated TF and coagulation. SUMMARY: Background There is extensive cross-talk between the complement system, the Toll-like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression. Objectives To study the relative roles of complement, TLRs and TF in Staphylococcus aureus-induced coagulation. Methods Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF1 + 2 ) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively. Results All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF1 + 2 , and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF1 + 2 levels to baseline levels. Conclusions S. aureus-induced coagulation in human whole blood was mainly attributable to C5a-induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation.

Expression of Toll-like receptor-3 is enhanced in active inflammatory bowel disease and mediates the excessive release of lipocalin 2.

Anti-microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the anti-microbial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in-situ hybridization. Moreover, we examined the regulation of LCN2 in HT-29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll-like receptor (TLR)-3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up-regulated genes in both active ulcerative colitis (UCa) and active Crohn's disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de-novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT-29 was enhanced by both interleukin (IL)-1ß and the TLR-3 ligand poly(I:C), and TLR-3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation.

Differential effect of inhibiting MD-2 and CD14 on LPS- versus whole E. coli bacteria-induced cytokine responses in human blood.

BACKGROUND: Sepsis is a major world-wide medical problem with high morbidity and mortality. Gram-negative bacteria are among the most important pathogens of sepsis and their LPS content is regarded to be important for the systemic inflammatory reaction. The CD14/myeloid differentiation factor 2 (MD-2)/TLR4 complex plays a major role in the immune response to LPS . The aim of this study was to compare the effects of inhibiting MD-2 and CD14 on ultra-pure LPS - versus whole E. coli bacteria-induced responses. METHODS: Fresh human whole blood was incubated with upLPS or whole E. coli bacteria in the presence of MD-2 or CD14 neutralizing monoclonal antibodies, or their respective controls, and/or the specific complement-inhibitor compstatin. Cytokines were measured by a multiplex (n = 27) assay. NFκB activity was examined in cells transfected with CD14, MD-2 and/or Toll-like receptors. RESULTS: LPS-induced cytokine response was efficiently and equally abolished by MD-2 and CD14 neutralization. In contrast, the response induced by whole E. coli bacteria was only modestly reduced by MD-2 neutralization, whereas CD14 neutralization was more efficient. Combination with compstatin enhanced the effect of MD-2 neutralization slightly. When compstatin was combined with CD14 neutralization, however, the response was virtually abolished for all cytokines, including IL-17, which was only inhibited by this combination. The MD-2-independent effect observed for CD14 could not be explained by TLR2 signaling. CONCLUSION: Inhibition of CD14 is more efficient than inhibition of MD-2 on whole E. coli-induced cytokine response, suggesting CD14 to be a better target for intervention in Gram-negative sepsis, in particular when combined with complement inhibition.

Toll-like receptor 2 (P631H) mutant impairs membrane internalization and is a dominant negative allele.

We have sequenced 416 Toll-like receptor-2 (TLR2) alleles in 208 subjects in a tuberculosis case-control study in Croatian Caucasian population. We found ten single nucleotide polymorphisms (SNP) among which three were novel (S97S, T138I and L266F). The genotype containing TLR2-P631H SNP was significantly overrepresented in patients with tuberculosis when compared to contact controls, suggesting a small yet increased risk to disease. The causative agent of tuberculosis is Mycobacterium tuberculosis, which can bind to TLR2 with its lipoprotein coat. The TLR2-P631H mutant has a dominant negative effect on the wild type TLR2 signalling in transfected HEK293 kidney cells using the NF-kappaB-driven luciferase as a reporter gene with ligands like M. avium extracts, Pam3CysSK4 or FSL-1 that bind TLR2/TLR1 or TLR2/TLR6 heterodimers, respectively. Studies on internalization from the Regular Madine Darby Canine Kidney cell surface into the early endosomal compartments showed a lower rate of the mutant compared to the wild type. Our data, in combination with a report by others show that the TLR2-P631H allele could be associated with protection to meningococcal meningitis, suggest that by dominantly inhibiting the response of cells important in the immune response this mutant might confer either protection or susceptibility to meningitis or tuberculosis, respectively.

Beryllium sensitivity among workers at a Norwegian aluminum smelter.

BACKGROUND: Sensitivity to beryllium was investigated among workers at an aluminum smelter in Norway as a consequence of the findings in an occupational exposure survey. METHODS: Three hundred and sixty-two employees and 31 reference persons were tested for sensitization to beryllium with the beryllium lymphocyte proliferation test (BeLPT) based on specifications by the US Department of Energy in 2001. The results are reported as abnormal, borderline, or normal. RESULTS: One person (0.28%) from the aluminum smelter was found to have abnormal results in two separate blood samples and is sensitized to beryllium. Three other persons had one abnormal test that was not confirmed by a second test. One person in the reference group had one abnormal and one normal test result. No borderline samples were detected. None of the employees with one or more abnormal sample results had pot room asthma. The sensitized individual worked in a Soederberg line in 1972-1974. The beryllium concentration in the work atmosphere is estimated to have been similar as today (0.1-0.3 microg/m(3)), but work routines, etc. would cause higher total exposures. CONCLUSIONS: Only one sensitized person of 362 is in line with what is found in other studies in the aluminum industry. The low number, compared with the beryllium handling industry, may be attributable to lower work atmosphere concentrations, beryllium speciation effects, or use of respiratory protection equipment. Pot room asthma does not appear to be associated with beryllium sensitization.

Innate immune responses to danger signals in systemic inflammatory response syndrome and sepsis.

The systemic immune response induced by non-infectious agents is called systemic inflammatory response syndrome (SIRS) and infection-induced systemic immune response is called sepsis. The host inflammatory response in SIRS and sepsis is similar and may lead to multiple organ dysfunction syndrome (MODS) and ultimately death. The mortality and morbidity in SIRS and sepsis (i.e. critical illness) remain high despite advances in diagnostic and organ supporting possibilities in intensive care units. In critical illness, the acute immune response is organized and executed by innate immunity influenced by the neuroendocrine system. This response starts with sensing of danger by pattern-recognition receptors on the immune competent cells and endothelium. The sensed danger signals, through specific signalling pathways, activate nuclear transcription factor kappaB and other transcription factors and gene regulatory systems which up-regulate the expression of pro-inflammatory mediators. The plasma cascades are also activated which together with the produced pro-inflammatory mediators stimulate further the production of inflammatory biomarkers. The acute inflammatory response underlies the pathophysiological mechanisms involved in the development of MODS. The inflammatory mediators directly affect organ function and cause a decline in remote organ function by mediating the production of nitric oxide leading to mitochondrial anergy and cytopathic hypoxia, a condition of cellular inability to use oxygen. Understanding the mechanisms of acute immune responses in critical illness is necessary for the development of urgently needed therapeutics. The aim of this review is to provide a description of the key components and mechanisms involved in the immune response in SIRS and sepsis.

Decreased serum lipocalin-2 levels in human immunodeficiency virus-infected patients: increase during highly active anti-retroviral therapy.

Although neutrophil gelatinase-associated lipocalin (NGAL) may play a pivotal role in the innate immune response, there are currently no data on NGAL levels in human immunodeficiency virus (HIV)-infected patients. In this study we aimed to examine the regulation of NGAL in HIV infection. The regulation of NGAL in HIV infection was examined by different experimental approaches, including studies in peripheral blood and mononuclear cells (MNC) from bone marrow aspirates before and during highly active anti-retroviral therapy (HAART). We found that: before initiating HAART, HIV-infected patients (n = 37) had significantly decreased serum NGAL levels compared with healthy controls (n = 26); (ii) during HAART, there was a gradual and significant increase in NGAL concentrations reaching levels comparable to those in healthy controls after 12 months; (iii) this increase was seen primarily in virological responders to HAART (HIV RNA level <200 copies/ml after 24 months); (iv) phytohaemagglutinin-stimulated NGAL release in MNC cells from bone marrow aspirates was decreased in untreated HIV-infected patients compared with healthy controls, but increased after 26 weeks on HAART; and (v) there was a significant positive correlation between neutrophil counts and NGAL levels at all time-points during HAART. We have shown decreased NGAL levels in HIV-infected patients, potentially reflecting decreased number and function of neutrophils as well as impaired bone marrow myelopoiesis. These abnormalities were reversed by successful HAART. Our findings underscore further the involvement of neutrophils and innate immunity in HIV-related immunodeficiency.

Toll-like receptors mediate proliferation and survival of multiple myeloma cells.

Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in bone marrow (BM) and recurrent or persistent infections. Toll-like receptors (TLRs) are essential in the host defense against infections and today 10 human TLRs (TLR1-TLR10) and one TLR-homolog (RP105) have been characterized. B cells express several TLRs (mainly TLR1, 6, 7, 9, 10 and RP105) and TLR-initiated responses in B cells include proliferation, anti-apoptosis effect and plasma cell (PC) differentiation. The present study was designed to analyze the role of TLRs in MM. We show that frequent expressions of TLRs were detected in cell lines from MM patients (minimum six TLRs in each). In comparison, only few TLRs (mainly TLR1 and or RP105) were found expressed in PCs from BM of healthy donors. In addition, TLR-specific ligands induce increased proliferation and survival of the MM cell lines, partially due to an autocrine interleukin-6 production. Importantly, we demonstrate that also PC from MM patients proliferates in response to TLR-specific ligands. In conclusion, TLR-ligands may contribute to increased growth and survival of MM cells in MM patients.

Modulatory effect of mannose-binding lectin on cytokine responses: possible roles in HIV infection.

BACKGROUND: Mannose-binding lectin (MBL) is a soluble receptor of the innate immune system, probably contributing to antimicrobial defence. The possible role of MBL in HIV infection is unclear. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) from 28 HIV-infected patients and 13 healthy controls were stimulated with MBL and costimulated with HIV-1 gp120 or mannan from Saccharomyces cerevisiae before inflammatory responses in PBMC cultures were examined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. HIV-1 RNA replication in vitro was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR) in supernatants from patients with measurable HIV-1 RNA levels. RESULTS: (i) Enhanced TNF-alpha responses were observed when PBMCs from healthy controls and HIV-infected patients were stimulated with MBL and costimulated with HIV-1 gp120 or mannan. (ii) MBL stimulation induced increased HIV RNA replication in culture supernatants when costimulated with mannan. CONCLUSIONS: The present study suggests a modulatory role of MBL on cytokine responses, and HIV replication after stimulation with microbial products. These effects of MBL on inflammatory responses and viral replication may be clinically relevant for HIV infection.

Myocardial ischaemia and the inflammatory response: release of heat shock protein 70 after myocardial infarction.

OBJECTIVES: To test the hypothesis that heat shock protein (Hsp) 70 may be released into the circulation after acute myocardial infarction (AMI) by exploring the kinetics of Hsp70 release and the relations between Hsp70 and markers of inflammation and myocardial damage in AMI. DESIGN: Blood samples from 24 patients were prospectively collected through to the first day after AMI. Hsp70, interleukin (IL) 6, IL-8, and IL-10 in serum were measured by enzyme linked immunosorbent assay (ELISA). RESULTS: Median Hsp70 concentrations in AMI patients measured at arrival, six hours thereafter, and the following morning were 686, 868, and 607 pg/ml, respectively. These concentrations were all significantly different from those of the control patients with angina with a median serum Hsp70 concentration of 306 pg/ml. Peak Hsp70 correlated with creatine kinase (CK) MB (r = 0.62, p < 0.01) and cardiac troponin T (r = 0.58, p < 0.01). Furthermore, serum Hsp70 correlated with IL-6 and IL-8 at six hours (r = 0.60, p < 0.01 and r = 0.59, p < 0.01, respectively). CONCLUSIONS: In this study, Hsp70 was rapidly released into the circulation after AMI. Circulating Hsp70 is suggested as a marker of myocardial damage. In addition, Hsp70 may have a role in the inflammatory response after AMI.

Stimulation of toll-like receptor 2 in mononuclear cells from HIV-infected patients induces chemokine responses: possible pathogenic consequences.

Toll-like receptor 2 (TLR2) stimulation in monocytes may contribute to enhanced inflammation and viral replication in HIV infection. In the present study we examined if TLR2 stimulation could modulate chemokine responses in peripheral blood mononuclear cells (PBMC) from HIV-infected patients and healthy controls. Our main findings were, with similar qualitative patterns in both healthy controls and HIV-infected patients: (1) TLR2 stimulation induced up-regulation of several chemokines at the mRNA level as well as increased protein levels of macrophage inflammatory protein (MIP)-1alpha, interleukin (IL)-8 and regulated on activation, normal T cell expressed and secreted (RANTES); (2) TLR2 stimulation induced enhanced protein expression of CCR5 (a receptor for MIP-1alpha and RANTES) on monocytes; (3) In vitro stimulation with RANTES induced release of MIP-1alpha, MCP-1, IL-8 and interferon-gamma from PBMC. While increased levels of beta-chemokines possibly have antiviral effects, TLR2 stimulation may also promote a chemokine-driven inflammatory loop, potentially contributing to the immunopathogenesis of HIV infection.

Monocytes stimulated with group B streptococci or interferons release tumour necrosis factor-related apoptosis-inducing ligand.

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a cytotoxic member of the TNF family. Some reports have shown that TRAIL is released from cells in a soluble form. In this work, we have investigated the mechanism of release of TRAIL from monocytes. First, we show that whole gram-positive, gram-negative and mycoplasmal bacteria as well as lipopolysaccharide (LPS), interferon-alpha (IFN-alpha), -beta and -gamma all induced upregulation of TRAIL on the surface of human monocytes. Next, we show that IFN-alpha, -beta and -gamma all induced a dose-dependent release of TRAIL, giving significant amounts of soluble TRAIL after 2 days. Of the bacteria, only the Group B streptococcus COH-1 (GBS) induced release of TRAIL and concomittantly induced IFN-alpha. Monocytes stimulated with GBS or IFN-alpha also showed extensive cell death. When monocyte apoptosis was prevented by interleukin-1, GM-CSF, LPS or the caspase inhibitor zVADfmk, the IFN-alpha-induced release of TRAIL was reduced, whereas agents inducing necrosis caused increased release of TRAIL. LPS also prevented release of TRAIL from GBS-stimulated monocytes. The release of TRAIL from IFN-alpha-stimulated monocytes was reduced by inhibitors of both cysteine and metalloproteases. We conclude that bacteria and IFN induce upregulation of membrane TRAIL and that release of TRAIL is associated with cell death.

Evaluation of different types of alginate microcapsules as bioreactors for producing endostatin.

The use of nonautologous cell lines producing a therapeutic substance encapsulated within alginate microcapsules could be an alternative way of treating different diseases in a cost-effective way. Malignant brain tumors have been proposed to be treated locally using engineered cells secreting proteins with therapeutic potential encapsulated within alginate microcapsules. Optimization of the alginate capsule bioreactors is needed before this treatment can be a reality. Recently, we have demonstrated that alginate-poly-L-lysine microcapsules made with high-G alginate and a gelled core disintegrated as cells proliferated. In this study we examined the growth and endostatin secretion of 293-EBNA (293 endo) cells encapsulated in six different alginate microcapsules made with native high-G alginate or enzymatically tailored alginate. Stability studies using an osmotic pressure test showed that alginate-poly-L-lysine-alginate microcapsules made with enzymatically tailored alginate was mechanically stronger than alginate capsules made with native high-G alginate. Growth studies showed that the proliferation of 293 endo cells was diminished in microcapsules made with enzymatically tailored alginate and gelled in a barium solution. Secretion of endostatin was detected in lower amounts from the enzymatically tailored alginate microcapsules compared with the native alginate microcapsules. The stability of the alginate microcapsules diminished as the 293 endo cells grew inside the capsules, while empty alginate microcapsules remained stable. By using microcapsules made of fluorescenamine-labeled alginate it was clearly visualized that cells perforated the alginate microcapsules as they grew, destroying the alginate network. Soluble fluorescence-labeled alginate was taken up by the 293 endo cells, while alginate was not detected in live spheroids within fluorescence-labeled alginate microcapsules. Despite that increased stability was achieved by using enzymatically tailored alginate, the cell proliferation destroyed the alginate microcapsules with time. It is therefore necessary to use cell lines that have properties more suited for alginate encapsulation before this technology can be used for therapy.

Immunohistochemical localization of Toll-like receptors 2 and 4 in gingival tissue from patients with periodontitis.

The present study investigated the expression of Toll-like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2-, TLR4-, CD14- and CD1a-positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2-positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4-positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14-positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down-regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.

Alginate-polylysine-alginate microcapsules: effect of size reduction on capsule properties.

Alginate-polylysine-alginate capsules containing insulin-producing cells have been used as a bio-artificial pancreas in the treatment of diabetes mellitus. In a search for microcapsules with improved diffusion characteristics, a high voltage system was developed that produces 250,000 beads/min with a diameter of 160 microm +/- 3-5%. The diameter of the beads could be varied between 160-700 microm depending on the needle diameter and construction, the voltage, the distance between the electrodes and the flow of alginate solution. Ca-alginate beads with diameters of 200 and 500 microm were produced by the high voltage electrostatic system. The 200 microm beads were sensitive to poly-L-lysine (PLL) exposure and had to be washed in ion-free solution to avoid collapse. The 200 microm beads swelled more than the 500 microm beads in the washing and PLL treatment. Also, the porosity of the capsules changed with size, but capsules impermeable to tumour necrosis factor (TNF) could be made by exchanging PLL with poly-D-lysine (PDL) for the 500 microm beads. The 200 microm beads were impermeable to IgG after PLL exposure. Islets of Langerhans were encapsulated in alginate-PLL-alginate capsules and evaluated by measuring protruding islets and insulin production. Islets in microcapsules made by the high voltage electrostatic system did not function differently from islets in larger microcapsules made by an air jet system. In conclusion, alginate capsules made by a high voltage electrostatic system enable large-scale production of small capsules with a narrow size distribution that can meet the functional properties of larger capsules by small changes in the encapsulation procedure.

Novel engagement of CD14 and multiple toll-like receptors by group B streptococci.

Group B streptococcus (GBS) imposes a major health threat to newborn infants. Little is known about the molecular basis of GBS-induced sepsis. Both heat-inactivated whole GBS bacteria and a heat-labile soluble factor released by GBS during growth (GBS-F) induce nuclear translocation of NF-kappaB, the secretion of TNF-alpha, and the formation of NO in mouse macrophages. Macrophages from mice with a targeted disruption of MyD88 failed to secrete TNF-alpha in response to both heat-inactivated whole bacteria and GBS-F, suggesting that Toll-like receptors (TLRs) are involved in different aspects of GBS recognition. Immune cell activation by whole bacteria differed profoundly from that by secreted GBS-F. Whole GBS activated macrophages independently of TLR2 and TLR6, whereas a response to the secreted GBS-F was not observed in macrophages from TLR2-deficient animals. In addition to TLR2, TLR6 and CD14 expression were essential for GBS-F responses, whereas TLR1 and TLR4 or MD-2 did not appear to be involved. Heat lability distinguished GBS-F from peptidoglycan and lipoproteins. GBS mutants deficient in capsular polysaccharide or beta-hemolysin had GBS-F activity comparable to that of wild-type streptococci. We suggest that CD14 and TLR2 and TLR6 function as coreceptors for secreted microbial products derived from GBS and that cell wall components of GBS are recognized by TLRs distinct from TLR1, 2, 4, or 6.

Poly-L-Lysine induces fibrosis on alginate microcapsules via the induction of cytokines.

Alginate-poly-L-lysine (PLL) microcapsules can be used for transplantation of insulin-producing cells for treatment of type I diabetes. In this work we wanted to study the inflammatory reactions against implanted microcapsules due to PLL. We have seen that by reducing the PLL layer, less overgrowth of the capsule is obtained. By incubating different cell types with PLL and afterwards measuring cell viability with MTT, we found massive cell death at concentrations of PLL higher than 10 microg/ml. Staining with annexin V and propidium iodide showed that PLL induced necrosis but not apoptosis. The proinflammatory cytokine, tumor necrosis factor (TNF), was detected in supernatants from monocytes stimulated with PLL. The TNF response was partly inhibited with antibodies against CD14, which is a well-known receptor for lipopolysaccharide (LPS). Bactericidal permeability increasing protein (BPI) and a lipid A analogue (B-975), which both inhibit LPS, did not inhibit PLL from stimulating monocytes to TNF production. This indicates that PLL and LPS bind to different sites on monocytes, but because they both are inhibited by a p38 MAP kinase inhibitor, they seem to have a common element in the signal transducing pathway. These results suggest that PLL may provoke inflammatory responses either directly or indirectly through its necrosis-inducing abilities. By combining soluble PLL and alginate both the toxic and TNF-inducing effects of PLL were reduced. The implications of these data are to use alginate microcapsules with low amounts of PLL for transplantation purposes.