Facilitated Subcutaneous Immunoglobulin Treatment in Patients with Immunodeficiencies: the FIGARO Study.

PURPOSE: The FIGARO study aims to provide insights on real-world utilization and tolerability of facilitated subcutaneous immunoglobulin (fSCIG) for primary immunodeficiency disease (PID) or secondary immunodeficiency disease (SID). METHODS: This prospective, multicenter, observational study, evaluated medical records, charts, and diaries of patients who had received at least 1 fSCIG infusion for PID or SID. Data were analyzed by cohort (PID, SID) and age groups (pediatric [< 18 years], adult [18-64 years], older adult [≥ 65 years]). Patients were followed up to 36 months. RESULTS: The study enrolled 156 patients: 15 pediatric, 120 adult, 21 older-adult. Twelve-month follow-up data were available for 128 patients. fSCIG was mainly prescribed for PID among patients aged < 65 years and for SID among older adults. At inclusion, 75.6% received their fSCIG infusion at home, and 78.7% self-administered. Adults were more likely to receive their initial infusion at home and self-administer (81.7% and 86.6%, respectively) than pediatric patients (53.3% each) and older adults (57.1% and 52.4%, respectively). At 12 months, the proportion of patients infusing at home and self-administering increased to 85.8% and 88.2%. Regardless of age, most patients self-administered the full fSCIG dose at home every 3-4 weeks and required a single infusion site. The tolerability profile was consistent with previous pivotal trials. Acute severe bacterial infections occurred in 0%-9.1% of patients during follow-up visits (full cohort). CONCLUSIONS: FIGARO confirms the feasibility, tolerability, and good infection control of fSCIG in PID and SID patients across the age spectrum in both the home-setting and medical facility. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03054181.

Facilitated subcutaneous immunoglobulin use in pediatric patients with primary or secondary immunodeficiency diseases.

Aim: While facilitated subcutaneous immunoglobulin (fSCIG) has been evaluated in pediatric patients with primary immunodeficiency diseases in clinical trials, real-world data are lacking. Materials & methods: This multicenter, retrospective, chart review study assessed fSCIG utilization in 30 patients less than 18 years old, with primary or secondary immunodeficiency diseases. Medical records were reviewed at fSCIG initiation and at 6 months. Results: Most (90%) patients received their first fSCIG infusion at a medical facility; by 6 months, all fSCIG infusions were administered at home by the patient/caregiver, the majority infusing every 3-4 weeks into a single site. No serious adverse drug reactions occurred. Conclusion: This study supports the feasibility and tolerability of administering fSCIG at home to pediatric patients with immunodeficiencies. Clinical Trial Registration: DRKS00015436 (German Clinical Trials Register).

Update on real-world use of facilitated subcutaneous immunoglobulin and immune globulin subcutaneous (human) 20% solution in patients with immunodeficiencies: poster extracts from the 19th Biennial Meeting of the European Society for Immunodeficiencies.

Immunoglobulin replacement therapy has been shown in clinical trials to be an important therapeutic option for reducing the incidence of serious bacterial infections and improving the quality of life in patients with primary and secondary immunodeficiencies (PID and SID, respectively). This article summarizes a poster series presented at the 19th Biennial Meeting of the European Society for Immunodeficiencies (October 14-17, 2020) further evaluating real-world usage and patient/physician experience with Immune Globulin Subcutaneous (Human) 20% Solution (Ig20Gly) in patients with PID and facilitated subcutaneous immunoglobulin (fSCIG) in patients with PID or SID.

IVIG induces apoptotic cell death in CD56dim NK cells resulting in inhibition of ADCC effector activity of human PBMC.

The mechanism of the efficacy of Intravenous immunoglobulins (IVIG) in autoimmune and inflammatory diseases is not well understood. This study aimed at understanding mechanisms of IVIG-mediated suppression of effector cell activities of peripheral blood mononuclear cells (PBMC) in antibody-dependent cellular cytotoxicity (ADCC). We were particularly interested in CD56dim NK cells, the main ADCC effector cells in PBMC. Exposure of PBMC to IVIG for at least 48 h induced a caspase-3-dependent apoptotic cell death of CD56dim NK cells without affecting CD56bright NK cells. Induction of apoptosis in CD56dim NK cells and concomitant suppression of ADCC effector activities of PBMC was associated with the monomer fraction of IVIG. Moreover, it was independent of IgG sialyation, did not depend on engagement of FcγRIII and could not be mimicked by IVIG (Fab')2 or IVIG Fc preparations. The described effect could contribute to the reduction of peripheral NK cells observed during IVIG therapy in patients.

Immunoglobulins stimulate cultured Schwann cell maturation and promote their potential to induce axonal outgrowth.

BACKGROUND: Schwann cells are the myelinating glial cells of the peripheral nervous system and exert important regenerative functions revealing them as central repair components of many peripheral nerve pathologies. Intravenous immunoglobulins (IVIG) are widely used to treat autoimmune and inflammatory diseases including immune-mediated neuropathies. Nevertheless, promotion of peripheral nerve regeneration is currently an unmet therapeutical goal. We therefore examined whether immunoglobulins affect glial cell homeostasis, differentiation, and Schwann cell dependent nerve regenerative processes. METHODS: The responses of different primary Schwann cell culture models to IVIG were investigated: immature or differentiation competent Schwann cells, myelinating neuron/glial cocultures, and dorsal root ganglion explants. Immature or differentiating Schwann cells were used to study cellular proliferation, morphology, and gene/protein expression. Myelination rates were determined using myelinating neuron/glia cocultures, whereas axonal outgrowth was assessed using non-myelinating dorsal root ganglion explants. RESULTS: We found that IVIG specifically bind to Schwann cells and detected CD64 Fc receptor expression on their surface. In response to IVIG binding, Schwann cells reduced proliferation rates and accelerated growth of cellular protrusions. Furthermore, we observed that IVIG treatment transiently boosts myelin gene expression and myelination-related signaling pathways of immature cells, whereas in differentiating Schwann cells, myelin expression is enhanced on a long-term scale. Importantly, myelin gene upregulation was not detected upon application of IgG1 control antibodies. In addition, we demonstrate for the first time that Schwann cells secrete interleukin-18 upon IVIG stimulation and that this cytokine instructs these cells to promote axonal growth. CONCLUSIONS: We conclude that IVIG can positively influence the Schwann cell differentiation process and that it enhances their regenerative potential.

Human anti-Aß IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

Soluble non-fibrillar assemblies of amyloid-beta (Aß) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aß immunotherapy is a promising and advanced therapeutic strategy, but the precise Aß species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aß conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aß dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aß extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aß's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aß monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aß NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aß NAbs are warranted.

Diesel exhaust particles impair platelet response to collagen and are associated with GPIbα shedding.

OBJECTIVE: Air pollution with fine particulates (PM(10) and PM(2.5)) is associated with an increased incidence of cardiovascular events. The proposed mechanisms include indirect proinflammatory and procoagulant reactions involving activation of pulmonary macrophages, endothelial cells and the TNF/TF pathway, or direct procoagulant effects. Our laboratory has observed a reduction of the platelet responsiveness to collagen after exposure to diesel exhaust particles (DEP). HYPOTHESIS: DEP directly interfere with platelet-collagen interactions by selectively inducing the shedding of platelet signaling receptors via metalloproteinases, which would represent a novel mechanism for DEP action on platelets. METHODS: Citrated blood from healthy volunteers was exposed to highly standardized DEP at concentrations of 0.1, 2.5 and 5.0µg/ml or ultrafine carbon black (ufCB, 0.1µg/ml) and the plasmatic and platelet response was analysed. The closure times with the PFA-100 device and the platelet aggregation in response to a variety of agonists were monitored. Interleukins (IL)-1ß and IL-8 levels were determined by ELISA and soluble P-selectin by the Luminex bead assay. Thrombin activity was measured as the endogenous thrombin potential (ETP) by fluorescence spectrometry. Soluble GPVI and GPIbα (glycocalicin) ectodomain fragments were measured by ELISA. ADAMTS13 activity was determined by a FRETS based assay and plasmin activity with Spectrozyme PL. RESULTS: Aggregation assays where platelets were treated with low dose DEP or ultrafine carbon black (ufCB) revealed a significantly increased response to low doses of collagen (p<0.05, n=5). At higher doses, however, collagen induced aggregation was suppressed by DEP treatment: at 2.5µg/ml, the inhibition was 34±12% (p<0.01, n=10). Aggregations with cross-linked collagen related peptide (CRPxl), convulxin and with the monoclonal antibody 9O12.2 (all known to specifically bind to and activate GPVI) were also diminished. Ristocetin, arachidonic acid and ADP responses were normal at all DEP concentrations. No cleavage of GPVI ectodomain was detected (soluble GPVI 27.8±3 vs. 28±4µg/ml mean±SEM, n=10); however increased plasma glycocalicin (GPIbα ectodomain) was detected upon diesel exposure (2.58±0.11 vs. 2.28±0.03µg/ml p<0.01, n=10). ADAMTS13 and plasmin activity remained unaffected by DEP under the conditions tested. Platelets were not activated by either DEP or ufCB as soluble P-selectin was insensitive to these. CONCLUSIONS: DEP specifically and directly interferes with platelet-collagen interactions. The functional consequences are biphasic and include enhance platelet aggregation at lower DEP concentrations and inhibition at a higher dose. Our data indicate that this interaction does not involve P-selectin or GPVI shedding. It is however associated with an increase in GPIb cleavage.

CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice.

Today it is generally accepted that B cells require cognate interactions with CD4(+) T cells to develop high-affinity antibodies against proteins. CD4(+) T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4(+) T cells that can bind to the MHC class II-peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4(+) T-cell epitopes presented by HLA-DRB1*1501 to CD4(+) T cells during immune responses against FVIII. CD4(+) T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

Antigens of persistent Chlamydia pneumoniae within coronary atheroma from patients undergoing heart transplantation.

AIMS: In order for Chlamydia pneumoniae to play a causative role in chronic human disease, it would need to persist within infected tissue for extended periods of time. Current theory suggests that C pneumoniae may persist at the site of infection via an alternative replicative form, known as an aberrant body. METHODS: A panel of C pneumoniae-specific antibodies upregulated by the aberrant body was used to probe tissue specimens from the coronary atheroma from 13 explanted hearts to identify patterns of reactivity in these tissues, as well as to determine the presence and prevalence of C pneumoniae aberrant bodies. RESULTS: Six of 13 patients had an ischaemic cardiomyopathy secondary to coronary atherosclerosis, while another six patients had an idiopathic, dilated cardiomyopathy. One additional patient, a young (24 years) woman with cardiomyopathy, had no history of atherosclerotic disease. Eleven patients were positive by immunohistochemistry with at least one antibody. Coronary arteries of the two other patients were negative by immunohistochemistry with all antibodies. One of these patients was the 24-year-old woman with grade I disease and no risk factors for coronary artery disease. CONCLUSIONS: The protein antigens of persistent C pneumoniae infection found in the atheromatous lesions from patients in this study could potentially be used as markers to detect such infections and some may be virulence factors or immunogens specific to C pneumoniae, thus serving as target molecules for diagnostic use or therapeutic intervention.

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model.

Replacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed.

Biomarker candidates of Chlamydophila pneumoniae proteins and protein fragments identified by affinity-proteomics using FTICR-MS and LC-MS/MS.

We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.

Anti-inflammatory capacity of selected lactobacilli in experimental colitis is driven by NOD2-mediated recognition of a specific peptidoglycan-derived muropeptide.

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) has been linked to a loss of tolerance towards the resident microflora. Therapeutic use of probiotics is known to be strain specific, but precise mechanisms remain unclear. The role of NOD2 signalling and the protective effect of Lactobacillus peptidoglycan (PGN) and derived muropeptides in experimental colitis were evaluated. METHODS: The anti-inflammatory capacity of lactobacilli and derived bacterial compounds was evaluated using the 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis model. The role of NOD2, MyD88 and interleukin 10 (IL-10) in this protection was studied using Nod2(-/-), MyD88(-/-) and Il10-deficient mice, while induction of regulatory dendritic cells (DCs) was monitored through the expansion of CD103(+) DCs in mesenteric lymph nodes or after adoptive transfer of bone marrow-derived DCs. The development of regulatory T cells was investigated by following the expansion of CD4(+)FoxP3(+) cells. High-performance liquid chromatography and mass spectrometry were used to analyse the PGN structural differences. RESULTS: The protective capacity of strain Lactobacillus salivarius Ls33 was correlated with a local IL-10 production and was abolished in Nod2-deficient mice. PGN purified from Ls33 rescued mice from colitis in an IL-10-dependent manner and favoured the development of CD103(+) DCs and CD4(+)Foxp3(+) regulatory T cells. In vitro Ls33 PGN induced IL-10-producing DCs able to achieve in vivo protection after adoptive transfer in a NOD2-dependent way. This protection was also correlated with an upregulation of the indoleamine 2,3-dioxygenase immunosuppressive pathway. The protective capacity was not obtained with PGN purified from a non-anti-inflammatory strain. Structural analysis of PGNs highlighted in Ls33 the presence of an additional muropeptide, M-tri-Lys. The synthesised ligand protected mice from colitis in a NOD2-dependent but MyD88-independent manner. CONCLUSIONS: The results indicated that PGN and derived muropeptides are active compounds in probiotic functionality and might represent a useful therapeutic strategy in IBD.

Growth temperature-dependent expression of structural variants of Listeria monocytogenes lipoteichoic acid.

Investigating the expression of lipoteichoic acid (LTA) from Listeria monocytogenes, we found two distinct structural variants of LTA (LTA1 and LTA2) using NMR and MS technology. While both LTA consisted of a poly-glycerophosphate backbone (differing in length) bound via a disaccharide to a diacyl-glycerol moiety, one LTA type (LTA2) possessed a second diacyl-glycerol moiety linked to the disaccharide via a phosphodiester. As examined in vitro, LTA2 in contrast to LTA1 failed to activate the L-ficolin dependent pathway of complement. Most interestingly, growth temperature had a strong influence on the expression levels of LTA1 and LTA2 in the cell wall: while the amount of LTA1 was comparable, the expression of LTA2 was low when Listeria had grown at room temperature (ratio of LTA1 to LTA2 was 1:0.06), but increased when Listeria had been cultivated at 37°C (ratio of LTA1 to LTA2 was 1:0.68). The observed shift in LTA expression, probably accompanying the switch from the saprophytic to the virulent entity, indicates an important adaptation to the different structural requirements inside the host cells.

Different Staphylococcus aureus whole bacteria mutated in putative pro-inflammatory membrane components have similar cytokine inducing activity.

The role of the different major cell wall components of Gram-positive bacteria for immune stimulation is controversial. We thus compared the cytokine inducing capacity of different Staphylococcus aureus (SA) mutants lacking either lipoproteins (SA 113Δlgt), or wall teichoic acids (WTA) (ΔTA), or possessing a reduced d-alanine content in lipoteichoic acid (LTA) (SA 113Δdlt) to its corresponding wildtype (SA 113wt). Inactivated whole bacteria and their purified cell wall components peptidoglycan and LTA, were used to stimulate human whole blood and macrophages from TLR2 knock-out mice. We found that all S. aureus strains induced similar amounts of TNF, IL-8 and IL-10 and none of them was dependent on the presence of TLR2. Surprisingly, in case of SA 113Δlgt a significant attenuated release of only IL-1ß protein and mRNA in human whole blood was observed. Highly purified peptidoglycan from all strains in contrast to LTA had a very low activity in stimulating cytokine release. Taken together these results demonstrate that major cell wall alterations like lack of WTA, lipoproteins or alterations in d-alanine content do not affect the overall cytokine inducing potential of whole bacteria and thus cytokine induction is initiated by redundant mechanisms.

Chlamydia pneumoniae-induced memory CD4+ T-cell activation in human peripheral blood correlates with distinct antibody response patterns.

Chlamydia pneumoniae is a frequent pathogen of the respiratory tract, and persistent infections with this obligate intracellular bacterium have been associated with different severe sequelae. Although T-cell activation during acute C. pneumoniae infections has been described, little is known about the frequency or the role of the C. pneumoniae-specific memory T cells that reside in the human body after the resolution of the infection. In the present study, the C. pneumoniae-induced T-cell responses in peripheral blood mononuclear cells of 56 healthy volunteers were analyzed and compared to the donor's serum antibody reactivity toward whole C. pneumoniae as well as recombinant C. pneumoniae antigens. Following short-term stimulation with C. pneumoniae, both gamma interferon (IFN-gamma)- and interleukin-2 (IL-2)-producing CD4(+) T-cell responses could be detected in 16 of 56 healthy individuals. C. pneumoniae-activated CD4(+) T cells expressed CD154, a marker for T-cell receptor-dependent activation, and displayed a phenotype of central memory T cells showing dominant IL-2 production but also IFN-gamma production. Interestingly, individuals with both IFN-gamma- and IL-2-producing responses showed significantly decreased immunoglobulin G reactivity toward C. pneumoniae RpoA and DnaK, antigens known to be strongly upregulated during chlamydial persistence, compared to IgG reactivity of seropositive individuals with no T-cell response or CD4(+) T-cell responses involving the production of a single cytokine (IFN-gamma or IL-2). Our results demonstrate that memory CD4(+) T cells responding to C. pneumoniae stimulation can be detected in the circulation of healthy donors. Furthermore, among seropositive individuals, the presence or the absence of dual IFN-gamma- and IL-2-producing T-cell responses was associated with distinct patterns of antibody responses toward persistence-associated C. pneumoniae antigens.

IL-10 release requires stronger toll-like receptor 4-triggering than TNF: a possible explanation for the selective effects of heterozygous TLR4 polymorphism Asp(299)Gly on IL-10 release.

The toll-like receptor 4 Asp(299)Gly polymorphism results in an inactive receptor. Heterozygosis is associated with reduced LPS-inducible IL-10 protein and IL-10 mRNA from blood leukocytes and isolated monocytes, while numerous other mediators are not affected. We could exclude that this effect is due to the differences in the kinetics of IL-10 release, in the expression of total surface TLR4 or in LPS-binding to monocytes between subjects heterozygous for the Asp(299)Gly polymorphism or homozygous carriers of the wild-type allele. Furthermore, we could show that IL-10 induction in general requires stronger LPS-triggering than TNF and is more sensitive to LPS inhibitors. The lower number of responsive wild-type TLR4 receptors on monocytes of heterozygotes may explain why only IL-10 release is affected.

Immunoproteomic identification and serological responses to novel Chlamydia pneumoniae antigens that are associated with persistent C. pneumoniae infections.

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.

Cytokine induction by Gram-positive bacteria.

Despite similar clinical relevance of Gram-positive and Gram-negative infections, immune activation by Gram-positive bacteria is by far less well understood than immune activation by Gram-negative bacteria. Our group has made available highly purified lipoteichoic acids (LTA) as a key Gram-positive immunostimulatory component. We have characterized the reasons for lower potency of LTA compared to Gram-negative lipopolysaccharide (LPS), identifying lack of IL-12/IFNgamma induction as a general characteristic of TLR2 agonists, and need for presentation of LTA on surfaces for enhanced immunostimulatory potency, as major aspects. Aspects of chemokine induction, where LTA is more potent than LPS, have been addressed. Furthermore, novel complement and plant defence activation, as well as CD36 as a new LTA receptor, were identified. The bacterial costimuli and modulators of LTA inducible responses are being investigated: LTA isolated from so far 16 bacterial species, although different in structure, behave remarkably similar while whole live and killed bacteria differ with regard to the pattern of induced responses. The purification and characterization of the respective components of the bacterial cell wall has begun.

Endothelial cells are protected against phagocyte-transmitted Chlamydophila pneumoniae infections by laminar shear stress Gueinzius: Shear stress protects from C. pneumoniae infection.

The respiratory pathogen Chlamydophila pneumoniae can be detected in atherosclerotic vessels, but the mechanism of dissemination from lung to vasculature remains unknown. Disturbance of vascular shear stress is a risk factor for atherosclerosis. We investigated whether polymorphonuclear neutrophils (PMN) might serve as carriers, transmitting C. pneumoniae to endothelial cells and how this is affected by shear stress. PMN were prepared from blood and incubated with C. pneumoniae. Real-time PCR and Pathfinder staining showed that after 1h, 20% of C. pneumoniae were ingested and started to form inclusions. When infected PMN were co-incubated with HUVEC for 96h, 10% of PMN-ingested C. pneumoniae were transmitted to HUVEC as shown by PCR and confocal microscopy. Infection of HEp-2 cells with C. pneumoniae harvested from HUVEC resulted in C. pneumoniae replication and confirmed that the bacteria remained infective. Exposure to laminar shear stress in a rotating cone-and-plate apparatus did not affect the transmission of C. pneumoniae from PMN to HUVEC, but led to a 75% reduction of inclusion formation. This can explain the focal distribution of C. pneumoniae in the vasculature and links two risk factors of atherosclerosis, i.e. the lack of laminar flow and infection.

Molecular basis for detection of invading pathogens in the brain.

Classical immunology textbooks have described the central nervous system as an immune-privileged site, i.e., as devoid of inflammatory and host-vs.-graft immunoreactions. This view has been refined, since we now know that hematopoietic cells infiltrate the CNS under certain circumstances and that CNS-resident cells are capable of launching an innate immune response. Microglia cells express an extensive repertoire of pattern-recognition receptors and act as sentinels surveilling the CNS for possible damage or infection. Astrocytes are the most abundant cell type in the brain, and they are capable of launching a strong supportive innate immune response. Novel findings show that both astrocytes and, surprisingly, even neurons express pattern-recognition receptors. Activation of these receptors leads to a functional response, indicating that cells other than microglia are capable of initiating a primary innate immune response against CNS-invading pathogens. Here, we put these findings into context with what has been learned from recent in vitro and in vivo experiments about the initiation of an innate immune response in the brain.