Complement-Coagulation Cross-talk: Factor H-mediated regulation of the Complement Classical Pathway activation by fibrin clots.

The classical pathway of the complement system is activated by the binding of C1q in the C1 complex to the target activator, including immune complexes. Factor H is regarded as the key downregulatory protein of the complement alternative pathway. However, both C1q and factor H bind to target surfaces via charge distribution patterns. For a few targets, C1q and factor H compete for binding to common or overlapping sites. Factor H, therefore, can effectively regulate the classical pathway activation through such targets, in addition to its previously characterized role in the alternative pathway. Both C1q and factor H are known to recognize foreign or altered-self materials, e.g., bacteria, viruses, and apoptotic/necrotic cells. Clots, formed by the coagulation system, are an example of altered self. Factor H is present abundantly in platelets and is a well-known substrate for FXIIIa. Here, we investigated whether clots activate the complement classical pathway and whether this is regulated by factor H. We show here that both C1q and factor H bind to the fibrin formed in microtiter plates and the fibrin clots formed under in vitro physiological conditions. Both C1q and factor H become covalently bound to fibrin clots, and this is mediated via FXIIIa. We also show that fibrin clots activate the classical pathway of complement, as demonstrated by C4 consumption and membrane attack complex detection assays. Thus, factor H downregulates the activation of the classical pathway induced by fibrin clots. These results elucidate the intricate molecular mechanisms through which the complement and coagulation pathways intersect and have regulatory consequences.

Nanoparticle-induced immune response: Health risk versus treatment opportunity?

Nanoparticles (NPs) are not only employed in many biomedical applications in an engineered form, but also occur in our environment, in a more hazardous form. NPs interact with the immune system through various pathways and can lead to a myriad of different scenarios, ranging from their quiet removal from circulation by macrophages without any impact for the body, to systemic inflammatory effects and immuno-toxicity. In the latter case, the function of the immune system is affected by the presence of NPs. This review describes, how both the innate and adaptive immune system are involved in interactions with NPs, together with the models used to analyse these interactions. These models vary between simple 2D in vitro models, to in vivo animal models, and also include complex all human organ on chip models which are able to recapitulate more accurately the interaction in the in vivo situation. Thereafter, commonly encountered NPs in both the environment and in biomedical applications and their possible effects on the immune system are discussed in more detail. Not all effects of NPs on the immune system are detrimental; in the final section, we review several promising strategies in which the immune response towards NPs can be exploited to suit specific applications such as vaccination and cancer immunotherapy.

Hb Nouakchott [α114(GH2)Pro→Leu; HBA1: c.344C>T], A Second and Third Case Described in Two Unrelated Dutch Families.

We report two families, members of which are carriers of a hemoglobin (Hb) variant previously described as Hb Nouakchott [α114(GH2)Pro→Leu; HBA1: c.344C>T; p.Pro115Leu]. In the first family of Dutch origin, the proband, a 32-year-old male and his 65-year-old father, were both carriers of Hb Nouakchott. Of the second family we tested, only the proband, a 56-year-old Dutch female was a Hb Nouakchott carrier. Hematological analyses of these cases showed the anomaly behaves as a silent Hb variant without clinical consequences. The Hb variant remained unnoticed using high performance liquid chromatography (HPLC), while an additional peak was detected by capillary electrophoresis (CE). These independent findings of Hb Nouakchott indicate that this Hb variant might not be very rare, but simply remains under diagnosed depending on the Hb separation technique used.

Accurate determination of the CYP2D6 (*1/*4)xN genotype by quantitative PCR.

BACKGROUND: CYP2D6 is responsible for the metabolism of approximately 25% of all drugs. The expression of cytochrome P450 2D6 (CYP2D6) is influenced by a combination of factors including polymorphisms in the CYP2D6 gene. Analysis of the CYP2D6 genotype is used to personalize the medication to a patient's metabolism. Although many genotypes can be determined using standard genotype analysis, in some cases, an incomplete analysis is performed. The CYP2D6 genotype *1/*4 often occurs in combination with a multiplication of the CYP2D6 gene, and is reported as (*1/*4)xN. Accurate determination of the multiplied gene is essential to provide a phenotype prediction for these patients. Duplication of the *1 gene leads to an extensive metabolizer genotype whereas multiplication of the *4 gene would not lead to extra functional enzyme and therefore provides an intermediate metabolizer phenotype. METHODS: Here, a technique is described in which the copy numbers of both the *4 and *1 genes are determined using quantitative PCR techniques. RESULTS AND CONCLUSIONS: This technique provides a method to predict the patient's CYP2D6 phenotype, and is therefore an important step toward personalized medicine.

Interactions of the innate immune system with carbon nanotubes.

The therapeutic application of nanomaterials requires that they are biocompatible and can reach the desired target. The innate immune system is likely to be the first defence machinery that would recognise the nanomaterials as 'non-self'. A number of studies have addressed the issue of how carbon nanotubes (CNTs) interact with phagocytic cells and their surface receptors that can impact on their intracellular processing and subsequent immune response. In addition, soluble innate immune factors also get involved in the recognition and clearance of CNTs. The interaction of CNTs with the complement system, the most potent and versatile innate immune mechanism, has shed interesting light on how complement activation on the surface of CNTs can modulate their phagocytosis and effector cytokine response. The charge or altered molecular pattern on the surface of CNTs due to functionalization and derivatization can also dictate the level of complement activation and subsequent inflammatory response. It is becoming evident that complement deposition may facilitate phagocytic uptake of CNTs through receptor routes that leads to dampening of pro-inflammatory response by complement-receptor bearing macrophages and B cells. Thus, recombinant complement regulators decorated on the CNT surface can constructively influence the therapeutic strategies involving CNTs and other nanoparticles.

Pulmonary surfactant protein SP-D opsonises carbon nanotubes and augments their phagocytosis and subsequent pro-inflammatory immune response.

Carbon nanotubes (CNTs) are increasingly being developed for use in biomedical applications, including drug delivery. One of the most promising applications under evaluation is in treating pulmonary diseases such as tuberculosis. Once inhaled or administered, the nanoparticles are likely to be recognised by innate immune molecules in the lungs such as hydrophilic pulmonary surfactant proteins. Here, we set out to examine the interaction between surfactant protein D (SP-D), a key lung pattern recognition molecule and CNTs, and possible downstream effects on the immune response via macrophages. We show here that a recombinant form of human SP-D (rhSP-D) bound to oxidised and carboxymethyl cellulose (CMC) coated CNTs via its C-type lectin domain and enhanced phagocytosis by U937 and THP-1 macrophages/monocytic cell lines, together with an increased pro-inflammatory response, suggesting that sequestration of SP-D by CNTs in the lungs can trigger an unwanted and damaging immune response. We also observed that functionalised CNTs, opsonised with rhSP-D, continued to activate complement via the classical pathway, suggesting that C1q, which is the recognition sub-component of the classical pathway, and SP-D have distinct pattern recognition sites on the CNTs. Consistent with our earlier reports, complement deposition on the rhSP-D opsonised CNTs led to dampening of the pro-inflammatory immune response by THP-1 macrophages, as evident from qPCR, cytokine array and NF-κB nuclear translocation analyses. This study highlights the importance of understanding the interplay between innate immune humoral factors including complement in devising nanoparticle based drug delivery strategies.

Complement Deposition on Nanoparticles Can Modulate Immune Responses by Macrophage, B and T Cells.

Nanoparticles are attractive drug delivery vehicles for targeted organ-specific as well as systemic therapy. However, their interaction with the immune system offers an intriguing challenge to the success of nanotherapeutics in vivo. Recently, we showed that pristine and derivatised carbon nanotubes (CNT) can activate complement mainly via the classical pathway leading to enhanced uptake by phagocytic cells, and transcriptional down-regulation of pro-inflammatory cytokines. Here, we report the interaction of complement-activating CC-CNT and RNA-CNT, and non-complement-activating gold-nickel (Au-Ni) nanowires with cell lines representing macrophage, B and T cells. Complement deposition considerably enhanced uptake of CNTs by immune cells known to overexpress complement receptors. Real-Time qPCR and multiplex array analyses showed complement-dependent down-regulation of TNF-α and IL-1ß and up-regulation of IL-12 by CMC- and RNA-CNTs, in addition to revealing IL-10 as a crucial regulator during nanoparticle-immune cell interaction. It appears that complement system can recognize molecular patterns differentially displayed by nanoparticles and thus, modulate subsequent processing of nanoparticles by antigen capturing and antigen presenting cells, which can shape innate and adaptive immune axes.

Innate immune humoral factors, C1q and factor H, with differential pattern recognition properties, alter macrophage response to carbon nanotubes.

Interaction between the complement system and carbon nanotubes (CNTs) can modify their intended biomedical applications. Pristine and derivatised CNTs can activate complement primarily via the classical pathway which enhances uptake of CNTs and suppresses pro-inflammatory response by immune cells. Here, we report that the interaction of C1q, the classical pathway recognition molecule, with CNTs involves charge pattern and classical pathway activation that is partly inhibited by factor H, a complement regulator. C1q and its globular modules, but not factor H, enhanced uptake of CNTs by macrophages and modulated the pro-inflammatory immune response. Thus, soluble complement factors can interact differentially with CNTs and alter the immune response even without complement activation. Coating CNTs with recombinant C1q globular heads offers a novel way of controlling classical pathway activation in nanotherapeutics. Surprisingly, the globular heads also enhance clearance by phagocytes and down-regulate inflammation, suggesting unexpected complexity in receptor interaction. FROM THE CLINICAL EDITOR: Carbon nanotubes (CNTs) maybe useful in the clinical setting as targeting drug carriers. However, it is also well known that they can interact and activate the complement system, which may have a negative impact on the applicability of CNTs. In this study, the authors functionalized multi-walled CNT (MWNT), and investigated the interaction with the complement pathway. These studies are important so as to gain further understanding of the underlying mechanism in preparation for future use of CNTs in the clinical setting.

Soft magnets from the self-organization of magnetic nanoparticles in twisted liquid crystals.

Organizing magnetic nanoparticles into long-range and dynamic assemblies would not only provide new insights into physical phenomena but also open opportunities for a wide spectrum of applications. In particular, a major challenge consists of the development of nanoparticle-based materials for which the remnant magnetization and coercive field can be controlled at room temperature. Our approach consists of promoting the self-organization of magnetic nanoparticles in liquid crystals (LCs). Using liquid crystals as organizing templates allows us to envision the design of tunable self-assemblies of magnetic nanoparticles, because liquid crystals are known to reorganize under a variety of external stimuli. Herein, we show that twisted liquid crystals can be used as efficient anisotropic templates for superparamagnetic nanoparticles and demonstrate the formation of hybrid soft magnets at room temperature.

Complement activation by carbon nanotubes and its influence on the phagocytosis and cytokine response by macrophages.

Carbon nanotubes (CNTs) have promised a range of applications in biomedicine. Although influenced by the dispersants used, CNTs are recognized by the innate immune system, predominantly by the classical pathway of the complement system. Here, we confirm that complement activation by the CNT used continues up to C3 and C5, indicating that the entire complement system is activated including the formation of membrane-attack complexes. Using recombinant forms of the globular regions of human C1q (gC1q) as inhibitors of CNT-mediated classical pathway activation, we show that C1q, the first recognition subcomponent of the classical pathway, binds CNTs via the gC1q domain. Complement opsonisation of CNTs significantly enhances their uptake by U937 cells, with concomitant downregulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines in both U937 cells and human monocytes. We propose that CNT-mediated complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response. FROM THE CLINICAL EDITOR: This study highlights the importance of the complement system in response to carbon nanontube administration, suggesting that the ensuing complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response.

Au coated Ni nanowires with tuneable dimensions for biomedical applications.

Due to their shape anisotropy, high aspect ratio magnetic nanoparticles offer many advantages in biomedical applications. For biocompatibility, it is essential to have full control over the dimensions and surface chemistry of the particles. The aim of this study was to synthesize biocompatible nanowires with tuneable dimensions. This was achieved by electrodeposition of Ni in polycarbonate membranes. To ensure biocompatibility, a continuous gold coating was deposited onto the Ni wires by a newly developed electroless deposition method. The coating was analysed using electron microscopy and X-ray diffraction. Magnetic properties, anisotropy and Au film thickness were studied using vibrating sample magnetometry. After biofunctionalization, no significant cytotoxic effects were found in studies involving a diverse range of primary and tumour cells exposed to increasing concentrations of nanowires for up to 7 days. These nanowires may thus be used for in vivo applications such as magnetic drug delivery.

MR-guided biopsy of the prostate: an overview of techniques and a systematic review.

CONTEXT: Systematic transrectal ultrasound-guided biopsy (TRUSBx) is the gold standard for detecting prostate cancer. This systematic approach is characterized by low sensitivity (39-52%) and high specificity (81-82%). Magnetic resonance (MR)-guided biopsy techniques are becoming more and more available, but there is no current consensus on the optimal technique. OBJECTIVE: This review presents an overview of MR-guided biopsy techniques for prostate cancer detection. EVIDENCE ACQUISITION: Current literature was reviewed regarding MR-guided biopsy for prostate cancer detection. A literature search was performed using the commercially available MedLine online search engine. Combinations of the following search and Medical Subject Headings terms were applied to retrieve relevant articles: "magnetic resonance," "prostatic neoplasms," and "biopsy." Review articles and studies describing techniques other than MR-guided biopsy were excluded. EVIDENCE SYNTHESIS: Biopsy of the prostate is an essential procedure for determining optimal treatment. Systematic TRUSBx is the gold standard, but it fails to detect numerous tumors. Diagnostic MR imaging provides more accurate selection of regions in which tumors are suspected. Using these diagnostic images during an MR-directed biopsy procedure improves quality of the biopsy. In open MR scanners, the prebiopsy images often must be registered to the real-time biopsy images because open MR scanners do not provide optimal tissue contrast; thus, the patient must first be examined in a closed MR scanner and then biopsied in an open scanner. The advantage of open MR over closed MR is that the physician has easy patient access. With special equipment, prostate MR-guided biopsy is also possible in a closed system. Closed MR scanners can be used for the prebiopsy scan as well as for the biopsy procedure. CONCLUSIONS: The combination of a diagnostic MR examination and MR-guided biopsy is a promising tool and may be used in patients with previous negative TRUSBx.