Jan R. Thiele

C-reactive protein: How conformational changes influence inflammatory properties

Recent evidence suggests that the prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP belongs to the family of pentraxins and as such consists of five identical non-covalently linked subunits. Contradictory data on the characteristics of CRP as either being pro- or anti-inflammatory may be explained by the existence of two conformations of the protein: the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. In vitro both isoforms exhibit a very distinct inflammatory profile. We recently identified a localized, physiologically relevant pCRP dissociation mechanism by activated platelets and apoptotic cells and showed the deposition of mCRP in inflamed tissue. Here we review the literature on the causal role of p- and mCRP in the light of our findings and critically analyze the current controversies around CRP. The novel understanding of the localized dissociation of circulating pentameric CRP to the distinctively pro-inflammatory monomeric CRP allows for a new view on CRP in inflammatory reactions and further highlights mCRP and the pCRP dissociation process as a potential therapeutic target.

Negative pressure wound therapy reduces the ischaemia/reperfusion-associated inflammatory response in free muscle flaps☆

BACKGROUND We recently established negative pressure wound therapy (NPWT) as a safe postoperative care concept for free muscle flaps; however, the molecular effects of NPWT on free muscle flaps remain elusive. Here we investigated the effects of NPWT on pathological changes associated with ischaemia/reperfusion injury in free flap tissue. METHODS From July 2008 to September 2010, 30 patients receiving skin-grafted free muscle transfer for defect coverage were randomly assigned to two treatment groups: In one group the skin-grafted free flap was covered by a vacuum dressing (NPWT); in the second group, flaps were covered by conventional petroleum gauze dressings (conv). Biopsies were taken intra-operatively prior to clipping of the pedicle and on postoperative day 5. Samples were analysed by immunohistochemistry for infiltration of inflammatory cells, real-time polymerase chain reaction (RT-PCR) for the analysis of expression levels of interleukin-1β (IL-1β) and tumour necrosis factor (TNF)-alpha as markers of inflammation. Histological samples were also examined for interstitial oedema formation, and apoptosis was detected by a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. RESULTS NPWT leads to a significantly reduced tissue infiltration of CD68 + macrophages and reduced expression of the inflammatory cytokines IL-1β and TNFα. None of these parameters was significantly elevated in the pre-ischaemic biopsies. Furthermore, NPWT reduced the interstitial oedema formation and the number of apoptotic cells in free flap tissue. CONCLUSION NPWT of skin-grafted free muscle flaps leads to a reduced inflammatory response following ischaemia/reperfusion, resulting in reduced oedema formation improving the microcirculation and ultimately reduced tissue damage. We thereby deliver new insight into the effects of NPWT.

MicroRNA-155 aggravates ischemia–reperfusion injury by modulation of inflammatory cell recruitment and the respiratory oxidative burst

The inflammatory sequelae of ischemia–reperfusion injury (IRI) are a major causal factor of tissue injury in various clinical settings. MicroRNAs (miRs) are short, non-coding RNAs, which regulate protein expression. Here, we investigated the role of miR-155 in IR-related tissue injury. Quantifying microRNA-expression levels in a human muscle tissue after IRI, we found miR-155 expression to be significantly increased and to correlate with the increased expression of TNF-α, IL-1β, CD105, and Caspase3 as well as with leukocyte infiltration. The direct miR-155 target gene SOCS-1 was downregulated. In a mouse model of myocardial infarction, temporary LAD ligation and reperfusion injury resulted in a smaller area of necrosis in miR-155−/− animals compared to wildtype animals. To investigate the underlying mechanisms, we evaluated the effect of miR-155 on inflammatory cell recruitment by intravital microscopy and on the generation of reactive oxygen species (ROS) of macrophages. Our intravital imaging results demonstrated a decreased recruitment of inflammatory cells in miR-155−/− animals during IRI. The generation of ROS in leukocytic cells of miR-155−/− animals was also reduced. RNA silencing of the direct miR-155 target gene SOCS-1 abrogated this effect. In conclusion, miR-155 aggravates the inflammatory response, leukocyte infiltration and tissue damage in IRI via modulation of SOCS-1-dependent generation of ROS. MiR-155 is thus a potential target for the treatment or prevention of IRI.

Targeting C-Reactive Protein in Inflammatory Disease by Preventing Conformational Changes

C-reactive protein (CRP) is a pentraxin that has long been employed as a marker of inflammation in clinical practice. Recent findings brought up the idea of CRP to be not only a systemic marker but also a mediator of inflammation. New studies focused on structural changes of the plasma protein, revealing the existence of two distinct protein conformations associated with opposed inflammatory properties. Native, pentameric CRP (pCRP) is considered to be the circulating precursor form of monomeric CRP (mCRP) that has been identified to be strongly proinflammatory. Recently, a dissociation mechanism of pCRP has been identified on activated platelets and activated/apoptotic cells associated with the amplification of the proinflammatory potential. Correspondingly, CRP deposits found in inflamed tissues have been identified to exhibit the monomeric conformation by using conformation-specific antibodies. Here we review the current literature on the causal role of the dissociation mechanism of pCRP and the genesis of mCRP for the amplification of the proinflammatory potential in inflammatory reactions such as atherosclerosis and ischemia/reperfusion injury. The chance to prevent the formation of proinflammatory mediators in ubiquitous inflammatory cascades has pushed therapeutic strategies by targeting pCRP dissociation in inflammation. In this respect, the development of clinically applicable derivatives of the palindromic compound 1,6-bis(phosphocholine)-hexane (1,6-bis PC) should be a major focus of future CRP research.

Dissociation of Pentameric to Monomeric C-Reactive Protein Localizes and Aggravates Inflammation

Background— The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo. Methods and Results— We identified mCRP in inflamed human striated muscle, human atherosclerotic plaque, and infarcted myocardium (rat and human) and its colocalization with inflammatory cells, which suggests a general causal role of mCRP in inflammation. This was confirmed in rat intravital microscopy of lipopolysaccharide-induced cremasteric muscle inflammation. Intravenous pCRP administration significantly enhanced leukocyte rolling, adhesion, and transmigration via localized dissociation to mCRP in inflamed but not noninflamed cremaster muscle. This was confirmed in a rat model of myocardial infarction. Mechanistically, this process was dependent on exposure of lysophosphatidylcholine on activated cell membranes, which is generated after phospholipase A2 activation. These membrane changes could be visualized intravitally on endothelial cells, as could the colocalized mCRP generation. Blocking of phospholipase A2 abrogated C-reactive protein dissociation and thereby blunted the proinflammatory effects of C-reactive protein. Identifying the dissociation process as a therapeutic target, we stabilized pCRP using 1,6-bis(phosphocholine)-hexane, which prevented dissociation in vitro and in vivo and consequently inhibited the generation and proinflammatory activity of mCRP; notably, it also inhibited mCRP deposition and inflammation in rat myocardial infarction. Conclusions— These results provide in vivo evidence for a novel mechanism that localizes and aggravates inflammation via phospholipase A2–dependent dissociation of circulating pCRP to mCRP. mCRP is proposed as a pathogenic factor in atherosclerosis and myocardial infarction. Most importantly, the inhibition of pCRP dissociation represents a promising, novel anti-inflammatory therapeutic strategy.Background— The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo. Methods and Results— We identified mCRP in inflamed human striated muscle, human atherosclerotic plaque, and infarcted myocardium (rat and human) and its colocalization with inflammatory cells, which suggests a general causal role of mCRP in inflammation. This was confirmed in rat intravital microscopy of lipopolysaccharide-induced cremasteric muscle inflammation. Intravenous pCRP administration significantly enhanced leukocyte rolling, adhesion, and transmigration via localized dissociation to mCRP in inflamed but not noninflamed cremaster muscle. This was confirmed in a rat model of myocardial infarction. Mechanistically, this process was dependent on exposure of lysophosphatidylcholine on activated cell membranes, which is generated after phospholipase A2 activation. These membrane changes could be visualized intravitally on endothelial cells, as could the colocalized mCRP generation. Blocking of phospholipase A2 abrogated C-reactive protein dissociation and thereby blunted the proinflammatory effects of C-reactive protein. Identifying the dissociation process as a therapeutic target, we stabilized pCRP using 1,6-bis(phosphocholine)-hexane, which prevented dissociation in vitro and in vivo and consequently inhibited the generation and proinflammatory activity of mCRP; notably, it also inhibited mCRP deposition and inflammation in rat myocardial infarction. Conclusions— These results provide in vivo evidence for a novel mechanism that localizes and aggravates inflammation via phospholipase A2–dependent dissociation of circulating pCRP to mCRP. mCRP is proposed as a pathogenic factor in atherosclerosis and myocardial infarction. Most importantly, the inhibition of pCRP dissociation represents a promising, novel anti-inflammatory therapeutic strategy. # CLINICAL PERSPECTIVE {#article-title-40}

Salvage Procedures After Failed Facial Reanimation Surgery Using the Masseteric Nerve as the Motor Nerve for Free Functional Gracilis Muscle Transfer

IMPORTANCE Free muscle transfer innervated by a cross-facial nerve graft represents the criterion standard for smile reconstruction in facial paralysis. If primary reconstruction fails, a second muscle transfer is usually needed. Herein, we investigated the possibility of avoiding a second free muscle transfer by in situ coaptation of the gracilis muscle to the masseteric nerve. OBSERVATIONS We report a series of 3 failed free muscle transfers for facial reanimation among 21 free flap transfers performed for facial reanimation between March 2008 and August 2013. To salvage the muscle, we performed coaptation of the neural pedicle from the cross-facial nerve graft to the masseteric nerve. This method allows for leaving the fixation sutures of the muscle at the oral commissure in place. All patients showed muscle contraction after 3 months and a smile with open mouth after 6 months. No significant difference in the range of commissure excursion was observed between the healthy and operated sides. CONCLUSIONS AND RELEVANCE Recoaptation of the neural pedicle from the cross-facial nerve graft to the masseteric nerve, leaving the muscle transplant in place, is a suitable salvage procedure after unsuccessful reconstruction with a cross-facial nerve graft, avoiding a second free muscle transfer. LEVEL OF EVIDENCE 4.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery. Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion. Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor. While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM). Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968. Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality. We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method. In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Single-stage Dynamic Reanimation of the Smile in Irreversible Facial Paralysis by Free Functional Muscle Transfer

Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic facial paralysis (Bells palsy) is the most common diagnosis, patients can also present with a history of physical trauma, infectious disease, tumor, or iatrogenic facial paralysis. Early repair within one year of injury can be achieved by direct nerve repair, cross-face nerve grafting or regional nerve transfer. It is due to muscle atrophy that in long lasting facial paralysis complex reconstructive methods have to be applied. Instead of one single procedure, different surgical approaches have to be considered to alleviate the various components of the paralysis. The reconstruction of a spontaneous dynamic smile with a symmetric resting tone is a crucial factor to overcome the functional deficits and the social handicap that are associated with facial paralysis. Although numerous surgical techniques have been described, a two-stage approach with an initial cross-facial nerve grafting followed by a free functional muscle transfer is most frequently applied. In selected patients however, a single-stage reconstruction using the motor nerve to the masseter as donor nerve is superior to a two-stage repair. The gracilis muscle is most commonly used for reconstruction, as it presents with a constant anatomy, a simple dissection and minimal donor site morbidity. Here we demonstrate the pre-operative work-up, the post-operative management, and precisely describe the surgical procedure of single-stage microsurgical reconstruction of the smile by free functional gracilis muscle transfer in a step by step protocol. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly comprehend the procedure. We further discuss indications and limitations of the technique and demonstrate representative results.

A Conformational Change in C-Reactive Protein Enhances Leukocyte Recruitment and Reactive Oxygen Species Generation in Ischemia/Reperfusion Injury

Introduction C-reactive protein circulates as a pentameric protein (pCRP). pCRP is a well-established diagnostic marker as plasma levels rise in response to tissue injury and inflammation. We recently described pro-inflammatory properties of CRP, which are mediated by conformational changes from pCRP to bioactive isoforms expressing pro-inflammatory neo-epitopes [pCRP* and monomeric C-reactive protein (mCRP)]. Here, we investigate the role of CRP isoforms in renal ischemia/reperfusion injury (IRI). Methods Rat kidneys in animals with and without intraperitoneally injected pCRP were subjected to IRI by the time of pCRP exposure and were subsequently analyzed for monocyte infiltration, caspase-3 expression, and tubular damage. Blood urea nitrogen (BUN) was analyzed pre-ischemia and post-reperfusion. CRP effects on leukocyte recruitment were investigated via intravital imaging of rat-striated muscle IRI. Localized conformational CRP changes were analyzed by immunohistochemistry using conformation specific antibodies. 1,6-bis(phosphocholine)-hexane (1,6-bisPC), which stabilizes CRP in its native pentameric form was used to validate CRP effects. Leukocyte activation was assessed by quantification of reactive oxygen species (ROS) induction by CRP isoforms ex vivo and in vitro through electron spin resonance spectroscopy. Signaling pathways were analyzed by disrupting lipid rafts with nystatin and subsequent ROS detection. In order to confirm the translational relevance of our findings, biopsies of microsurgical human free tissue transfers before and after IRI were examined by immunofluorescence for CRP deposition and co-localization of CD68+ leukocytes. Results The application of pCRP aggravates tissue damage in renal IRI. 1,6-bisPC reverses these effects via inhibition of the conformational change that leads to exposure of pro-inflammatory epitopes in CRP (pCRP* and mCRP). Structurally altered CRP induces leukocyte–endothelial interaction and induces ROS formation in leukocytes, the latter can be abrogated by blocking lipid raft-dependent signaling pathways with Nystatin. Stabilizing pCRP in its native pentameric state abrogates these pro-inflammatory effects. Importantly, these findings are confirmed in human IRI challenged muscle tissue. Conclusion These results suggest that CRP is a potent modulator of IRI. Stabilizing the native pCRP conformation represents a promising anti-inflammatory therapeutic strategy by attenuation of leukocyte recruitment and ROS formation, the primary pathomechanisms of IRI.

Comparison of symmetry after smile reconstruction for flaccid facial paralysis with combined fascia lata grafts and functional gracilis transfer for static suspension or gracilis transfer alone

Facial paralysis has a profound impact on functionality and esthetics of the oral region. In patients with strong skin laxity and soft tissue ptosis, functional smile reconstruction is challenging due to the accentuated asymmetry at rest. Thus, the purpose of the study was to analyze facial symmetry in this patient clientele following a combination of dynamic reanimation with fascial strips for static suspension compared to functional gracilis transfer alone.