Suicide by the intraoral blast of firecrackers - experimental simulation using a skull simulant model.

Suicides committed by intraorally placed firecrackers are rare events. Given to the use of more powerful components such as flash powder recently, some firecrackers may cause massive life-threatening injuries in case of such misuse. Innocuous black powder firecrackers are subject to national explosives legislation and only have the potential to cause harmless injuries restricted to the soft tissue. We here report two cases of suicide committed by an intraoral placement of firecrackers, resulting in similar patterns of skull injury. As it was first unknown whether black powder firecrackers can potentially cause serious skull injury, we compared the potential of destruction using black powder and flash powder firecrackers in a standardized skull simulant model (Synbone, Malans, Switzerland). This was the first experiment to date simulating the impacts resulting from an intraoral burst in a skull simulant model. The intraoral burst of a "D-Böller" (an example of one of the most powerful black powder firecrackers in Germany) did not lead to any injuries of the osseous skull. In contrast, the "La Bomba" (an example of the weakest known flash powder firecrackers) caused complex fractures of both the viscero- and neurocranium. The results obtained from this experimental study indicate that black powder firecrackers are less likely to cause severe injuries as a consequence of intraoral explosions, whereas flash powder-based crackers may lead to massive life-threatening craniofacial destructions and potentially death.

Pulmonary artery perforation and coronary air embolism-two fatal outcomes in percutaneous left atrial appendage occlusion.

Percutaneous left atrial appendage (LAA) closure is a routinely performed method to reduce the risk of stroke in patients suffering from atrial fibrillation, when an oral anticoagulation is no longer indicated due to relevant bleeding complications. Currently, the Amplatzer Amulet and the Watchman system are two equally used systems. While there is an acute success rate of more than 95 per cent for this intervention, several minor and major complications such as pericardial effusions, air embolism, vascular lesions in proximity to the heart or even death can occur. Here, we report two cases of very rare fatal outcomes in percutaneous LAA occlusion. Eight hours after deployment of an Amplatzer Amulet a patient died, after the pulmonary trunk was perforated by a hook of the occluder device causing pericardial tamponade. In the second case during final radiological position control of the deployed Watchman occluder air was injected accidentally. The patient immediately died due to coronary air embolism. Forensic autopsies are necessary to solve the cause and manner of death, to evaluate and develop medical devices and to rule out medical malpractice. Thus, a close collaboration of legal medicine and the various cardiologic departments is proposed.

On the mechanical significance of vascular imprints of the human neurocranium when impacted at 11 m/s.

The course of the middle meningeal vessels can be traced through imprints on the inner table of the human neurocranium. It is as yet unexplored whether these notches lower the load-bearing capacity of the bone when compared to areas that are free of vascular imprints. Here, 310 temporo-parietal samples with and without vascular imprints, from 52 human Crosado-embalmed cadavers, were tested in a three-point bending setup with a half-cylindrical impactor (1 mm radius of curvature) contacting the sample at 11 m/s. The maximum forces before breaking, and the thicknesses of the samples, were statistically compared, including comparing the avascular group to several groups with vascular imprints of different orientations. Furthermore, the influence of sample length and impact location were investigated. To investigate structure and mechanical function of vascular imprints concomitantly, scanning electron microscopy was performed on selected samples in two different planes. The results showed that avascular samples were on average thicker (p < 0.001) and stronger (p ≤ 0.050) compared to samples with vascular imprints. When only thickness-matched samples were analysed, the observed maximum forces of vascular and avascular samples were statistically similar (p ≥ 0.531). Regarding the load-bearing capacity of samples with vascular imprints, it was irrelevant whether the imprint was placed parallel to and directly underneath the impactor, parallel to and offset from the impactor, or perpendicular to the impactor (p > 0.999). The overall results of this study were statistically unrelated to both sample length (p ≥ 0.720) and impact location (p > 0.999). Scanning electron microscopy revealed that vascular imprints are formed through a curve of the inner table. Perforating holes of the inner table are present in avascular areas, however, they are considerably larger in size and higher in number within vascular imprints. In conclusion, vascular imprints are formed through curving of the inner table. In numerical models of human head mechanics, vascular imprints can be accounted for through a simple thinning of the bone assuming the same load-bearing capacity as for the surrounding imprint-free areas.

Effect of Blood Decontamination Procedures on the Microshear Bond Strength of Resin-modified Glass Ionomer Cement to Resin Composite.

OBJECTIVE: To investigate the effect of decontamination procedures on the microshear bond strength (µSBS) of blood-contaminated resin-modified glass ionomer cement (RMGIC) bonded to resin composite (RC). METHODS: Eighty RMGIC disc specimens were allocated into 5 groups (n=16). All groups except Group 2 were contaminated with blood. Group 1 had no decontamination procedure, Group 3 was decontaminated by rinsing, Group 4 was decontaminated by 34% phosphoric acid etching, and Group 5 was decontaminated by 5% sodium hypochlorite application. RMGIC specimens were subsequently bonded with RC using a universal adhesive in self-etch mode. µSBS tests were conducted using a universal testing machine at a crosshead speed of 1 mm/min. Failure mode analysis was conducted on RMGIC fracture surfaces under a scanning electron microscope. RESULTS: µSBS results indicated that Group 4 had the highest mean µSBS value of 6.22 ± 2.14 MPa, while Group 1 had the lowest mean µSBS value of 3.53 ±1.67 MPa. Significant differences were observed in the µSBS of Group 2 with no contamination (p=0.023) and Group 4 with decontamination by phosphoric acid-etching (p=0.003) when compared to Group 1 with blood contamination. No statistically significant differences (p>0.05) were observed between all other groups' µSBS. For all groups, the predominant mode of failure was adhesive failure between the RMGIC-RC interface, with a few mixed failures in RMGIC for Groups 2-5. CONCLUSIONS: Blood contamination before adhesive application significantly reduced the µSBS between RMGIC and RC. Phosphoric acid etching was the most effective blood decontamination procedure to improve the µSBS.

The Influence of Saliva and Blood Contamination on Bonding Between Resin-modified Glass Ionomer Cements and Resin Composite.

OBJECTIVE: To investigate the influence of blood and saliva contamination on the microshear bond strength (µSBS) between resin-modified glass ionomer cement (RMGIC) and resin composite (RC). METHODS AND MATERIALS: Eighty RMGIC discs were allocated into four groups (n=20). Group 1 received universal dental adhesive application in a self-etch mode followed by a build-up with RC. Group 2 received saliva as a contaminant, Group 3 received blood as a contaminant, Group 4 received a 1:1 blood-saliva mixture as a contaminant. Specimens from Groups 2, 3, and 4 were submerged into their respective contaminants for 15 seconds and dried prior to the adhesive application, followed by the protocol for Group 1. All specimens were stored in distilled water for 24 hours. Subsequently, the bonded specimens were subjected to µSBS testing using a universal testing machine. Failure mode of the debonded RMGIC surfaces was examined using scanning electron microscopy. RESULTS: The µSBS from groups 1-4 were 10.76 ± 3.03 MPa, 9.36 ± 2.54 MPa, 6.55 ± 1.67 MPa and 8.42 ± 2.79 MPa, respectively. Contamination by blood and blood-saliva significantly decreased the µSBS (p<0.001, p=0.029). Saliva contamination alone had no statistically significant effect on the µSBS (p=0.524). A statistically significant difference in the mode of failure was detected between the experimental groups (p=0.012). CONCLUSION: Saliva contamination has no influence on µSBS between RMGIC and RC when it is dried thoroughly, while blood and blood-saliva contamination reduced µSBS between RMGIC and RC even when dried thoroughly.

Dynamic load response of human dura mater at different velocities.

Despite of its assumed role to mitigate brain tissue response under dynamic loading conditions, the human dura mater is frequently neglected in computational and physical human head models. A reason for this is the lack of load-deformation data when the dura mater is loaded dynamically. To date, the biomechanical characterization of the human dura mater predominantly involved quasi-static testing setups. This study aimed to investigate the strain rate-dependent mechanical properties of the human dura mater comparing three different velocities of 0.3, 0.5 and 0.7 m/s. Samples were chosen in a perpendicular orientation to the visible main fiber direction on the samples' surface, which was mostly neglected in previous studies. The elastic modulus of dura mater significantly increased at higher velocities (5.16 [3.38; 7.27] MPa at 0.3 m/s versus 44.38 [35.30; 74.94] MPa at 0.7 m/s). Both the stretch at yield point λf (1.148 [1.137; 1.188] for 0.3 m/s, 1.062 [1.054; 1.066] for 0.5 m/s and 1.015 [1.012; 1.021] for 0.7 m/s) and stress at yield point σf of dura mater (519.14 [366.74; 707.99] kPa for 0.3 m/s versus 300.52 [245.31; 354.89] kPa at 0.7 m/s) significantly decreased with increasing velocities. Conclusively, increasing the load application velocity increases stiffness and decreases tensile strength as well as straining potential of human dura mater between 0.3 and 0.7 m/s. The elastic modulus of human dura mater should be adapted to the respective velocities in computational head impact simulations.

The relationship between the pH value of a hydration solution and the biomechanical properties of Crosado-embalmed human iliotibial bands.

Determining the biomechanical properties of human tissues commonly involves the immersion or spraying of the tissues to maintain them in a hydrated state. However, the influence of the pH value of these solutions on the biomechanical properties of the tissues is not well understood. This study investigated the effects of the pH value on the biomechanical properties of the collagen-rich human iliotibial band (ITB). A total of 124 samples were allocated to polyethylene glycol (PEG) solutions of pH values between 3 and 13 for 24 h, which is a frequently used immersion time prior to biomechanical tests. After this, the samples were biomechanically tested in a uniaxial tensile testing setup using an established testing routine. Similarly, 69 samples were allocated to pH groups of 6, 7 and 8 and biomechanically tested after 1, 2 and 3 weeks. The cross-sectional area of all samples was determined after immersion into the PEG solutions for the specified time frames. In the 24-h experiment, the elastic modulus (pH 12: p ≤ 0.045; pH 13: p ≤ 0.020) and the ultimate tensile strength (pH 12: p ≤ 0.031; pH 13: p ≤ 0.026) of the pH groups 12 and 13 were significantly lower and their cross-sectional areas were higher (pH 12: p ≤ 0.005; pH 13: p ≤ 0.003) compared to several groups of acidic to alkaline pH values. There was no difference in the maximum forces between the different groups within a 24-h immersion time (p > 0.999). In the 3-week-test, a decrease of the ultimate tensile strength was noted between the 24-h and 3 week values for the pH groups 7 (p = 0.034) and 8 (p = 0.029). It is concluded that pH-dependent tissue swelling influences the cross-sectional area-dependent biomechanical properties of the human ITB. Therefore, the pH value of storage and hydration solutions for the preparation of biomechanical tests should be recorded. From a biomechanical perspective, the collagen stability of the human ITB is largely unaltered in PEG solutions with pH values between 3 and 13 over 24 h.

On the correlations of biomechanical properties of super-imposed temporal tissue layers and their age-, sex-, side- and post-mortem interval dependence.

Obtaining biomechanical properties of biological tissues for simulation purposes or graft developments is time and resource consuming. The number of samples required for biomechanical tests could be reduced if the load-deformation properties of a given tissue layer could be estimated from adjacent layers or if the biomechanical parameters were unaffected by age, bodyside, sex or post-mortem interval. This study investigates for the first time potential correlations of multiple super-imposed tissue layers using the temporal region of the human head as an area of broad interest in biomechanical modelling. Spearman correlations between biomechanical properties of the scalp, muscle fascia, muscle, bone and dura mater from up to 83 chemically unfixed cadavers were investigated. The association with age, sex and post-mortem interval was assessed. The results revealed sporadic correlations between the corresponding layers, such as the maximum force (r = 0.43) and ultimate tensile strength (r = 0.33) between scalp and muscle. Side- and age-dependence of the biomechanical properties were different between the tissue types. Strain at maximum force of fascia (r = -0.37) and elastic modulus of temporal muscle (r = 0.26) weakly correlated with post-mortem interval. Only strain at maximum force of scalp differed significantly between sexes. Uniaxial biomechanical properties of individual head tissue layers can thus not be estimated solely based on adjacent layers. Therefore, correlations between the tissues' biomechanical properties, anthropometric data and post-mortem interval need to be established independently for each layer. Sex seems not to be a relevant influencing factor for the passive tissue mechanics of the here investigated temporal head tissue layers.

Biomechanics of vascular areas of the human cranial dura mater.

Accurate biomechanical properties of the human cranial dura mater are paramount for computational head models, artificial graft developments and biomechanical basic research. Yet, it is unclear whether areas of the dura containing meningeal vessels biomechanically differ from avascular areas. Here, 244 dura mater samples with or without vessels from 32 cadavers were tested in a quasi-static uniaxial tensile testing setup. The thicknesses of the meningeal and periosteal dura in vascular and avascular areas were histologically investigated in 36 samples using van Gieson staining. The elastic modulus of 112 MPa from dura samples containing vessels running transversely was significantly lower than samples with vessels running longitudinally (151 MPa; p < 0.001). The ultimate tensile strength of dura samples with transversely running vessels (11.1 MPa) was significantly lower in comparison to both avascular samples (14.9 MPa; p < 0.001) and samples with a longitudinally running vessel (15.0 MPa; p < 0.001). The maximum force of dura samples with longitudinally running vessels was 37 N (p < 0.001), this was significantly higher compared to the other groups which were 23 N (p < 0.001). The meningeal and periosteal dura layer thicknesses were not statistically different in avascular areas (p > 0.222). However, around the vessels, the meningeal dura layer was significantly thicker compared to the periosteal layer (p ≤ 0.019). The sum of the meningeal and periosteal layers was similar between vascular and avascular areas (p ≥ 0.071). Vascular areas of the human cranial dura mater withstand the same forces as avascular areas when being stretched. When stretched along the vessel, the dura-vessel composite can withstand even higher tensile forces compared to avascular areas. Vascular areas of the cranial dura mater seem to be similar when compared to avascular areas making their separate simulation in computational models non-essential.

On the morphological relations of the Achilles tendon and plantar fascia via the calcaneus: a cadaveric study.

Current treatments of plantar fasciitis are based on the premise that the Achilles tendon (AT) and plantar fascia (PF) are mechanically directly linked, which is an area of debate. The aim of this study was to assess the morphological relationship between the AT and PF. Nineteen cadaveric feet were x-ray imaged, serially sectioned and plastinated for digital image analyses. Measurements of the AT and PF thicknesses and cross-sectional areas (CSA) were performed at their calcaneal insertion. The fiber continuity was histologically assessed in representative subsamples. Strong correlations exist between the CSA of the AT and PF at calcaneal insertion and the CSA of PF's insertional length (r = 0.80), and between the CSAs of AT's and PF's insertional lengths. Further correlations were observed between AT and PF thicknesses (r = 0.62). This close morphological relationship could, however, not be confirmed through x-ray nor complete fiber continuity in histology. This study provides evidence for a morphometric relationship between the AT and PF, which suggests the presence of a functional relationship between these two structures following the biological key idea that the structure determines the function. The observed morphological correlations substantiate the existing mechanical link between the AT and PF via the posterior calcaneus and might explain why calf stretches are a successful treatment option for plantar heel pain.

Assessment of plantaris and peroneus tertius tendons as graft materials for ankle ligament reconstructions - A cadaveric biomechanical study.

Both the plantaris tendon and the peroneus tertius tendon are used as auto- and allogenous graft materials to reconstruct the ankle ligament complex. However, it is unclear to what extent these graft materials resemble the load-deformation behavior of the ankle ligaments. A total of 34 human ankle ligaments and 35 tendons were assessed mechanically deploying a quasi-static tensile testing setup. Tendons were significantly stiffer (median elastic moduli: plantaris tendon = 465.7 MPa, peroneus tertius tendon = 338.5 MPa, medial ligament = 61.4 MPa, lateral ligament = 49.3 MPa; p ≤ 0.035), but more distensible (median strain at maximum force: plantaris tendon = 15.1%, peroneus tertius tendon = 15.3%, medial ligament = 9.3%, lateral ligament = 9.6%; p ≤ 0.008) and mechanically tougher (median ultimate tensile strength: plantaris tendon = 51.0 MPa, peroneus tertius tendon = 40.5 MPa, medial ligament = 4.1 MPa, lateral ligament = 3.5 MPa; p ≤ 0.033) when compared to medial and lateral ankle ligaments. The lateral ligaments of the right ankle were significantly tougher compared to the left side (p = 0.015). The elastic modulus of the medial ligament (r = 0.489, p = 0.045) and the peroneus tertius tendon (r = 0.517, p = 0.014) yielded an age-dependent increase. Both tendons seem biomechanically suitable graft materials to replace the medial and lateral ankle ligaments during physiological loading. The age-dependent increase in tissue elastic properties of the medial vs. lateral ankle ligaments, and differences in ultimate tensile strength between the lateral ligaments left vs. right, may reflect the complex asymmetric loading behavior of both ankle ligaments.

Computed tomography osteoabsorptiometry-based investigation on subchondral bone plate alterations in sacroiliac joint dysfunction.

Sacroiliac joint dysfunction (SIJD) is an underappreciated source of back pain. Mineralization patterns of the sacroiliac (SIJ) subchondral bone plate (SCB) may reflect long-term adaptations to the loading of the joint. Mineralization densitograms of 27 SIJD patients and 39 controls, were obtained using CT osteoabsorptiometry. Hounsfield unit (HU) values of the SCB mineralization of superior, anterior and inferior regions on the iliac and sacral auricular surfaces were derived and statistically compared between SIJD-affected and control cohorts. Healthy controls showed higher HU values in the iliac; 868 ± 211 (superior), 825 ± 121 (anterior), 509 ± 114 (inferior), than in the sacral side; 541 ± 136 (superior), 618 ± 159 (anterior), 447 ± 91 (inferior), of all regions (p < 0.01). This was similar in SIJD; ilium 908 ± 170 (superior), 799 ± 166 (anterior), 560 ± 135 (inferior), sacrum 518 ± 150 (superior), 667 ± 151 (anterior), 524 ± 94 (inferior). In SIJD, no significant HU differences were found when comparing inferior sacral and iliac regions. Furthermore, HU values in the inferior sacral region were significantly higher when compared to the same region of the healthy controls (524 ± 94 vs. 447 ± 91, p < 0.01). Region mineralization correlated negatively with age (p < 0.01). SIJD-affected joints reflect a high mineralization of the sacral inferior region, suggesting increased SIJD-related mechanical stresses. Age-related SCB demineralization is present in all individuals, regardless of dysfunction.

Passive load-deformation properties of human temporal muscle.

The passive load-deformation properties of the human temporal muscle applicable to computer simulations of the human head or the comparison of the temporal muscle to other graft materials are unexplored to date and it is unclear, if these properties depend on age, sex, post-mortem interval or body side. Eighty-eight fresh temporal muscle samples from 69 human cadavers (age range 4 months - 94 years) were investigated in a quasi-static tensile setup. For comparative reasons, 20 age-matched human temporal muscle fascia and scalp samples were tested in the same manner as the temporal muscle. Human temporal muscle showed an elastic modulus of 1.58 ± 0.64 MPa, an ultimate tensile strength of 0.26 ± 0.11 MPa and a strain at maximum force of 26.21 ± 12.48%. These parameters were independent of sex (p > 0.88), side (p > 0.92) and post-mortem interval (p > 0.09). All passive load-deformation parameters of the human temporal muscle differed from temporal muscle fascia and scalp except for the strain at maximum force of the temporal muscle and scalp. Significantly different load-deformation properties of the human temporal muscle from temporal muscle fascia and scalp indicate the need for a separate simulation of these soft tissue layers in computational head models to reflect lifelike conditions. Contrary to other tissues such as scalp or temporal muscle fascia the biomechanical temporal muscle properties in head models may not require adjustments for sex, side and age based on the here-presented findings.

Mechanical properties of native and acellular temporal muscle fascia for surgical reconstruction and computational modelling purposes.

The temporal muscle fascia (TMF) is a widely used graft material and of interest for computational simulations of the temporomandibular joint as well as computational and physical human head models in general. However, reliable biomechanical properties of the TMF are lacking to date. This study provides tensile data of 52 TMFs at an age range of 18 to 94 years. It further investigates, if acellular fascia scaffolds differ from native counterparts in their biomechanical behaviour. Native TMF has a median elastic modulus of 26.2 MPa (acellular: 24.5 MPa), an ultimate tensile strength of 2.9 MPa (acellular: 2.1 MPa), a maximum force of 12.6 N (acellular: 9.9 N) and a strain at failure of 14.1% (acellular: 14.8%). No significant difference was found regarding the properties of native and acellular samples. Elastic modulus and the ultimate tensile strength increased with age but only in the acellular group (p < 0.01). Decorin and fibronectin seemed to be washed out by the acellularization procedure. The absence of cells in acellular TMF samples is not of biomechanical relevance compared to the native state. Acellular TMF is a biomechanically promising scaffold material for graft purposes, which can be retrieved easily due to its superficial location.

Nucleotide and corrected amino acid sequence of the functional recombinant rat anaphylatoxin C5a.

For bacterial expression of rat anaphylatoxin C5a, the cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using rat liver RNA and degenerate primers designed according to the published amino acid sequence [1]. Surprisingly, the amino acid sequence deduced from cDNA differed at positions 55 (N for K), 63 (K for H), 67 (E for N), 68 (S for E) and 69 (H for S) from the published sequence. The overall amino acid composition, however, was unchanged because these 5 amino acids were located at different positions compared to the published sequence. As a consequence, the proposed N-glycosylation site was absent, suggesting O-glycosylation of the mature molecule. Recombinant rat C5a with a 6 histidine tag at the N-terminus was expressed in bacteria, purified and renatured. The peptide was as potent as recombinant human C5a in eliciting lysosomal enzyme release from human granulocytes.

Expression of the anaphylatoxin C5a receptor in non-myeloid cells.

C5, a 74 amino acid peptide cleaved from the complement protein C5, represents the most potent anaphylatoxin and possesses inflammatory as well as immunomodulatory activities. In the past, expression of the receptor for the anaphylatoxin C5a (C5aR) has been thought to be restricted to cells of myeloid origin. However, recent evidence suggests that the C5aR is constitutively expressed in non-myeloid cells including epithelial, endothelial and smooth muscle cells in the human liver and lung. These findings are contrasted by results from our laboratory which demonstrated that in the normal human liver and lung C5aR expression is detectable exclusively in macrophages and macrophage-derived cells (Kupffer cells). Interestingly, we found evidence that C5aR expression may be inducible in epithelial cells as C5aR mRNA was observed in vivo in human keratinocytes of the inflamed but not of the normal skin. Herein we review the work of our laboratory and others on the expression of the C5aR in various human non-myeloid cells types. A better understanding of the expression patterns of this important anaphylatoxin receptor may provide new insights in the pathophysiological role of C5a in vivo.

Molecular cloning and expression of the functional rat C5a receptor.

The C5a receptor belongs to the superfamily of G-protein coupled receptors with seven transmembrane segments. In this study we report on the cloning of the rat C5a receptor (ratC5aR). We used a hybridization probe produced by PCR utilizing degenerate primers which corresponded to conserved parts of the human, canine and murine C5a receptor nucleotide sequences and to the published partial amino acid sequence of the rat C5a receptor to screen a rat macrophage cDNA library. We found two overlapping clones containing an open reading frame of 1056 bp, a 3'untranslated region of 683 bp and a 5'untranslated region of 27 bp. The overall nucleotide acid sequence identity, compared to the murine, human and canine C5a receptor sequences, was 85.8, 70.5 and 68.9%, respectively. The greatest diversity exists in the putative extracellular domains, especially in the aminoterminal domain which is assumed to be involved in ligand binding. An N-glycosylation site is present within the N-terminal sequence at residue 6. One of the cDNA containing the 5'untranslated region, the coding sequence and part of the 3'untranslated region was cloned into an eucaryotic expression vector and stably transfected into the rat basophilic leukemia cell line RBL-2H3. Expression of the rat C5a receptor on the surface of these cells could be demonstrated by flow cytometric analysis using FITC-labeled recombinant rat C5a (rrC5a). By measuring the release of calcium from intracellular stores after stimulation with rrC5a it could further be shown that the receptor is functionally coupled. Receptor binding assays showed that rrC5a specifically binds to the ratC5aR with a KD of 0.91 +/- 0.36 and to the human C5a receptor (huC5aR) with a KD of 7.19 +/- 1.56. The determined KD for binding of human C5a (huC5a) to the huCSaR was 2.16 +/- 0.65. No binding of huC5a to the ratC5aR could be observed although high concentrations of this ligand (> 60 nM) promoted chemotaxis of RBL cells transfected with the huC5aR.

Stimulation of glycogen phosphorylase in rat hepatocytes via prostanoid release from Kupffer cells by recombinant rat anaphylatoxin C5a but not by native human C5a in hepatocyte/Kupffer cell co-cultures.

Human anaphylatoxin C3a had previously been shown to increase glycogenolysis in perfused rat liver and prostanoid formation in rat liver macrophages. Surprisingly, human C5a, which in other systems elicited stronger responses than C3a, did not increase glycogenolysis in perfused rat liver. Species incompatibilities within the experimental system had been supposed to be the reason. The current study supports this hypothesis: (1) In rat liver macrophages that had been maintained in primary culture for 72 h recombinant rat anaphylatoxin C5a in concentrations between 0.1 and 10 micrograms/ml increased the formation of thromboxane A2, prostaglandin D2, E2 and F2 alpha 6- to 12-fold over basal within 10 min. In contrast, human anaphylatoxin C5a did not increase prostanoid formation in rat Kupffer cells. (2) The increase in prostanoid formation by recombinant rat C5a was specific. It was inhibited by a neutralizing monoclonal antibody. (3) In co-cultures of rat hepatocytes and rat Kupffer cells but not in hepatocyte mono-cultures recombinant rat C5a increased glycogen phosphorylase activity 3-fold over basal. This effect was inhibited by incubation of the co-cultures with 500 microM acetylsalicyclic acid. Thus, C5a generated either locally in the liver or systemically e.g. in the course of sepsis, may increase hepatic glycogenolysis by a prostanoid-mediated intercellular communication between Kupffer cells and hepatocytes.

A novel ELISA for the assessment of classical pathway of complement activation in vivo by measurement of C4-C3 complexes.

Measurements of complement split products by enzyme-linked immunosorbent assays (ELISA) are well established for the assessment of in vivo complement activation. We have combined two monoclonal antibodies (mAb) with specificities for C3b/iC3b/C3dg (mAb I3/15) and C4/C4b/C4d (mAb M4d2), respectively, in a sandwich ELISA to quantitate C4-C3 complexes as an indicator of complement activation. Serum incubated with heat aggregated IgG (HAG) was used as a standard and the C4-C3 levels expressed as microgram equivalent HAG/ml (microgram HAG-equ/ml). Normal values of C4-C3 complexes in plasma (EDTA) of healthy probands (n = 11) were 6.3 micrograms HAG-equ/ml +/- 1.5 (mean +/- 1 standard deviation (SD), with a range from 3.6 to 9.1). In patients with systemic lupus erythematosus (SLE, n = 23) C4-C3 values were clearly elevated (48.8 micrograms HAG-equ/ml +/- 52.9, range 7.5-184.7) as compared to samples from patients with idiopathic hypertension (IDH, n = 10) (6.5 micrograms HAG-equ/ml +/- 1.7, range 4.1-9.4). For SLE patients C4-C3 levels significantly correlated with values for C3b/iC3b/C3d (r = 0.69, p < 0.001) and C3 containing immune complexes (r = 0.68, p < 0.001), but not with the C4d fragment (r = 0.26). C4-C3 levels of 96% of the studied SLE patients were increased more than 2 SD above the normal mean as compared to 74% of C4d and activated C3 values, respectively. Serum treated with zymosan as an activator of the alternative pathway of complement did not exhibit higher C4-C3 values. These results demonstrate that the quantitation of in vivo generated C4-C3 complexes by ELISA provide a novel, sensitive parameter for classical pathway of complement activation.