Visual inspection versus spectrophotometry for xanthochromia detection in patients with sudden onset severe headache-A diagnostic accuracy study.

OBJECTIVE: There is still disagreement about whether to routinely use spectrophotometry to detect xanthochromia in cerebrospinal fluid (CSF) or whether visual inspection is adequate. We aimed to evaluate the diagnostic accuracy of these methods in detecting an aneurysmal subarachnoid hemorrhage in patients with sudden onset severe headache. BACKGROUND: When a patient presents to the emergency department with a headache for which there is suspicion of a subarachnoid hemorrhage, the gold standard to rule this out is to perform a CSF analysis for xanthochromia with or without spectrophotometry if the cranial non-contrast computed tomography (CT) upon admission is negative. METHODS: Having applied the gold standard, we retrospectively included patients with acute headache who underwent both CT scan and CSF spectrophotometry at our hospital in the period 2002-2020. Patients were excluded if the cranial CT was interpreted as positive, there was a bloody CSF, or if visual assessment data of the CSF was unavailable. We scrutinized the patients' medical records and evaluated the benefit of spectrophotometry compared to visual inspection. The net bilirubin absorbance cut-off for support of subarachnoid hemorrhage was set at >0.007 absorbance units. The spectrophotometry was also considered positive if the net bilirubin absorbance was ≤0.007 and net oxyhemoglobin absorbance was ≥0.1 absorbance units. We calculated and compared the sensitivity and specificity of CSF spectrophotometry and visual inspection of the CSF. RESULTS: In total, 769 patients, with a mean age of 42.3 ± (standard deviation [SD] = 17.3) years, were included. The headache onset was classified as a thunderclap headache in 41.5%, and 4.7% had a sudden loss of consciousness. Fifteen patients (2%) were finally diagnosed with a subarachnoid hemorrhage, six (0.8%) had an aneurysmal subarachnoid hemorrhage, seven (0.9%) had a perimesencephalic hemorrhage, one (0.1%) had a cortical cerebral sinus venous thrombosis, and one (0.1%) had a spinal epidural hematoma. Four patients (0.5%) had a subarachnoid hemorrhage that was not detected by visual inspection, and two were caused by an aneurysmal rupture. One of these two patients died just before intervention, and the other underwent coiling for an anterior communicating aneurysm. The number needed for lumbar puncture to detect a subarachnoid hemorrhage was 51, but 128 to detect an aneurysmal hemorrhage. The corresponding numbers needed for CSF spectrophotometric analysis were 192 and 385, respectively. Spectrophotometry was positive in 31 patients (4.0%), of whom 18 (2.3%) also had visually detected xanthochromia (11 true positive). The mean net bilirubin absorbance in the 13 samples with visually clear CSF was 0.0111 ± (SD = 0.0103) absorbance units, compared to 0.0017 ± (SD = 0.0013) in the CSF with negative spectrophotometry. The corresponding figures for net oxyhemoglobin absorbance were 0.0391 ± (SD = 0.0522) versus 0.0057 ± (SD = 0.0081). The sensitivity of spectrophotometric xanthochromia detection was 100% (95% confidence interval [CI], 78-100), compared to 73% (95% CI, 45-92) for visual xanthochromia detection. The specificity of spectrophotometric xanthochromia detection was 98% (95% CI, 97-99) compared to 99% (95% CI, 98-100) for visual xanthochromia detection. Both methods had high negative predictive values: 100% (95% CI, 99.5-100) versus 99.5% (95% CI, 98.6-99.9), respectively. CONCLUSIONS: Both visual inspection and spectrophotometry have high diagnostic accuracy for detecting CSF xanthochromia, but the lower sensitivity of visual assessment makes it unreliable, and we recommend the use of spectrophotometry in clinical practice.

Cytokines in hepatitis C-infected patients with or without opioid maintenance therapy.

OBJECTIVE: Both chronic hepatitis C virus (HCV) infection and opioids cause altered blood levels of cytokines. Previous studies have investigated levels of selected groups of cytokines in patients on opioid maintenance treatment. Little is known about the levels of multiple cytokines in patients with chronic HCV infection on opioid maintenance treatment. Our aim was to investigate the cytokine profile in patients with active HCV infection with and without opioid maintenance treatment. METHODS: We conducted a cross-sectional study in an out-patients population included upon referral for antiviral hepatitis C infection treatment. The level of 27 cytokines was measured in serum using multiplex technology. Patients were interviewed using a modified version of the European addiction severity index. Data pertaining to weight, height, current medication, smoking habits, allergies, previous medical history and ongoing withdrawal symptoms were collected. Non-parametric testing was used to investigate differences in levels of cytokines between the two groups. A 3-model hierarchical regression analysis was used to analyse associations between cytokines and confounding variables. RESULTS: Out of 120 included patients, 53 were on opioid maintenance treatment. Median duration of opioid treatment was 68.4 months. There were no demographical differences between the two groups other than age. IL-1ß was lower and eotaxin-1 higher in the group on opioid maintenance treatment than in the non-opioid group. No other inter-group differences in the remaining cytokine levels were found. CONCLUSION: In HCV infection patients, the impact of chronic opioid administration on peripheral circulating cytokine level is minimal.

Air Bubbles Activate Complement and Trigger Hemostasis and C3-Dependent Cytokine Release Ex Vivo in Human Whole Blood.

Venous air embolism, which may complicate medical and surgical procedures, activates complement and triggers thromboinflammation. In lepirudin-anticoagulated human whole blood, we examined the effect of air bubbles on complement and its role in thromboinflammation. Whole blood from 16 donors was incubated with air bubbles without or with inhibitors of C3, C5, C5aR1, or CD14. Complement activation, hemostasis, and cytokine release were measured using ELISA and quantitative PCR. Compared with no air, incubating blood with air bubbles increased, on average, C3a 6.5-fold, C3bc 6-fold, C3bBbP 3.7-fold, C5a 4.6-fold, terminal complement complex sC5b9 3.6-fold, prothrombin fragments 1+2 (PTF1+2) 25-fold, tissue factor mRNA (TF-mRNA) 26-fold, microparticle tissue factor 6.1-fold, ß-thromboglobulin 26-fold (all p < 0.05), and 25 cytokines 11-fold (range, 1.5-78-fold; all p < 0.0001). C3 inhibition attenuated complement and reduced PTF1+2 2-fold, TF-mRNA 5.4-fold, microparticle tissue factor 2-fold, and the 25 cytokines 2.7-fold (range, 1.4-4.9-fold; all p < 0.05). C5 inhibition reduced PTF1+2 2-fold and TF-mRNA 12-fold (all p < 0.05). C5 or CD14 inhibition alone reduced three cytokines, including IL-1ß (p = 0.02 and p = 0.03). Combined C3 and CD14 inhibition reduced all cytokines 3.9-fold (range, 1.3-9.5-fold; p < 0.003) and was most pronounced for IL-1ß (3.2- versus 6.4-fold), IL-6 (2.5- versus 9.3-fold), IL-8 (4.9- versus 8.6-fold), and IFN-γ (5- versus 9.5-fold). Antifoam activated complement and was avoided. PTF1+2 was generated in whole blood but not in plasma. In summary, air bubbles activated complement and triggered a C3-driven thromboinflammation. C3 inhibition reduced all mediators, whereas C5 inhibition reduced only TF-mRNA. Combined C5 and CD14 inhibition reduced IL-1ß release. These data have implications for future mechanistic studies and possible pharmacological interventions in patients with air embolism.

A Conformational Change of Complement C5 Is Required for Thrombin-Mediated Cleavage, Revealed by a Novel Ex Vivo Human Whole Blood Model Preserving Full Thrombin Activity.

Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH ≤ 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.

Association between brain-derived neurotropic factor (BDNF), high-sensitivity C-reactive protein (hs-CRP) and psychiatric symptoms in medicated and unmedicated patients.

BACKGROUND: There is evidence that brain-derived neurotropic factor (BDNF) plays a protective role in the brain. Peripheral levels of BDNF correlate with its concentration in the brain. Previous studies have revealed lower serum BDNF levels in patients with mental illnesses. In most studies serum BDNF correlates negatively with psychiatric disorders and disease severity. Most studies in this field are on psychiatric diagnosis and personality traits. The aim of our study is to explore associations between general psychiatric symptoms, independent of diagnostic groups, and serum BDNF as well as the inflammatory biomarker high-sensitive CRP (hs-CRP). Comparison between the group regularly using psychotropic medication and those not using psychotropic medication is conducted. METHODS: The study is a cross sectional study with 132 participants from a general open inpatient psychiatric ward at the Nordland Hospital Trust, Bodoe, Norway. Participants were assessed on serum levels of BDNF and hs-CRP. Psychiatric symptoms were assessed by a self-rating scale (Symptom check list, SCL-90- R). Multiple linear regression model was used for statistical analyses of associations between levels of BDNF, hs-CRP and symptoms. RESULTS: We found a positive association (p < 0.05), for most SCL-90 symptom clusters with BDNF in the psychotropic medication-free group. No associations were found in the group of patients using psychotropic medication, except one, the paranoid ideation cluster (p 0.022). No associations were found between hs-CRP and symptom clusters. CONCLUSION: We found no relation between symptom clusters and the inflammatory biomarker hs-CRP. Serum BDNF levels were positively associated with intensity of psychiatric symptoms in the group of patients not using psychotropic medication. Our findings are in conflict with several previous studies reporting increased hs-CRP as well as decreased rather than increased BDNF in mental suffering. Patients on psychotropic medication may not require the same upregulation because the medication is modulating the underlying biological pathology.

Cholesterol Crystals Induce Coagulation Activation through Complement-Dependent Expression of Monocytic Tissue Factor.

Cholesterol crystals (CC) are strong activators of complement and could potentially be involved in thromboinflammation through complement-coagulation cross-talk. To explore the coagulation-inducing potential of CC, we performed studies in lepirudin-based human whole blood and plasma models. In addition, immunohistological examinations of brain thrombi and vulnerable plaque material from patients with advanced carotid atherosclerosis were performed using polarization filter reflected light microscopy to identify CC. In whole blood, CC exposure induced a time- and concentration-dependent generation of prothrombin fragment 1+2 (PTF1.2), tissue factor (TF) mRNA synthesis, and monocyte TF expression. Blocking Abs against TF abolished CC-mediated coagulation, thus indicating involvement of the TF-dependent pathway. Blockade of FXII by corn trypsin inhibitor had a significant inhibitory effect on CC-induced PTF1.2 in platelet-free plasma, although the overall activation potential was low. CC exposure did not induce platelet aggregation, TF microparticle induction, or TF on granulocytes or eosinophils. Inhibition of complement C3 by CP40 (compstatin), C5 by eculizumab, or C5aR1 by PMX53 blocked CC-induced PTF1.2 by 90% and reduced TF+ monocytes from 18-20 to 1-2%. The physiologic relevance was supported by birefringent CC structures adjacent to monocytes (CD14), TF, and activated complement iC3b and C5b-9 in a human brain thrombus. Furthermore, monocyte influx and TF induction in close proximity to CC-rich regions with activated complement were found in a vulnerable plaque. In conclusion, CC could be active, releasable contributors to thrombosis by inducing monocyte TF secondary to complement C5aR1 signaling.

Effect of the anticoagulant, storage time and temperature of blood samples on the concentrations of 27 multiplex assayed cytokines - Consequences for defining reference values in healthy humans.

Cytokines are potentially useful biomarkers of sepsis and other inflammatory conditions. Many cytokines can be released by leukocytes and platelets after sampling. The sampling and processing techniques are consequently critically important to measure the in vivo levels. We therefore examined the effects of four different anticoagulants, EDTA, citrate, lepirudin, heparin compared to serum, on the levels of 27 different cytokines. The effects of storage temperature, freezing and thawing on the plasma cytokines were examined. Cytokines were analysed using a multiplex immunoassay. The cytokine levels in serum were significantly higher compared with plasma, consistent with release of cytokines in vitro during coagulation. In general, the lowest values for all cytokines were found in EDTA samples, stored on crushed ice, centrifuged within 4h and thereafter stored at -80°C. MCP-1 and MIP-1ß levels were highest in heparin plasma and storage of blood for up to 4h at room temperature significantly increased the interleukin (IL)-2, IL-6, IL-8, IFN-γ and GM-CSF levels in EDTA plasma, indicating post-sampling release. In contrast, the IP-10 levels were unaffected by sample storage at both temperatures. Our results indicate that the cytokines were more stable in plasma than in whole blood after sampling. Thus, cytokines should be analysed in EDTA plasma samples stored on ice and centrifuged within 4h. Based on these data, the reference ranges of 27 cytokines in EDTA plasma in 162 healthy human donors were calculated.

Cholesterol crystals induce complement-dependent inflammasome activation and cytokine release.

Inflammation is associated with development of atherosclerosis, and cholesterol crystals (CC) have long been recognized as a hallmark of atherosclerotic lesions. CC appear early in the atheroma development and trigger inflammation by NLRP3 inflammasome activation. In this study we hypothesized whether CC employ the complement system to activate inflammasome/caspase-1, leading to release of mature IL-1ß, and whether complement activation regulates CC-induced cytokine production. In this study we describe that CC activated both the classical and alternative complement pathways, and C1q was found to be crucial for the activation. CC employed C5a in the release of a number of cytokines in whole blood, including IL-1ß and TNF. CC induced minimal amounts of cytokines in C5-deficient whole blood, until reconstituted with C5. Furthermore, C5a and TNF in combination acted as a potent primer for CC-induced IL-1ß release by increasing IL-1ß transcripts. CC-induced complement activation resulted in upregulation of complement receptor 3 (CD11b/CD18), leading to phagocytosis of CC. Also, CC mounted a complement-dependent production of reactive oxygen species and active caspase-1. We conclude that CC employ the complement system to induce cytokines and activate the inflammasome/caspase-1 by regulating several cellular responses in human monocytes. In light of this, complement inhibition might be an interesting therapeutic approach for treatment of atherosclerosis.

Effects on muscle strength, maximal jump height, flexibility and postural sway after soccer and Zumba exercise among female hospital employees: a 9-month randomised controlled trial.

This 9-month randomised controlled workplace physical activity trial investigated the effects of soccer and Zumba exercise, respectively, on muscle strength, maximal jump height, sit-and-reach flexibility and postural sway among female workers. A total of 107 female hospital employees aged 25-63 were cluster-randomised to a soccer group, a Zumba group or a control group. Training was conducted outside working hours as two to three 1-h weekly sessions the first 3 months and once a week the last 6 months. Tests were conducted at baseline, after 3 and 9 months. The soccer group improved maximal neck extension strength both after 3 (1.2 kg; P < 0.05) and 9 months (1.7 kg; P < 0.01) compared to the control group. The Zumba group improved maximal trunk extension strength (3.1 kg; P = 0.04) after 3 months, with improvements in postural sway velocity moment (-9.2 mm(2)/s; P < 0.05) and lower limb lean mass (0.4 kg; P < 0.05) after 9 months. No significant intervention effects were revealed in vertical jump height or sit-and-reach flexibility. The present study indicates that workplace-initiated soccer and Zumba exercise may be beneficial for improvement of the neck and trunk strength, which may have preventive effects with regard to future perceived muscle pain in the respective body regions. Furthermore, the Zumba group revealed positive effects on lower limb lean mass and postural sway compared to the control group.

Chimeric anti-CD14 IGG2/4 Hybrid antibodies for therapeutic intervention in pig and human models of inflammation.

CD14 is a key recognition molecule of innate immune responses, interacting with several TLRs. TLR signaling cross-talks extensively with the complement system, and combined CD14 and complement inhibition has been proved effective in attenuating inflammatory responses. Pig models of human diseases have emerged as valuable tools to study therapeutic intervention, but suitable neutralizing Abs are rare. Undesired Fc-mediated functions, such as platelet activation and IL-8 release induced by the porcine CD14-specific clone Mil2, limit further studies. Therefore, an inert human IgG2/IgG4 hybrid C region was chosen for an rMil2. As revealed in ex vivo and in vivo pig experiments, rMil2 inhibited the CD14-mediated proinflammatory cytokine response similar to the original clone, but lacked the undesired Fc-effects, and inflammation was attenuated further by simultaneous complement inhibition. Moreover, rMil2 bound porcine FcRn, a regulator of t1/2 and biodistribution. Thus, rMil2, particularly combined with complement inhibitors, should be well suited for in vivo studies using porcine models of diseases, such as sepsis and ischemia-reperfusion injury. Similarly, the recombinant anti-human CD14 IgG2/4 Ab, r18D11, was generated with greatly reduced Fc-mediated effects and preserved inhibitory function ex vivo. Such Abs might be drug candidates for the treatment of innate immunity-mediated human diseases.

Combined inhibition of complement and CD14 improved outcome in porcine polymicrobial sepsis.

INTRODUCTION: Sepsis is an exaggerated and dysfunctional immune response to infection. Activation of innate immunity recognition systems including complement and the Toll-like receptor family initiate this disproportionate inflammatory response. The aim of this study was to explore the effect of combined inhibition of the complement component C5 and the Toll-like receptor co-factor CD14 on survival, hemodynamic parameters and systemic inflammation including complement activation in a clinically relevant porcine model of polymicrobial sepsis. METHODS: Norwegian landrace piglets (4 ± 0.5 kg) were blindly randomized to a treatment group (n = 12) receiving the C5 inhibitor coversin (OmCI) and anti-CD14 or to a positive control group (n = 12) receiving saline. Under anesthesia, sepsis was induced by a 2 cm cecal incision and the piglets were monitored in standard intensive care for 8 hours. Three sham piglets had a laparotomy without cecal incision or treatment. Complement activation was measured as sC5b-9 using enzyme immunoassay. Cytokines were measured with multiplex technology. RESULTS: Combined C5 and CD14 inhibition significantly improved survival (p = 0.03). Nine piglets survived in the treatment group and four in the control group. The treatment group had significantly lower pulmonary artery pressure (p = 0.04) and ratio of pulmonary artery pressure to systemic artery pressure (p < 0.001). Plasma sC5b-9 levels were significantly lower in the treatment group (p < 0.001) and correlated significantly with mortality (p = 0.006). IL-8 and IL-10 were significantly (p < 0.05) lower in the treatment group. CONCLUSIONS: Combined inhibition of C5 and CD14 significantly improved survival, hemodynamic parameters and inflammation in a blinded, randomized trial of porcine polymicrobial sepsis.

Establishing IMMULITE® 2000 cut-off values for serum allergen-specific immunoglobulin and exploring their relationship to exhaled nitric oxide.

AIM: Paediatric cut-off values for serum allergen-specific IgE (sIgE) using the Siemens IMMULITE(®) 2000 system to diagnose allergic rhinoconjunctivitis have not been established. We aimed to determine cut-off levels for sIgE for 10 common inhalant allergens and to study the relationship between sIgE, total IgE and fractional exhaled nitric oxide (FENO ). METHODS: We enrolled 243 schoolchildren, including 164 with allergic rhinoconjunctivitis. Parental interviews, skin prick tests, sIgE, total IgE, FENO measurements, spirometry and exercise tests were performed. RESULTS: Cut-off values with the best combined sensitivity and specificity were above the detection limit of the assay for seven of the ten allergens (0.23-1.1 kU/L). The overall accuracy of the IMMULITE(®) in detecting allergic rhinoconjunctivitis was good. sIgE was superior to total IgE and FENO in predicting allergic rhinoconjunctivitis to timothy, birch, mugwort, cat, dog and house dust mite. FENO was elevated in children with allergic rhinoconjunctivitis, irrespective of asthma. CONCLUSION: Cut-off values for sIgE were dependent on the allergic phenotype and were above the IMMULITE(®) detection limit for seven of ten inhalant allergens. Consequently, using the detection limit for sIgE as the decision point would result in over-diagnosing allergic rhinoconjunctivitis. When measuring elevated FENO in children, allergic rhinoconjunctivitis should be suspected.

Soccer and Zumba as health-promoting activities among female hospital employees: a 40-weeks cluster randomised intervention study.

The aim of this study was to evaluate the physiological effects of soccer and Zumba among female hospital employees during a 40-week intervention period. Hospital employees (n = 118) were cluster-randomised to either a soccer group (n = 41), a Zumba group (n = 38) or a control group (n = 39). Both training groups were encouraged to perform 1-h training sessions twice a week outside working hours throughout the 40 weeks. Maximal oxygen uptake (VO2 max), blood pressure and body composition were measured and blood samples collected before and after the intervention period. Using intention-to-treat analyses, the Zumba group improved VO2 max compared to the control group (2.2 mL · kg(-1) · min(-1), 95% CI, 0.9, 3.5, P = 0.001), with no significant increase in the soccer group (1.1 mL · kg(-1) · min(-1), 95% CI, -0.2, 2.4, P = 0.08). Both intervention groups reduced total body fat mass and fat percentage compared to the control group (P < 0.01). In the soccer group, but not the Zumba group, a significant difference in lower limb bone mineral density and bone mineral content was observed in comparison to the control group (P < 0.01). Furthermore, the soccer group, but not the Zumba group, had increased plasma osteocalcin (6.6 µg · L(-1), 95% CI, 2.2, 11.0, P < 0.01) and decreased plasma leptin (-6.6 µg · L(-1), 95% CI, -12.5, -0.7, P < 0.05) compared to the control group. The present study suggests that workplace-initiated soccer and Zumba training comprising 1-2 sessions per week outside working hours may promote physiological health among female hospital employees.

Transdiagnostic Associations between Anger Hostility and Chemokine Interferon-gamma Inducible Protein 10.

Objective: Many psychiatric disorders are linked to low grade systemic inflammation as measured by systemic cytokine levels. Exploration of cytokines and immune activity and their role in psychiatric symptoms may inform pathobiology and treatment opportunities. The aim of this study is to explore if there are associations between cytokines and psychiatric symptom clusters. Comparison between patients regularly using and those not using psychotropic medication is also conducted. Methods: This was a cross sectional naturalistic study with 132 participants from a general open inpatient psychiatric ward at the Nordland Hospital Trust, Norway. Serum levels of 28 different cytokines were assessed. Psychiatric symptoms the last week were assessed by a self-rating scale (Symptom check list, SCL-90-R) and grouped in defined clusters. Multiple linear regression model was used for statistical analyses of associations between levels of cytokines and symptoms, adjusting for possible confounding factors. Results: We found a positive association (p = 0.009) between the chemokine interferon-gamma inducible protein 10 (CXCL 10; IP-10) and the anger hostility cluster. No associations were found between the other symptom clusters and cytokines. IP-10 and the anger hostility cluster were positively associated (p = 0.002) in the subgroup of patients using psychotropic medication, not in the subgroup not using psychotropic medication. Conclusion: Our analyses revealed a significant positive association between the symptom cluster anger hostility in SCL-90-R and the chemokine IP-10 in the subgroup of patients using psychotropic medications.

Potential Biomarkers for the Earlier Diagnosis of Kidney and Liver Damage in Acute Intermittent Porphyria.

Acute intermittent porphyria (AIP) is an inherited metabolic disorder associated with complications including kidney failure and hepatocellular carcinoma, probably caused by elevations in the porphyrin precursors porphobilinogen (PBG) and delta-aminolevulinic acid (ALA). This study explored differences in modern biomarkers for renal and hepatic damage between AIP patients and controls. Urine PBG testing, kidney injury panels, and liver injury panels, including both routine and modern biomarkers, were performed on plasma and urine samples from AIP cases and matched controls (50 and 48 matched pairs, respectively). Regarding the participants' plasma, the AIP cases had elevated kidney injury marker-1 (KIM-1, p = 0.0002), fatty acid-binding protein-1 (FABP-1, p = 0.04), and α-glutathione S-transferase (α-GST, p = 0.001) compared to the matched controls. The AIP cases with high PBG had increased FABP-1 levels in their plasma and urine compared to those with low PBG. In the AIP cases, KIM-1 correlated positively with PBG, CXCL10, CCL2, and TCC, and the liver marker α-GST correlated positively with IL-13, CCL2, and CCL4 (all p < 0.05). In conclusion, KIM-1, FABP-1, and α-GST could represent potential early indicators of renal and hepatic damage in AIP, demonstrating associations with porphyrin precursors and inflammatory markers.

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood.

Introduction: Platelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy. Methods: In this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate® aggregometry, flow cytometry, and confocal microscopy. Results and Discussion: We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process.

Blood Transcriptome Analysis of Septic Patients Reveals a Long Non-Coding Alu-RNA in the Complement C5a Receptor 1 Gene.

Many severe inflammation conditions are complement-dependent with the complement component C5a-C5aR1 axis as an important driver. At the RNA level, the blood transcriptome undergoes programmed expression of coding and long non-coding RNAs to combat invading microorganisms. Understanding the expression of long non-coding RNAs containing Alu elements in inflammation is important for reconstructing cell fate trajectories leading to severe disease. We have assembled a pipeline for computation mining of new Alu-containing long non-coding RNAs by intersecting immune genes with known Alu coordinates in the human genome. By applying the pipeline to patient bulk RNA-seq data with sepsis, we found immune genes containing 48 Alu insertion as robust candidates for further study. Interestingly, 1 of the 48 candidates was located within the complement system receptor gene C5aR1 and holds promise as a target for RNA therapeutics.

A Novel Porcine Model of Ischemia-Reperfusion Injury After Cross-Clamping the Thoracic Aorta Revealed Substantial Cardiopulmonary, Thromboinflammatory and Biochemical Changes Without Effect of C1-Inhibitor Treatment.

Ischemic injury worsens upon return of blood and innate immunity including the complement system play a central role in ischemia-reperfusion injury (IRI) as in thoracic aortic surgery. Complement component1 inhibitor (C1-INH) has been shown to reduce IRI and is a broad-acting plasma cascade inhibitor. We established a new porcine model of IRI by cross-clamping the thoracic aorta and evaluated the global changes occurring in organ function, systemic inflammatory response and organ damage with or without treatment with C1-INH-concentrate. Twenty-four piglets (8.8-11.1 kg) underwent 45 minutes clamping of the thoracic aorta at the Th8 level. Upfront 12 piglets received human saline and 12 received C1-INH (250 IU/kg) intravenously. Three sham animals received thoracic opening without clamping. Reperfusion lasted 5 hours. We studied ten cardiorespiratory markers, three hematologic markers, eleven inflammatory markers, and twelve organ damage markers over the whole experimental period. Postmortem tissue homogenates from seven organs were examined for inflammatory markers and analysed by two-way repeated-measures ANOVA, area under the curve or unpaired t-tests. By excluding sham and combining treated and untreated animals, the markers reflected a uniform, broad and severe organ dysfunction. The mean and range fold change from before cross-clamp onset to maximum change for the different groups of markers were: cardiorespiratory 1.4 (0.2-3.7), hematologic 1.9 (1.2-2.7), plasma inflammatory 19.5 (1.4-176) and plasma organ damage 2.9 (1.1-8.6). Treatment with C1-INH had only a marginal effect on the IRI-induced changes, reaching statistical significance only for the plasma complement activation product TCC (p=0.0083) and IL-4 (p=0.022) and INF-α (p=0.016) in the colon tissue. In conclusion, the present novel model of porcine global IRI is forceful with regards to central markers and could generally be applicable for pathophysiological studies. C1-INH treatment had no significant effect, but the model allows for future testing of other drugs attenuating IRI globally.

Dental and Periodontal Health in Acute Intermittent Porphyria.

In the inherited metabolic disorder acute intermittent porphyria (AIP), high sugar intake prevents porphyric attacks due to the glucose effect and the following high insulin levels that may lower AIP disease activity. Insulin resistance is a known risk factor for periodontitis and sugar changes diabetogenic hormones and affects dental health. We hypothesized differences in homeostasis model assessment (HOMA) scores for insulin resistance in AIP cases vs. controls and in those with periodontitis. Our aim was to systematically study dental health in AIP as poor dental health was previously only described in case reports. Further, we aimed to examine if poor dental health and kidney failure might worsen AIP as chronic inflammation and kidney failure might increase disease activity. In 47 AIP cases and 47 matched controls, X-rays and physical examination of clinical attachment loss (CAL), probing pocket depth (PPD), and decayed missing filled teeth (DMFT) were performed. Dietary intake was evaluated through a diet logbook. Plasma cytokines and diabetogenic hormones were measured using multiplex technology and urine porphobilinogen and kidney and liver function by routine methods. An excel spreadsheet from the University of Oxford was used to estimate HOMA scores; beta cell function, HOMA%B (%B), insulin sensitivity, HOMA%S (%S), and insulin resistance HOMA-IR (IR), based on glucose and plasma (P) C-peptide. The Wilcoxon matched-pairs signed rank test, the Mann−Whitney U-test, and Spearman's non-parametric correlation were used. Insulin (p = 0.007) and C-peptide (p = 0.006) were higher in the AIP cases with periodontitis versus those without. In AIP patients, the liver fibrosis index 4 correlated with DMFT (p < 0.001) and CAL ≥4 mm (p = 0.006); the estimated glomerular filtration rate correlated with DMFT (p < 0.001) and CAL ≥4 mm (p = 0.02). CAL ≥4 mm was correlated with chemokine ligand 11 and interleukin (IL)-13 (p = 0.04 for both), and PPD >5 mm was correlated with plasminogen activator inhibitor-1 (p = 0.003) and complement component 3 (p = 0.02). In conclusion, dental health in AIP cases was correlated with insulin resistance, inflammatory markers, and biomarkers of kidney and liver function, demonstrating that organ damage in the kidney and liver are associated with poorer dental health.