Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity.

BACKGROUND: Interferon-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1-73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. METHODS: Relative levels of CXCL10(1-73) and intact CXCL10(1-77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1-73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1-73) was compared to intact CXCL10(1-77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cell migration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1-73) was also evaluated. RESULTS: Natural CXCL10(1-73) was more abundantly present compared to intact CXCL10(1-77) in synovial fluids of patients with RA. CXCL10(1-73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. CONCLUSION: Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.

Integrability of a Globally Coupled Complex Riccati Array: Quadratic Integrate-and-Fire Neurons, Phase Oscillators, and All in Between.

We present an exact dimensionality reduction for dynamics of an arbitrary array of globally coupled complex-valued Riccati equations. It generalizes the Watanabe-Strogatz theory [Integrability of a globally coupled oscillator array, Phys. Rev. Lett. 70, 2391 (1993).PRLTAO0031-900710.1103/PhysRevLett.70.2391] for sinusoidally coupled phase oscillators and seamlessly includes quadratic integrate-and-fire neurons as the real-valued special case. This simple formulation reshapes our understanding of a broad class of coupled systems-including a particular class of phase-amplitude oscillators-which newly fall under the category of integrable systems. Precise and rigorous analysis of complex Riccati arrays is now within reach, paving a way to a deeper understanding of emergent behavior of collective dynamics in coupled systems.

Bifurcations in adaptive vascular networks: Toward model calibration.

Transport networks are crucial for the functioning of natural and technological systems. We study a mathematical model of vascular network adaptation, where the network structure dynamically adjusts to changes in blood flow and pressure. The model is based on local feedback mechanisms that occur on different time scales in the mammalian vasculature. The cost exponent γ tunes the vessel growth in the adaptation rule, and we test the hypothesis that the cost exponent is γ=1/2 for vascular systems [D. Hu and D. Cai, Phys. Rev. Lett. 111, 138701 (2013)]. We first perform bifurcation analysis for a simple triangular network motif with a fluctuating demand and then conduct numerical simulations on network topologies extracted from perivascular networks of rodent brains. We compare the model predictions with experimental data and find that γ is closer to 1 than to 1/2 for the model to be consistent with the data. Our study, thus, aims at addressing two questions: (i) Is a specific measured flow network consistent in terms of physical reality? (ii) Is the adaptive dynamic model consistent with measured network data? We conclude that the model can capture some aspects of vascular network formation and adaptation, but also suggest some limitations and directions for future research. Our findings contribute to a general understanding of the dynamics in adaptive transport networks, which is essential for studying mammalian vasculature and developing self-organizing piping systems.

Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients.

The human chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is involved in several homeostatic processes and pathologies through interaction with its cognate G protein-coupled receptor CXCR4. Recent research has shown that CXCL12 is present in the lungs and circulation of patients with coronavirus disease 2019 (COVID-19). However, the question whether the detected CXCL12 is bioactive was not addressed. Indeed, the activity of CXCL12 is regulated by NH2- and COOH-terminal post-translational proteolysis, which significantly impairs its biological activity. The aim of the present study was to characterize proteolytic processing of CXCL12 in broncho-alveolar lavage (BAL) fluid and blood plasma samples from critically ill COVID-19 patients. Therefore, we optimized immunosorbent tandem mass spectrometry proteoform analysis (ISTAMPA) for detection of CXCL12 proteoforms. In patient samples, this approach uncovered that CXCL12 is rapidly processed by site-specific NH2- and COOH-terminal proteolysis and ultimately degraded. This proteolytic inactivation occurred more rapidly in COVID-19 plasma than in COVID-19 BAL fluids, whereas BAL fluid samples from stable lung transplantation patients and the non-affected lung of lung cancer patients (control groups) hardly induced any processing of CXCL12. In COVID-19 BAL fluids with high proteolytic activity, processing occurred exclusively NH2-terminally and was predominantly mediated by neutrophil elastase. In low proteolytic activity BAL fluid and plasma samples, NH2- and COOH-terminal proteolysis by CD26 and carboxypeptidases were observed. Finally, protease inhibitors already approved for clinical use such as sitagliptin and sivelestat prevented CXCL12 processing and may therefore be of pharmacological interest to prolong CXCL12 half-life and biological activity in vivo.

Physiological fibrin hydrogel modulates immune cells and molecules and accelerates mouse skin wound healing.

Introduction: Wound healing is a complex process to restore homeostasis after injury and insufficient skin wound healing is a considerable problem in medicine. Whereas many attempts of regenerative medicine have been made for wound healing with growth factors and cell therapies, simple pharmacological and immunological studies are lagging behind. We investigated how fibrin hydrogels modulate immune cells and molecules in skin wound healing in mice. Methods: Physiological fibrin hydrogels (3.5 mg/mL fibrinogen) were generated, biophysically analyzed for stiffness and protein contents and were structurally studied by scanning electron microscopy. Physiological fibrin hydrogels were applied to full thickness skin wounds and, after 3 days, cells and molecules in wound tissues were analyzed. Leukocytes, endothelial cells, fibroblasts and keratinocytes were explored with the use of Flow Cytometry, whereas cytokines and matrix metalloproteinases were analyzed with the use of qPCR, ELISAs and zymography. Skin wound healing was analyzed microscopically at day 3, macroscopically followed daily during repair in mice and compared with commercially available fibrin sealant Tisseel. Results: Exogenous fibrin at physiological concentrations decreased neutrophil and increased non-classical Ly6Clow monocyte and resolutive macrophage (CD206+ and CX3CR1+) populations, at day 3 after injury. Fibrin hydrogel reduced the expression of pro-inflammatory cytokines and increased IL-10 levels. In line with these findings, gelatinase B/MMP-9 was decreased, whereas gelatinase A/MMP-2 levels remained unaltered. Frequencies of dermal endothelial cells, fibroblasts and keratinocytes were increased and keratinocyte migration was enhanced by fibrin hydrogel. Importantly, physiological fibrin accelerated the healing of skin wounds in contrast to the highly concentrated fibrin sealant Tisseel, which delayed wound repair and possessed a higher fiber density. Conclusion: Collectively, we show that adding a tailored fibrin hydrogel scaffold to a wound bed positively influences the healing process, modulating leukocyte populations and inflammatory responses towards a faster wound repair.

Complex dynamics in adaptive phase oscillator networks.

Networks of coupled dynamical units give rise to collective dynamics such as the synchronization of oscillators or neurons in the brain. The ability of the network to adapt coupling strengths between units in accordance with their activity arises naturally in a variety of contexts, including neural plasticity in the brain, and adds an additional layer of complexity: the dynamics on the nodes influence the dynamics of the network and vice versa. We study a minimal model of Kuramoto phase oscillators including a general adaptive learning rule with three parameters (strength of adaptivity, adaptivity offset, adaptivity shift), mimicking learning paradigms based on spike-time-dependent plasticity. Importantly, the strength of adaptivity allows to tune the system away from the limit of the classical Kuramoto model, corresponding to stationary coupling strengths and no adaptation and, thus, to systematically study the impact of adaptivity on the collective dynamics. We carry out a detailed bifurcation analysis for the minimal model consisting of N=2 oscillators. The non-adaptive Kuramoto model exhibits very simple dynamic behavior, drift, or frequency-locking; but once the strength of adaptivity exceeds a critical threshold non-trivial bifurcation structures unravel: A symmetric adaptation rule results in multi-stability and bifurcation scenarios, and an asymmetric adaptation rule generates even more intriguing and rich dynamics, including a period-doubling cascade to chaos as well as oscillations displaying features of both librations and rotations simultaneously. Generally, adaptation improves the synchronizability of the oscillators. Finally, we also numerically investigate a larger system consisting of N=50 oscillators and compare the resulting dynamics with the case of N=2 oscillators.

Novel method to quantify peptidylarginine deiminase activity shows distinct citrullination patterns in rheumatoid and juvenile idiopathic arthritis.

Introduction: Peptidylarginine deiminases (PADs) mediate citrullination, an irreversible posttranslational modification that converts arginine to citrulline residues in proteins. Rheumatoid arthritis (RA) is characterized by unique autoantibodies that recognize citrullinated peptides, which are highly specific for this disease. However, the mechanism preceding the anti-citrulline response remains largely unclear. PAD enzymes are known to fuel the autoimmune response by generating autoreactive epitopes, and sustain local synovial inflammation through neutrophil extracellular trap formation. Therefore, detecting endogenous PAD activity is important to understand the pathogenesis of arthritis. Methods: In this study, we improved a fluorescent in vitro assay to enable endogenous PAD activity characterization in complex samples. We combine the use of an in-house synthetic, arginine-rich substrate and a negatively charged dye molecule to visualize enzyme activity. Results: This pioneering PAD assay allowed profiling of active citrullination in leukocytes and in local and systemic samples of an arthritis cohort. Our results reveal that RA and juvenile idiopathic arthritis (JIA) synovial fluids display similar levels of PAD activity. In contrast, citrullination was limited in joints of patients suffering from gout or Lyme's disease. Interestingly, in blood, a higher level of extracellular citrullination was only found in anti-CCP-positive RA patients. Discussion: Our finding suggests that enhanced synovial PAD activity drives the loss in tolerance towards citrullinated proteins and that systemic citrullination may indicate the risk for developing citrulline-specific autoimmunity.

Graphop mean-field limits and synchronization for the stochastic Kuramoto model.

Models of coupled oscillator networks play an important role in describing collective synchronization dynamics in biological and technological systems. The Kuramoto model describes oscillator's phase evolution and explains the transition from incoherent to coherent oscillations under simplifying assumptions, including all-to-all coupling with uniform strength. Real world networks, however, often display heterogeneous connectivity and coupling weights that influence the critical threshold for this transition. We formulate a general mean-field theory (Vlasov-Focker Planck equation) for stochastic Kuramoto-type phase oscillator models, valid for coupling graphs/networks with heterogeneous connectivity and coupling strengths, using graphop theory in the mean-field limit. Considering symmetric odd-valued coupling functions, we mathematically prove an exact formula for the critical threshold for the incoherence-coherence transition. We numerically test the predicted threshold using large finite-size representations of the network model. For a large class of graph models, we find that the numerical tests agree very well with the predicted threshold obtained from mean-field theory. However, the prediction is more difficult in practice for graph structures that are sufficiently sparse. Our findings open future research avenues toward a deeper understanding of mean-field theories for heterogeneous systems.

Symmetry breaking yields chimeras in two small populations of Kuramoto-type oscillators.

Despite their simplicity, networks of coupled phase oscillators can give rise to intriguing collective dynamical phenomena. However, the symmetries of globally and identically coupled identical units do not allow solutions where distinct oscillators are frequency-unlocked-a necessary condition for the emergence of chimeras. Thus, forced symmetry breaking is necessary to observe chimera-type solutions. Here, we consider the bifurcations that arise when full permutational symmetry is broken for the network to consist of coupled populations. We consider the smallest possible network composed of four phase oscillators and elucidate the phase space structure, (partial) integrability for some parameter values, and how the bifurcations away from full symmetry lead to frequency-unlocked weak chimera solutions. Since such solutions wind around a torus they must arise in a global bifurcation scenario. Moreover, periodic weak chimeras undergo a period-doubling cascade leading to chaos. The resulting chaotic dynamics with distinct frequencies do not rely on amplitude variation and arise in the smallest networks that support chaos.

Chlorite-Oxidized Oxyamylose (COAM) Has Antibacterial Activity and Positively Affects Skin Wound Healing.

Purpose: To verify the antibacterial and immunomodulatory effects of the amylose derivative - chlorite-oxidized oxyamylose (COAM) - in a skin wound setting. Methods: In vitro antibacterial effects of COAM against opportunistic bacterial pathogens common to skin wounds, including Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), were determined by cultivation methods. The effects of COAM on myeloid cell infiltration into full thickness skin wounds were investigated in wild-type and in transgenic CX3CR1-GFP mice. Results: On the basis of in vitro experiments, an antibacterial effect of COAM against Staphylococcus species including MRSA was confirmed. The minimum inhibitory concentration of COAM was determined as 2000 µg/mL against these bacterial strains. Control full thickness skin wounds yielded maximal neutrophil influxes and no additive effect on neutrophil influx was observed following topical COAM-treatment. However, COAM administration increased local CX3CR1 macrophage counts at days 3 and 4 and induced a trend towards better wound healing. Conclusion: Aside from its known broad antiviral impact, COAM possesses in vitro antibacterial effects specifically against Gram-positive opportunistic pathogens of the skin and modulates in vivo macrophage contents in mouse skin wounds.

Alpha-amylase as the culprit in an occupational mealworm allergy case.

Background: Occupational allergy has been described in employees working in contact with mealworms in pet stores, live fish bait or infested stored grains and recently, in mealworm farming for animal feed and human consumption. Mealworm allergens linked to occupational allergy are troponin C, cockroach-like allergen, tropomyosin, arginine kinase, early-staged encapsulation inducing- and larval cuticle proteins. Objective: We report a case of occupational mealworm allergy and studied the culprit component. Methods: Diagnosis was done by skin prick, specific IgE, basophil activation and lung function testing. Allergen purification was performed by anion-exchange chromatography and immunoblotting with patient IgE. Allergens were identified by in-gel trypsin digest and tandem mass spectrometry. Allergenicity and specificity further confirmed by IgE inhibition and passive basophil activation experiments. Results: We describe a new case of occupational mealworm allergy in a laboratory worker, with sensitization to different developmental stages and derivates of the mealworm. In basophil activation tests, the majority of patient's basophils (69%-91%) degranulated upon stimulation with the lowest concentration of mealworm extracts (0.16 µg/ml). Despite strong sensitization to mites, the patient did not show cross-reactivity to other insects. We were able to identify alpha-amylase as the main allergen and through inhibition experiments, we demonstrated that low amounts (0.1 µg/ml) of this allergen could strongly inhibit mealworm specific IgE by 79.1%. Moreover, passive BAT experiments demonstrated the IgE-alpha-amylase interaction to be functional, inducing up to 25.5% degranulation in healthy donor basophils. Conclusion: Alpha-amylase can be identified as the responsible allergen in this specific case of occupational mealworm allergy.

The glymphatic system: Current understanding and modeling.

We review theoretical and numerical models of the glymphatic system, which circulates cerebrospinal fluid and interstitial fluid around the brain, facilitating solute transport. Models enable hypothesis development and predictions of transport, with clinical applications including drug delivery, stroke, cardiac arrest, and neurodegenerative disorders like Alzheimer's disease. We sort existing models into broad categories by anatomical function: Perivascular flow, transport in brain parenchyma, interfaces to perivascular spaces, efflux routes, and links to neuronal activity. Needs and opportunities for future work are highlighted wherever possible; new models, expanded models, and novel experiments to inform models could all have tremendous value for advancing the field.

First-order like phase transition induced by quenched coupling disorder.

We investigate the collective dynamics of a population of X Y model-type oscillators, globally coupled via non-separable interactions that are randomly chosen from a positive or negative value and subject to thermal noise controlled by temperature T. We find that the system at T = 0 exhibits a discontinuous, first-order like phase transition from the incoherent to the fully coherent state; when thermal noise is present ( T > 0 ), the transition from incoherence to the partial coherence is continuous and the critical threshold is now larger compared to the deterministic case ( T = 0 ). We derive an exact formula for the critical transition from incoherent to coherent oscillations for the deterministic and stochastic case based on both stability analysis for finite oscillators as well as for the thermodynamic limit ( N → ∞) based on a rigorous mean-field theory using graphons, valid for heterogeneous graph structures. Our theoretical results are supported by extensive numerical simulations. Remarkably, the synchronization threshold induced by the type of random coupling considered here is identical to the one found in studies, which consider uniform input or output strengths for each oscillator node [H. Hong and S. H. Strogatz, Phys. Rev. E 84(4), 046202 (2011); Phys. Rev. Lett. 106(5), 054102 (2011)], which suggests that these systems display a "universal" character for the onset of synchronization.

Cerebrospinal fluid is a significant fluid source for anoxic cerebral oedema.

Cerebral oedema develops after anoxic brain injury. In two models of asphyxial and asystolic cardiac arrest without resuscitation, we found that oedema develops shortly after anoxia secondary to terminal depolarizations and the abnormal entry of CSF. Oedema severity correlated with the availability of CSF with the age-dependent increase in CSF volume worsening the severity of oedema. Oedema was identified primarily in brain regions bordering CSF compartments in mice and humans. The degree of ex vivo tissue swelling was predicted by an osmotic model suggesting that anoxic brain tissue possesses a high intrinsic osmotic potential. This osmotic process was temperature-dependent, proposing an additional mechanism for the beneficial effect of therapeutic hypothermia. These observations show that CSF is a primary source of oedema fluid in anoxic brain. This novel insight offers a mechanistic basis for the future development of alternative strategies to prevent cerebral oedema formation after cardiac arrest.

Proteoform Analysis of Matrix Metalloproteinase-9/Gelatinase B and Discovery of Its Citrullination in Rheumatoid Arthritis Synovial Fluids.

Objectives: To explore posttranslational modifications (PTMs), including proteolytic activation, multimerization, complex formation and citrullination of gelatinases, in particular of gelatinase B/MMP-9, and to detect in gelatin-Sepharose affinity-purified synovial fluids, the presence of specific MMP proteoforms in relation to arthritis. Methods: Latent, activated, complexed and truncated gelatinase-A/MMP-2 and gelatinase B/MMP-9 proteoforms were detected with the use of zymography analysis to compare specific levels, with substrate conversion assays, to test net proteolytic activities and by Western blot analysis to decipher truncation variants. Citrullination was detected with enhanced sensitivity, by the use of a new monoclonal antibody against modified citrullines. Results: All MMP-9 and MMP-2 proteoforms were identified in archival synovial fluids with the use of zymography analysis and the levels of MMP-9 versus MMP-2 were studied in various arthritic diseases, including rheumatoid arthritis (RA). Secondly, we resolved misinterpretations of MMP-9 levels versus proteolytic activities. Thirdly, a citrullinated, truncated proteoform of MMP-9 was discovered in archival RA synovial fluid samples and its presence was corroborated as citrullinated hemopexin-less MMP-9 in a small prospective RA sample cohort. Conclusion: Synovial fluids from rheumatoid arthritis contain high levels of MMP-9, including its truncated and citrullinated proteoform. The combination of MMP-9 as analyte and its PTM by citrullination could be of clinical interest, especially in the field of arthritic diseases.

A two-frequency-two-coupling model of coupled oscillators.

We considered the phase coherence dynamics in a Two-Frequency and Two-Coupling (TFTC) model of coupled oscillators, where coupling strength and natural oscillator frequencies for individual oscillators may assume one of two values (positive/negative). The bimodal distributions for the coupling strengths and frequencies are either correlated or uncorrelated. To study how correlation affects phase coherence, we analyzed the TFTC model by means of numerical simulations and exact dimensional reduction methods allowing to study the collective dynamics in terms of local order parameters [S. Watanabe and S. H. Strogatz, Physica D 74(3-4), 197-253 (1994); E. Ott and T. M. Antonsen, Chaos 18(3), 037113 (2008)]. The competition resulting from distributed coupling strengths and natural frequencies produces nontrivial dynamic states. For correlated disorder in frequencies and coupling strengths, we found that the entire oscillator population splits into two subpopulations, both phase-locked (Lock-Lock) or one phase-locked, and the other drifting (Lock-Drift), where the mean-fields of the subpopulations maintain a constant non-zero phase difference. For uncorrelated disorder, we found that the oscillator population may split into four phase-locked subpopulations, forming phase-locked pairs which are either mutually frequency-locked (Stable Lock-Lock-Lock-Lock) or drifting (Breathing Lock-Lock-Lock-Lock), thus resulting in a periodic motion of the global synchronization level. Finally, we found for both types of disorder that a state of Incoherence exists; however, for correlated coupling strengths and frequencies, incoherence is always unstable, whereas it is only neutrally stable for the uncorrelated case. Numerical simulations performed on the model show good agreement with the analytic predictions. The simplicity of the model promises that real-world systems can be found which display the dynamics induced by correlated/uncorrelated disorder.

Kinetics of peripheral blood neutrophils in severe coronavirus disease 2019.

OBJECTIVES: Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID-19). We isolated neutrophils from the blood of COVID-19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation. METHODS: Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co-culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allowed us to investigate viral replication in neutrophils. RESULTS: Upon ICU admission, patients displayed high plasma concentrations of granulocyte-colony-stimulating factor (G-CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10-, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non-metalloproteinase-derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID-19 neutrophils were hyper-responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS-CoV-2 failed to replicate inside human neutrophils. CONCLUSION: Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID-19 patients, and supports the concept of an increased neutrophil activation state in the circulation.

From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8.

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and -activating properties, occurs in different NH2-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.