Tomislav Kelava

Induction of osteoclast progenitors in inflammatory conditions: key to bone destruction in arthritis

The inflammatory milieu favors recruitment and activation of osteoclasts, and leads to bone destruction as a serious complication associated with arthritis and with other inflammatory processes. The frequency and activity of osteoclast progenitors (OCPs) correspond to arthritis severity, and may be used to monitor disease progression and bone resorption, indicating the need for detailed characterization of the discrete OCP subpopulations. Collectively, current studies suggest that the most potent murine bone marrow OCP population can be identified among lymphoid negative population within the immature myeloid lineage cells, as B220−CD3−CD11b–/loCD115+CD117+CX3CR1+ and possibly also Ter119−CD11c−CD135loLy6C+RANK−. In peripheral blood the OCP population bears the monocytoid phenotype B220−CD3−NK1.1−CD11b+Ly6ChiCD115+CX3CR1+, presumably expressing RANK in committed OCPs. Much less is known about human OCPs and their regulation in arthritis, but the circulating OCP subset is, most probably, comprised among the lymphoid negative population (CD3−CD19−CD56−), within immature monocyte subset (CD11b+CD14+CD16−), expressing receptors for M-CSF and RANKL (CD115+RANK+). Our preliminary data confirmed positive association between the proportion of peripheral blood OCPs, defined as CD3−CD19−CD56−CD11b+CD14+, and the disease activity score (DAS28) in the follow-up samples from patients with psoriatic arthritis receiving anti-TNF therapy. In addition, we reviewed cytokines and chemokines which, directly or indirectly, activate OCPs and enhance their differentiation potential, thus mediating osteoresorption. Control of the activity and migratory behaviour of OCPs as well as the identification of crucial bone/joint chemotactic mediators represent promising therapeutic targets in arthritis.

Effects of α-Melanocortin Enantiomers on Acetaminophen- Induced Hepatotoxicity in CBA Mice

Proteins and peptides in mammals are based exclusively on l-amino acids. Recent investigations show that d-amino acids exhibit physiological effects in vivo, despite of their very small quantities. We have investigated the hepatoprotective effects of the l- and d-enantiomers of α-melanocortin peptide (α-MSH). The results showed that peptide-enantiomerism is related to the protective effects of melanocortin peptides in vivo. l-α-MSH exhibited potent hepatoprotective effect in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice, while its d-mirror image was inefficient. Furthermore, the antibody to the l-peptide did not recognize the d-structure. The results indicate that the opposite peptide configuration may be used to modulate its function and metabolism in vivo and in vitro.

The Influence of α-, β-, and γ-Melanocyte Stimulating Hormone on Acetaminophen Induced Liver Lesions in Male CBA Mice

Research over the past decade has indicated that melanocortin peptides are potent inhibitors of inflammation and a promising source of new anti-inflammatory and cytoprotective therapies. The purpose of the present paper is to compare protective effects of α-, β-, and γ-melanocyte stimulating hormone on acetaminophen induced liver lesions in male CBA mice. Acetaminophen was applied intragastrically in a dose of 150 mg/kg, and tested substances were applied intraperitoneally 1 hour before acetaminophen. Mice were sacrificed after 24 hours and intensity of liver injury was estimated by measurement of plasma transaminase activity (AST and ALT) and histopathological grading of lesions. It was found that α-, β-, and γ-MSH decrease intensity of lesions by both criteria in a dose-dependent manner.

Modulation of γ2-MSH Hepatoprotection by Antisense Peptides and Melanocortin Subtype 3 and 4 Receptor Antagonists

Melanocortins, i.e., melanocyte stimulating hormones (MSH) are peptides with strong antiinflammatory effects. The most investigated aspects of γ2-MSH are related to cardiovascular effects and natriuresis, with limited research available about its anti-inflammatory and cytoprotective effects. The aims of this study were: 1) to examine the effects of γ2-MSH and its derivative [D-Trp(8)]-γ2-MSH on the acetaminophen model of liver damage in CBA mice; 2) to evaluate the modulation of γ2-MSH hepatoprotection by melanocortin subtypes 3 and 4 receptor antagonists SHU 9119 and HS 024; 3) to define the importance of central MSH pharmacophore region (HFRW) by using antisense peptides LVKAT and VKAT. In this study, specific antagonists and antisense peptides were used to target central pharmacophore region of γ2-MSH and [D-Trp(8)]-γ2-MSH, enabling the evaluation of hepatoprotection from the standpoint of the receptor and pharmacophore blockade. The criteria for monitoring the effects of the hormones on the liver damage were alanine transaminase, aspartate transaminase activities (U/L), and pathohistological scoring of liver necrosis (scale 0-5). γ2-MSH (0.24 mg/kg) indicated hepatoprotective effects in comparison to control (p < 0.001). In contrast, [D-Trp(8)]-γ2-MSH did not show any hepatoprotective effects. Application of antagonists SHU 9119 and HS 024, and antisense peptides LVKAT and VKAT, also did not show any hepatoprotective effects. In fact, when combined with γ2-MSH, it annulled its hepatoprotective effect. The results provide evidence for hepatoprotective and antiinflammatory effects of the γ2-MSH in the liver.

The effect of glucagon and cyclic adenosine monophosphate on acute liver damage induced by acetaminophen

Recent investigations suggest that glucagon might have a potentially important hepatoprotective activity. We investigated the effect of glucagon in a model of acetaminophen-induced liver injury. CBA male mice were injected intraperitoneally with a lethal (300 mg/kg) or sublethal (150 mg/kg) dose of acetaminophen. The liver injury was assessed by observing the survival of mice, by liver histology and by measuring the concentration of alanine-aminotransferase (ALT). Inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-κB) protein expressions were determined immunohistochemically. Hepatic levels of reduced glutathione (GSH) and cyclic adenosine monophosphate (cAMP) were also measured. Results show that glucagon, dose and time dependently, protects against acetaminophen-induced hepatotoxicity. This protection was achieved with a dose of 0.5 mg/kg of glucagon given intraperitoneally 15 min before or 1 h after acetaminophen. Treatment of animals with acetaminophen elevated ALT and nitrite/nitrate concentration in the plasma, enhanced iNOS and NF-κB expression and reduced GSH and cAMP concentration in the liver. Animals treated with glucagon had higher hepatic cAMP level, lower ALT and nitrite/nitrate concentration in plasma and lower expression of iNOS in liver cells than animals in control group, whereas there was no difference in the expression of NF-κB. Glucagon did not prevent the loss of GSH content caused by acetaminophen. Our investigation indicates that glucagon has a moderately protective effect against acetaminophen-induced liver injury, which is, at least partially, mediated through the downregulation of iNOS and through the increase in hepatic cAMP content, but it is not mediated through the modulation of NF-κB activity.

Hepatoprotective action of Panaxatriol saponins against acetaminophen‐induced liver injury: what is the mechanism?

To the Editor: We read with great interest a recent article published in Liver International by Wang et al., who reported on the powerful protective effect of Panaxatriol saponins (ginsenosides) extracted from Panax notoginseng against acetaminophen hepatotoxicity. The authors concluded that the protection is mediated through suppression of tumour necrosis factor alpha (TNF-a) and upregulation of thioredoxin-1 (TRX1) (1). However, a review of the published literature suggests a more plausible mechanism of protection. Toxicity of acetaminophen depends on its metabolic activation to N-acetyl-p-benzoquinoneimine (NAPQI) by cytochrome P450 enzymes. It is well known that many substances exert powerful protective effect against acetaminophen hepatotoxicity by inhibiting its cytochrome P450mediated biotransformation (2). Therefore, when performing a study on a hepatoprotective substance, it is critical to investigate its effect on acetaminophen activation, at least by monitoring the dynamic of GSH liver content loss or, in more precise way, by monitoring the levels of acetaminophen metabolites. The authors did not provide such data, although the literature suggests that ginsenosides might have inhibitory effect on several CYP isoforms, including the CYP1A2, CYP3A4 and CYP2E1 (3, 4), important for acetaminophen bioactivation (5). Therefore, treatment with Panaxatriol saponins could suppress the production of NAPQI and subsequently acetaminophen toxicity. Since NAPQI overproduction leads to oxidative stress and increases TNF-a production (6), the differences in levels of TRX1 and TNF-a between the control and Panaxatriol saponins-treated group might have been caused by differences in NAPQI production. Furthermore, literature data do not support authors’ conclusions regarding the role of TNF-a and TRX1 in acetaminophen hepatotoxicity:

Continuous remote monitoring of COPD patients—justification and explanation of the requirements and a survey of the available technologies

AbstractRemote patient monitoring should reduce mortality rates, improve care, and reduce costs. We present an overview of the available technologies for the remote monitoring of chronic obstructive pulmonary disease (COPD) patients, together with the most important medical information regarding COPD in a language that is adapted for engineers. Our aim is to bridge the gap between the technical and medical worlds and to facilitate and motivate future research in the field. We also present a justification, motivation, and explanation of how to monitor the most important parameters for COPD patients, together with pointers for the challenges that remain. Additionally, we propose and justify the importance of electrocardiograms (ECGs) and the arterial carbon dioxide partial pressure (PaCO2) as two crucial physiological parameters that have not been used so far to any great extent in the monitoring of COPD patients. We cover four possibilities for the remote monitoring of COPD patients: continuous monitoring during normal daily activities for the prediction and early detection of exacerbations and life-threatening events, monitoring during the home treatment of mild exacerbations, monitoring oxygen therapy applications, and monitoring exercise. We also present and discuss the current approaches to decision support at remote locations and list the normal and pathological values/ranges for all the relevant physiological parameters. The paper concludes with our insights into the future developments and remaining challenges for improvements to continuous remote monitoring systems. Graphical abstractᅟ

Chemokine signals are crucial for enhanced homing and differentiation of circulating osteoclast progenitor cells.

BackgroundThe peripheral blood (PB) monocyte pool contains osteoclast progenitors (OCPs), which contribute to osteoresorption in inflammatory arthritides and are influenced by the cytokine and chemokine milieu. We aimed to define the importance of chemokine signals for migration and activation of OCPs in rheumatoid arthritis (RA) and psoriatic arthritis (PsA).MethodsPB and, when applicable, synovial fluid (SF) samples were collected from 129 patients with RA, 53 patients with PsA, and 110 control patients in parallel to clinical parameters of disease activity, autoantibody levels, and applied therapy. Receptors for osteoclastogenic factors (CD115 and receptor activator of nuclear factor-κB [RANK]) and selected chemokines (CC chemokine receptor 1 [CCR1], CCR2, CCR4, CXC chemokine receptor 3 [CXCR3], CXCR4) were determined in an OCP-rich subpopulation (CD3−CD19−CD56−CD11b+CD14+) by flow cytometry. In parallel, levels of CC chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, CXC chemokine ligand 9 (CXCL9), CXCL10, and CXCL12 were measured using cytometric bead array or enzyme-linked immunosorbent assay. Sorted OCPs were stimulated in culture by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, and they were differentiated into mature osteoclasts that resorb bone. Selected chemokines (CCL2, CCL5, CXCL10, and CXCL12) were tested for their osteoclastogenic and chemotactic effects on circulatory OCPs in vitro.ResultsThe OCP population was moderately enlarged among PB cells in RA and correlated with levels of tumor necrosis factor-α (TNF-α), rheumatoid factor, CCL2, and CCL5. Compared with PB, the RANK+ subpopulation was expanded in SF and correlated with the number of tender joints. Patients with PsA could be distinguished by increased RANK expression rather than total OCP population. OCPs from patients with arthritis had higher expression of CCR1, CCR2, CCR4, CXCR3, and CXCR4. In parallel, patients with RA had increased levels of CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10, with significant elevation in SF vs PB for CXCL10. The subset expressing CXCR4 positively correlated with TNF-α, bone resorption marker, and rheumatoid factor, and it was reduced in patients treated with disease-modifying antirheumatic drugs. The CCR4+ subset showed a significant negative trend during anti-TNF treatment. CCL2, CCL5, and CXCL10 had similar osteoclastogenic effects, with CCL5 showing the greatest chemotactic action on OCPs.ConclusionsIn our study, we identified distinct effects of selected chemokines on stimulation of OCP mobilization, tissue homing, and maturation. Novel insights into migratory behaviors and functional properties of circulatory OCPs in response to chemotactic signals could open ways to new therapeutic targets in RA.

Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis.

Our study aimed to determine the functional activity of different osteoclast progenitor (OCP) subpopulations and signals important for their migration to bone lesions, causing local and systemic bone resorption during the course of collagen‐induced arthritis in C57BL/6 mice. Arthritis was induced with chicken type II collagen (CII), and assessed by clinical scoring and detection of anti‐CII antibodies. We observed decreased trabecular bone volume of axial and appendicular skeleton by histomorphometry and micro‐computed tomography as well as decreased bone formation and increased bone resorption rate in arthritic mice in vivo. In the affected joints, bone loss was accompanied with severe osteitis and bone marrow hypercellularity, coinciding with the areas of active osteoclasts and bone erosions. Flow cytometry analysis showed increased frequency of putative OCP cells (CD3–B220–NK1.1–CD11b–/loCD117+CD115+ for bone marrow and CD3–B220–NK1.1–CD11b+CD115+Gr‐1+ for peripheral haematopoietic tissues), which exhibited enhanced differentiation potential in vitro. Moreover, the total CD11b+ population was expanded in arthritic mice as well as CD11b+F4/80+ macrophage, CD11b+NK1.1+ natural killer cell and CD11b+CD11c+ myeloid dendritic cell populations in both bone marrow and peripheral blood. In addition, arthritic mice had increased expression of tumour necrosis factor‐α, interleukin‐6, CC chemokine ligand‐2 (Ccl2) and Ccl5, with increased migration and differentiation of circulatory OCPs in response to CCL2 and, particularly, CCL5 signals. Our study characterized the frequency and functional properties of OCPs under inflammatory conditions associated with arthritis, which may help to clarify crucial molecular signals provided by immune cells to mediate systemically enhanced osteoresorption.

Levels of Selected Aqueous Humor Mediators (IL-10, IL-17, CCL2, VEGF, FasL) in Diabetic Cataract

Abstract Purpose: To compare levels of selected mediators in serums and aqueous humor (AH) of type 2 diabetes mellitus cataract patients with senile cataract patients, and to determine their association with postoperative corneal edema (CE). Methods: Patients (32 senile and 29 diabetic cataract) undergoing standardized phacoemulsification combined with intraocular lens implantation were recruited. CE was assessed using an ordinal scale (grade 0 to 3). IL-10, CCL2, IL-17, FasL, and VEGF were measured by ELISA. Results: Diabetic patients had higher AH levels of VEGF (p = .042) and IL-10 (p = .021), lower AH levels of FasL (p = .048), and higher serum levels of CCL2 (p = .002). AH levels of CCL2 were higher in diabetic patients with more severe CE at the first postoperative day (p = .012). Conclusions: We found disturbed AH microenvironment in diabetic cataract, with significant changes for VEGF, IL-10, and FasL. Higher CCL2 was associated with the development of early postoperative CE in diabetic patients.