Alternative Ligand Research Articles

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in invitro and invivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase.

Protein engineering to alter recognition underlying ligand binding and activity has enormous potential. Here, ligand binding for Escherichia coli phosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into CO2 and phosphoenolpyruvate as the first committed step in gluconeogenesis, was engineered to accommodate alternative ligands as an exemplary system with structural information. From our identification of bicarbonate binding in the PEPCK active site at the supposed CO2 binding site, we probed binding of nonnative ligands with three oxygen atoms arranged to resemble the bicarbonate geometry. Crystal structures of PEPCK and point mutants with bound nonnative ligands thiosulfate and methanesulfonate along with strained ATP and reoriented oxaloacetate intermediates and unexpected bicarbonate were determined and analyzed. The mutations successfully altered the bound ligand position and orientation and its specificity: mutated PEPCKs bound either thiosulfate or methanesulfonate but never both. Computational calculations predicted a methanesulfonate binding mutant and revealed that release of the active site ordered solvent exerts a strong influence on ligand binding. Besides nonnative ligand binding, one mutant altered the Mn2+ coordination sphere: instead of the canonical octahedral ligand arrangement, the mutant in question had an only five-coordinate arrangement. From this work, critical features of ligand binding, position, and metal ion cofactor geometry required for all downstream events can be engineered with small numbers of mutations to provide insights into fundamental underpinnings of protein-ligand recognition. Through structural and computational knowledge, the combination of designed and random mutations aids in the robust design of predetermined changes to ligand binding and activity to engineer protein function.

The PqsE and RhlR proteins are an autoinducer synthase–receptor pair that control virulence and biofilm development in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading cause of life-threatening nosocomial infections. Many virulence factors produced by P. aeruginosa are controlled by the cell-to-cell communication process called quorum sensing (QS). QS depends on the synthesis, release, and groupwide response to extracellular signaling molecules called autoinducers. P. aeruginosa possesses two canonical LuxI/R-type QS systems, LasI/R and RhlI/R, that produce and detect 3OC12-homoserine lactone and C4-homoserine lactone, respectively. Previously, we discovered that RhlR regulates both RhlI-dependent and RhlI-independent regulons, and we proposed that an alternative ligand functions together with RhlR to control the target genes in the absence of RhlI. Here, we report the identification of an enzyme, PqsE, which is the alternative-ligand synthase. Using biofilm analyses, reporter assays, site-directed mutagenesis, protein biochemistry, and animal infection studies, we show that the PqsE-produced alternative ligand is the key autoinducer that promotes virulence gene expression. Thus, PqsE can be targeted for therapeutic intervention. Furthermore, this work shows that PqsE and RhlR function as a QS-autoinducer synthase-receptor pair that drives group behaviors in P. aeruginosa.

Conformational signatures in \u03b2-arrestin2 reveal natural biased agonism at a G-protein-coupled receptor

Discovery of biased ligands and receptor mutants allows characterization of G-protein- and β-arrestin-mediated signaling mechanisms of G-protein-coupled receptors (GPCRs). However, the structural mechanisms underlying biased agonism remain unclear for many GPCRs. We show that while Galanin induces the activation of the galanin receptor 2 (Galr2) that leads to a robust stimulation toward Gαq-protein and β-arrestin1/2, an alternative ligand Spexin and its analog have biased agonism toward G-protein signaling relative to Galanin. We used intramolecular fluorescein arsenical hairpin bioluminescence resonance energy transfer-based biosensors of β-arrestin2 combined with NanoBit technology to measure β-arrestin2–Galr2 interactions in real-time living systems. We found that Spexin and Galanin induce specific active conformations of Galr2, which may lead to different internalization rates of the receptor as well as different signaling outputs. This work represents an additional pharmacological evidence of endogenous G-protein-biased agonism at a GPCR.

Fatty-acylation target sequence in the ligand-binding domain of vertebrate steroid receptors demarcates evolution from estrogen-related receptors

Present-day nuclear receptors (NRs) responding to adrenal and sex steroids are key regulators of reproduction and growth in mammals, and are thought to have evolved from an ancestral NR most closely related to extant estrogen-related receptors (ERRs). The molecular events (and ligands) that distinguish steroid-activated NRs (SRs) from their inferred ancestor, that gave rise to both the ERRs and SRs, remain unknown. We report that target sequences for fatty-acylation (palmitoylation) at a key cysteine residue (corresponding to Cys447 in human estrogen receptor ERα) in helix 8 of the ligand-binding domain accurately demarcate SRs from ERRs. Docking studies are consistent with the hypothesis that palmitate embeds into a key groove in the receptor surface. The implications of lipidation, and of potential alternative ligands for the key cysteine residue, for receptor function and the evolution of SRs are discussed.

Engineering Botulinum Toxins to Improve and Expand Targeting and SNARE Cleavage Activity.

Botulinum neurotoxins (BoNTs) are highly successful protein therapeutics. Over 40 naturally occurring BoNTs have been described thus far and, of those, only 2 are commercially available for clinical use. Different members of the BoNT family present different biological properties but share a similar multi-domain structure at the molecular level. In nature, BoNTs are encoded by DNA in producing clostridial bacteria and, as such, are amenable to recombinant production through insertion of the coding DNA into other bacterial species. This, in turn, creates possibilities for protein engineering. Here, we review the production of BoNTs by the natural host and also recombinant production approaches utilised in the field. Applications of recombinant BoNT-production include the generation of BoNT-derived domain fragments, the creation of novel BoNTs with improved performance and enhanced therapeutic potential, as well as the advancement of BoNT vaccines. In this article, we discuss site directed mutagenesis, used to affect the biological properties of BoNTs, including approaches to alter their binding to neurons and to alter the specificity and kinetics of substrate cleavage. We also discuss the target secretion inhibitor (TSI) platform, in which the neuronal binding domain of BoNTs is substituted with an alternative cellular ligand to re-target the toxins to non-neuronal systems. Understanding and harnessing the potential of the biological diversity of natural BoNTs, together with the ability to engineer novel mutations and further changes to the protein structure, will provide the basis for increasing the scope of future BoNT-based therapeutics.

Abstract 5466: CXCL14 suppresses the aggressive phenotype in triple-negative breast cancer (TNBC) through CXCR4

Abstract The chemokine network is involved in cancer metastasis by modulating cell migration and angiogenesis. CXCL12-CXCR4 is a common signaling axis that is associated with cancer metastasis. Metastatic breast cancers often overexpress the CXCR4 receptor, whereas lung tissues express high levels of CXCL12; consequently, this is a proposed mechanism for breast cancer metastasis to the lung. CXCL14, an anti-inflammatory chemokine, has been postulated to be an alternative ligand for CXCR4, though a formal assignment of CXCR4 as the receptor for CXCL14 has not been made. Interestingly, CXCL14 competes with CXCL12, a main ligand for CXCR4, to modulate CXCL12-CXCR4 downstream activation of ERK. CXCL14 expression is downregulated in oral, colon, prostate, lung, pancreatic, cervical, kidney cancers, and in TNBC when compared to other subtypes of breast cancer. Overexpression of CXCL14 in cancer cell lines in vivo and in vitro leads to decrease in tumor metastasis. CXCL14 overexpression in the MDA-MB-231 breast cancer cell line leads to a decrease in cell growth, invasion and migration. To further explore a role for CXCL14 in breast cancer aggressiveness, we examined the effects of CXCL14 on the cell migration and metastasis of additional breast cancer cell lines. Here we performed cell migration and MTT cytotoxicity assays using MDA-MB-231 and BT-549 TNBC cell lines and the MCF-7 estrogen receptor+ cell line. Our results demonstrate TNBC cells are significantly more sensitive to CXCL14 compared to non-TNBC MCF7 cells (in Boyden chamber migration assay). Also, in our experiments CXCL14 did not alter proliferation in either TNBC cells or non-TNBC cells. However, when we generated CXCR4-overexpressing breast cancer cell lines using parental BT-549, MDA-MB-231, and MCF-7 cells, we observed the changes in the phosphorylation of ERK1/2 (MAPK targets) and pAKT (PI3K downstream effector protein). Based on these data, we propose CXCL14 as a negative modifier of the CXCL12-CXCR4 axis in TNBC. Based on these data, we propose a negative role for CXCL14 in the modulation of TNBC aggressiveness. Citation Format: Carla R. Gibbs, Rosa Mistica C. Ignacio, Ann Richmond, Deok-Soo Son. CXCL14 suppresses the aggressive phenotype in triple-negative breast cancer (TNBC) through CXCR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5466.

IMP3 Stabilization of WNT5B mRNA Facilitates TAZ Activation in Breast Cancer

SUMMARYInsulin-like growth factor-2 mRNA-binding protein 3 (IMP3) is an oncofetal protein associated with many aggressive cancers and implicated in the function of breast cancer stem cells (CSCs). The mechanisms involved, however, are poorly understood. We observed that IMP3 facilitates the activation of TAZ, a transcriptional co-activator of Hippo signaling that is necessary for the function of breast CSCs. The mechanism by which IMP3 activates TAZ involves both mRNA stability and transcriptional regulation. IMP3 stabilizes the mRNA of an alternative WNT ligand (WNT5B) indirectly by repressing miR145-5p, which targets WNT5B, resulting in TAZ activation by alternative WNT signaling. IMP3 also facilitates the transcription of SLUG, which is necessary for TAZ nuclear localization and activation, by a mechanism that is also mediated by WNT5B. These results demonstrate that TAZ can be regulated by an mRNA-binding protein and that this regulation involves the integration of Hippo and alternative WNT-signaling pathways.

Abstract 031: A Ligand-specific Blockade of the Integrin Mac-1 Selectively Targets Pathologic Vascular Inflammation While Maintaining Protective Host-defense

Background: Integrins, such as Mac-1 (alphaMbeta2, CD11b/CD18), drive myeloid cell recruitment and inflammation in cardiovascular disease. Although integrins have generated interest as therapeutic targets in acute and chronic inflammation, they also contribute to host defense, regeneration, and haemostasis. To overcome these limitations, we have designed a novel antibody that targets a distinct region within the Mac-1 major ligand-binding (I-) domain required for binding of its ligand CD40L. Here, we describe the consequences of a ligand-specific inhibition and its efficacy in mice. Methods and Results: To generate an antibody specifically targeting the interaction of Mac-1 with CD40L, mice were immunized with a peptide containing the CD40L-binding motif EQLKKSKTL. Antibody clones were screened and tested for specificity. One clone, termed anti-M7, showed specific peptide binding and blocked the binding of the activated, open conformation of Mac-1 to CD40L, but not to alternative Mac-1 ligands suggesting ligand specificity. Anti-M7 highly effectively limited leukocyte recruitment in vitro and to the mesentery and the omentum in intravital confocal microscopy. While conventional anti-Mac-1 antibodies induced outside-in activation of the integrin, anti-M7 did not provoke MAP-kinase dependent activation and expression of inflammatory cytokines in macrophages. Following cecal-ligation and puncture (CLP) sepsis in mice, a conventional anti-Mac-1 antibody inhibited leukocyte accumulation, but potentiated bacteremia and limited overall survival. In contrast, anti-M7 limited peritoneal leukocyte accumulation, reduced bacterial titers, and improved survival during CLP. Conclusion: We present proof-of-concept demonstrating selective inhibition of the interaction between the leukocyte integrin Mac-1 and CD40L by a novel monoclonal antibody. This antibody shows several advantages over conventional anti-integrin therapy, and prevented inflammation and sepsis-related mortality in mice. This novel approach gives new insight into ligand-specific integrin pathways and merits further evaluation as a selective anti-inflammatory therapy in cardiovascular disease.

Investigation of chelating agents/ligands for Fricke gel dosimeters

The Fricke solution is an absolute dosimeter which can be combined with some metal-binding organic ligands and thus show measured radiation doses through visible light absorption effects. Adding the solution to a gel matrix also allows mapping doses three-dimensionally, and is known as Fricke-gel dosimetry. The latter has some drawbacks that hinder its practical usage for 3D mapping, namely signal diffusion effects and spontaneous color changes due to auto-oxidation. Ligands play a major role in this respect and xylenol orange has been used extensively. Several alternative ligands and their interactions with Fricke solutions and gels were investigated in this study. Among these ligands, calcichrome (CH), eriochrome blue (EB), methylthymol blue (MTB), and naphthol green (NG) showed the required differentiation between optical absorbance of ferrous vs ferric ions. While our study confirmed the overall advantages of using xylenol orange, it also suggests that methylthymol blue offers potential worthy of further investigation.

Medicinal Applications of Gold(I/III)-Based Complexes Bearing N-Heterocyclic Carbene and Phosphine Ligands

Phosphines are the most studied ligands on gold-based metallodrugs, whereas N-heterocyclic carbenes (NHCs) have been emerging in the past years as potential alternative ligands for medicinal applications. Indeed, in both cases, phosphine and NHC allow the preparation of a plethora of transition metal-based complexes with attractive properties for medicinal applications. This review offers an update on phosphine and NHC ligands used in gold complexes with potential anti-cancer, anti-bacterial, anti-parasitic (malaria, leishmaniasis and trypanosomasis) and anti-viral (HIV-1) activity. Structural information about diverse ligands is discussed and the biological activity of the corresponding homo-metallic Au(I), Au(III) and hetero-metallic complexes is summarized in tables and discussed as well. Additionally, these complexes are compared with reference compounds and facts about mechanistic studies and luminescence properties are highlighted. The main target of this review is to find trends in ligand design for medicinal purposes. Particularly, questions about the balance between lipophilicity and hydrophilicity, the presence of functional groups, the utilization of multinuclear complexes and the comparison of neutral versus cationic complexes will be addressed.

An Initiation Kinetics Prediction Model Enables Rational Design of Ruthenium Olefin Metathesis Catalysts Bearing Modified Chelating Benzylidenes.

Rational design of second-generation ruthenium olefin metathesis catalysts with desired initiation rates can be enabled by a computational model that depends on a single thermodynamic parameter. Using a computational model with no assumption about the specific initiation mechanism, the initiation kinetics of a spectrum of second-generation ruthenium olefin metathesis catalysts bearing modified chelating ortho-alkoxy benzylidenes were predicted in this work. Experimental tests of the validity of the computational model were achieved by the synthesis of a series of ruthenium olefin metathesis catalysts and investigation of initiation rates by UV/Vis kinetics, NMR spectroscopy, and structural characterization by X-ray crystallography. Included in this series of catalysts were thirteen catalysts bearing alkoxy groups with varied steric bulk on the chelating benzylidene, ranging from ethoxy to dicyclohexylmethoxy groups. The experimentally observed initiation kinetics of the synthesized catalysts were in good accordance with computational predictions. Notably, the fast initiation rate of the dicyclohexylmethoxy catalyst was successfully predicted by the model, and this complex is believed to be among the fastest initiating Hoveyda-Grubbs-type catalysts reported to date. The compatibility of the predictive model with other catalyst families, including those bearing alternative NHC ligands or disubstituted alkoxy benzylidenes, was also examined.

Effect of double knockout of cartilage-specific mitogen-inducible gene 6 and transforming growth factor-alpha on joint homeostasis

Purpose: Osteoarthritis (OA) is a prevalent joint disease that involves the degeneration of smooth articular cartilage found at interfacing bone surfaces in synovial joints. The mRNA for transforming growth factor-alpha (Tgfa, a potential ligand of epidermal growth factor receptor (EGFR)), has been seen upregulated in a subset of OA cases, both in rat models and human patients (Appleton et al., 2007). Mitogen-inducible gene 6 (Mig-6) is induced by EGFR signaling and acts as a negative feedback regulator by sterically inhibiting additional downstream signaling and targeting EGFR for internalization. In previous studies, mice with a cartilage-specific knockout (KO) of Mig-6 have exhibited anabolic increases in articular cartilage thickness, suggesting that decreased EGFR signaling can mitigate OA effects (Pest et al, 2014). However, development of chondro-osseous nodules (CONs) within the joint capsule in this phenotype restricts movement of the joint, which can be debilitating. We aim to attenuate EGFR signaling by inhibiting Tgfa production in Mig-6-KO mice to further understand the role of this pathway in joint homeostasis. Methods: Male mice displaying cartilage-specific Mig-6 deletion with both homozygous Tgfa wild type (Mig-6fl/flCol2a1-Cre+ Tgfa+/+) and Tgfa-null (Mig-6fl/flCol2a1-Cre+ Tgfa-/-) genotypes, along with male mice displaying control genotypes (Mig-6fl/flCol2a1-Cre- Tgfa+/+ and Mig-6fl/flCol2a1-Cre- Tgfa-/-), were generated and raised to 12 weeks of age. Mice from different litters were age-matched in order to achieve N ≥ 5. Joint structure was then assessed using toluidine blue histological staining. Average articular cartilage thickness and cell density were obtained from sagittal sections of the elbow and knee joint, as well as from frontal sections of the knee. Immunohistochemical analysis was also performed for SOX9, a transcription factor and key marker for both chondrocyte development and phenotype maintenance. Results: Regardless of the absence or presence of TGFa, a significant increase in articular cartilage thickness was found at the surfaces of the humerus, ulna, femur, and tibia in Mig-6-KO models compared to controls. Similarly, cartilage-specific Mig-6-KO, but not whole-body TGFa KO, significantly increased cell density in mice 12 weeks of age. Formation of CONs were found in the knee joints of Mig-6-KO mice regardless of TGFa presence. Unexpectedly, Tgfa-null Mig-6-KO mice appeared to develop more severe CONs that were larger in area with increased proteoglycan staining compared to Mig-6-KO mice with TGFα. Immunochemical staining for SOX9 also appeared more intense in Mig-6-KO mice without TGFα compared to controls. Conclusions: This study indicates that MIG-6 plays an integral role in regulating articular cartilage growth in synovial joints and deletion of Mig-6 results in an anabolic phenotype. However, at the time point examined, TGFα did not have an effect on articular cartilage thickness or cell density. This suggests that the anabolic phenotype in Mig6-deficient mice arises from the role of MIG-6 in other non-EGFR specific mechanisms, or through alternate ligand activation of EGFR.

Influence of OLA and FA ligands on the optical and electronic properties of Cu2ZnSn(S, Se)4 thin films and solar cells prepared from nanoparticle inks

The use of formamide (FA) as an alternative capping ligand to oleylamine (OLA) during the preparation of Cu2ZnSnS4 (CZTS) nanoparticle inks was investigated. Photoluminescence properties of the selenised CZTS absorber layers revealed quasi-donor acceptor pairs (QDAP) as the main transition for carrier recombination with similar electrostatic potential fluctuations in both type of ligands. In OLA layers the transition shifts from QDAP to band-impurity as temperature rises. Solar cell devices fabricated from FA Cu2ZnSn(Se,S)4 were shunted due to the high porosity of the film while in contrast, the combination of FA + OLA absorber yielded reduced back contact barrier height, lower series resistance and higher fill factor and efficiency compared to OLA devices. This is a direct result of the removal of the fine grain carbon rich layer from the back contact interface usually present in films prepared with OLA only.

A ligand-specific blockade of the integrin Mac-1 selectively targets pathologic inflammation while maintaining protective host-defense

Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.

The Stability, Reduction Potential and Ligand State of Two Conformations of a C-Type Cytochrome from the Diatom Thalassiosira Pseudonana

In this study we investigate the structure and function of a c-type cytochrome from the diatom Thalassiosira pseudonana, Tp34211. The axial iron ligand of a c-type cytochrome is often either a methionine or histidine residue. The axial ligand plays an important role in determining the midpoint potential of the heme cofactor, which in turn determines the possible physiological electron donors and acceptors. Initial experiments suggested that recombinantly expressed Tp34211 forms two alternative conformations that can be differentiated by Soret band changes upon imidazole binding or quenching of the tryptophan fluorescence emission signal. Based on sequence alignments of Tp34211 and known cytochrome c6 proteins, we predict that Tp34211 has an axial histidine ligand (H78), but a neighboring lysine residue (K79) could serve as an alternate ligand. To test the possible role of ligand switching in the two alternative conformations observed for Tp34211, we recombinantly expressed and purified both wild-type Tp34211 and a K79A variant that should force bis-His ligation. Following purification by ion-exchange and size-exclusion chromatogrpahy, guanidine induced unfolding assays were performed on the various protein samples, with extent of unfolding measured via intrinsic tryptophan fluorescence. The ΔG°unfolding of the unquenched conformation of WT Tp34211 and the K79A mutant were determined to be the same within error, suggesting that histidine coordination is favored under normal solution conditions. Midpoint potentials of the K79A variant and the two conformations of the wild-type protein will also be measured to further identify the role of histidine versus lysine ligation in Tp34211.

N-Heterocyclic Carbenes as a Robust Platform for Surface-Enhanced Raman Spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) underpins a wide range of commercial and fundamental applications. SERS often relies on ligands, usually thiols, bound to a noble metal surface. The difficulty of straightforward thiol synthesis combined with their instability on surfaces highlights the need for alternative ligand design. We present the first example of SERS utilizing N-heterocyclic carbene ligands. A general three step synthesis is presented for functionalized NHC-CO2 adducts. These ligands are deposited on SERS-active gold film-over-nanosphere substrates (AuFONs) in solvent-free and base-free conditions, which prevents fouling. The resulting films are found to be robust and capable of postsynthetic modifications.

Influence of Smoking on Interleukin-34 Levels in Gingival Crevicular Fluid and Plasma in Periodontal Health and Disease: A Clinico-biochemical Study.

Interleukin-34 (IL-34), an alternative ligand for macrophage colony-stimulating factor receptor, plays an important role in osteoclastogenesis. The aim of this study was to analyze the effect of smoking on IL-34 levels in gingival crevicular fluid (GCF) and plasma in individuals with healthy periodontium and chronic generalized periodontitis (CP). A total of 60 individuals ranging in age from 25 to 55 years were enrolled in the study. The participants were divided into 4 groups: Group A, 30 samples (15 GCF and 15 plasma) obtained from 15 non-smokers with healthy periodontium; Group B, 30 samples (15 GCF and 15 plasma) from 15 smokers with healthy periodontium; Group C, 30 samples (15 GCF and 15 plasma) from 15 non-smokers with CP; and Group D, 30 samples (15 GCF and 15 plasma) from 15 smokers with CP. The Gingival Index and probing depth scores, together with the Clinical Attachment Level, were assessed in each group as clinical periodontal parameters. Levels of IL-34 in GCF and plasma were quantified using enzyme linked immunosorbent assay. The results showed that the mean IL-34 concentrations in GCF and plasma were highest in Group D, followed by Group C, Group B, and Group A, and the difference among them was statistically significant (p<0.05). The relatively elevated IL-34 levels observed here in smokers with CP suggest that this cytokine offers a potential inflammatory marker of periodontal disease in smokers.

Effect of nonsurgical periodontal therapy on interleukin-34 levels in periodontal health and disease.

Interleukin-34 (IL-34) is a recently identified alternative ligand for colony-stimulating factor-1 receptor plays an important role in osteoclastogenesis. The aim of this study was to evaluate the IL-34 levels in gingival crevicular fluid (GCF) and plasma in subjects with chronic periodontitis and to evaluate the effect of nonsurgical periodontal therapy on the GCF and plasma IL-34 levels. Thirty individuals (age range: 30-56 years) were selected and divided into groups based on the gingival index, probing pocket depth, clinical attachment level, and radiologic parameters (bone loss): Group I (15 individuals with healthy periodontium), Group II (15 individuals with chronic generalized periodontitis) while Group II patients after 8 weeks of the treatment (scaling and root planning) constituted Group III. GCF samples and plasma samples were collected to estimate the levels of IL-34 using enzyme-linked immunosorbent assay kit. The mean IL-34 concentration in GCF and plasma was highest for Group II compared to Group I and decreased after nonsurgical periodontal therapy in chronic generalized periodontitis group. The difference between them was statistically significant (P < 0.05). IL-34 can be considered as an "inflammatory marker" of periodontal disease and can be explored in the future as a potential therapeutic target in the treatment of periodontal disease.

Interleukin-34 Levels in Gingival Crevicular Fluid and Plasma in Healthy and Diseased Periodontal Tissue in Presence or Absence of Obesity: A Clinico-biochemical Study.

Interleukin (IL)-34 has recently been identified as an alternative ligand for colonystimulating factor-1 receptor and plays an important role in osteoclastogenesis. The aim of this study was to assess and compare IL-34 levels in gingival crevicular fluid (GCF) and plasma in obese individuals in the presence or absence of periodontal disease and to determine whether they showed a correlation with disease severity. Forty patients aged between 25 and 40 yr were enrolled and categorized into 4 groups: 10 non-obese patients with healthy periodontium (Group I); 10 obese patients with healthy periodontium (Group II); 10 non-obese patients with chronic periodontitis (Group III); and 10 obese patients with chronic periodontitis (Group IV). Demographic variables such as age and body mass index score were recorded and assessed, together with clinical periodontal parameters such as the gingival index, probing pocket depth, and clinical attachment level scores in all groups. The GCF and plasma levels of IL-34 were quantified using enzyme-linked immunosorbent assay. The results showed that the mean IL-34 concentrations in GCF or plasma were highest in Group IV, followed by Group III, Group II, and Group I, with the difference among them being statistically significant (p<0.05). These results suggest that obese individuals with periodontitis have higher GCF and plasma IL-34 levels than non-obese individuals with healthy periodontium. This suggests IL-34 as a potential inflammatory marker of periodontal disease and obesity.