Browsing by Author "Kamocki, Krzysztof"
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Item Bimix antimicrobial scaffolds for regenerative endodontics(Elsevier, 2014-11) Palasuk, Jadesada; Kamocki, Krzysztof; Hippenmeyer, Lauren; Platt, Jeffrey A.; Spolnik, Kenneth J.; Gregory, Richard L.; Bottino, Marco C.; Department of Restorative Dentistry, IU School of DentistryINTRODUCTION: Eliminating and/or inhibiting bacterial growth within the root canal system has been shown to play a key role in the regenerative outcome. The aim of this study was to synthesize and determine in vitro both the antimicrobial effectiveness and cytocompatibility of bimix antibiotic-containing polydioxanone-based polymer scaffolds. METHODS: Antibiotic-containing (metronidazole [MET] and ciprofloxacin [CIP]) polymer solutions (distinct antibiotic weight ratios) were spun into fibers as a potential mimic to the double antibiotic paste (DAP, a MET/CIP mixture). Fiber morphology, chemical characteristics, and tensile strength were evaluated by scanning electron microscopy, Fourier transform infrared spectroscopy, and tensile testing, respectively. Antimicrobial efficacy was tested over time (aliquot collection) against Enterococcus faecalis (Ef), Porphyromonas gingivalis (Pg), and Fusobacterium nucleatum (Fn). Similarly, cytotoxicity was evaluated in human dental pulp stem cells. Data were statistically analyzed (P < .05). RESULTS: Scanning electron microscopy and Fourier transform infrared spectroscopy confirmed that electrospinning was able to produce antibiotic-containing fibers with a diameter mostly in the nanoscale. The tensile strength of 1:1MET/CIP scaffolds was significantly (P < .05) higher than pure polydioxanone (control). Meanwhile, all other groups presented similar strength as the control. Aliquots obtained from antibiotic-containing scaffolds inhibited the growth of Ef, Pg, and Fn, except pure MET, which did not show an inhibitory action toward Pg or Fn. Antibiotic-containing aliquots promoted slight human dental pulp stem cell viability reduction, but none of them were considered to be cytotoxic. CONCLUSIONS: Our data suggest that the incorporation of multiple antibiotics within a nanofibrous scaffold holds great potential toward the development of a drug delivery system for regenerative endodontics.Item Biodegradable nanofibrous drug delivery systems: effects of metronidazole and ciprofloxacin on periodontopathogens and commensal oral bacteria(Springer-Verlag, 2014-12) Bottino, Marco C.; Arthur, Rodrigo A.; Waeiss, R. Aaron; Kamocki, Krzysztof; Gregson, Karen S.; Gregory, Richard L.; Department of Restorative Dentistry, IU School of DentistryOBJECTIVES: The purposes of this study were to fabricate biodegradable polydioxanone (PDS II®) electrospun periodontal drug delivery systems (hereafter referred to as matrices) containing either metronidazole (MET) or ciprofloxacin (CIP) and to investigate the effects of antibiotic incorporation on both periodontopathogens and commensal oral bacteria. MATERIALS AND METHODS: Fibrous matrices were processed from PDS polymer solution by electrospinning. Antibiotic-containing PDS solutions were prepared to obtain four distinct groups: 5 wt.% MET, 25 wt.% MET, 5 wt.% CIP, and 25 wt.% CIP. Pure PDS was used as a control. High-performance liquid chromatography (HPLC) was done to evaluate MET and CIP release. Dual-species biofilms formed by Lactobacillus casei (Lc) and Streptococcus salivarius (Ss) were grown on the surface of all electrospun matrices. After 4 days of biofilm growth, the viability of bacteria on biofilms was assessed. Additionally, antimicrobial properties were evaluated against periodontopathogens Fusobacterium nucleatum (Fn) and Aggregatibacter actinomycetemcomitans (Aa) using agar diffusion assay. RESULTS: A three-dimensional interconnected porous network was observed in the different fabricated matrices. Pure PDS showed the highest fiber diameter mean (1,158 ± 402 nm) followed in a descending order by groups 5 wt.% MET (1,108 ± 383 nm), 25 wt.% MET (944 ± 392 nm), 5 wt.% CIP (871 ± 309 nm), and 25 wt.% CIP (765 ± 288 nm). HPLC demonstrated that groups containing higher amounts (25 wt.%) of incorporated drugs released more over time, while those with lower levels (5 wt.%) the least. No inhibitory effect of the tested antibiotics was detected on biofilm formation by the tested commensal oral bacteria. Meanwhile, CIP-containing matrices inhibited growth of Fn and Aa. CONCLUSION: CIP-containing matrices led to a significant inhibition of periodontopathogens without negatively impairing the growth of periodontal beneficial bacteria. CLINICAL RELEVANCE: Based on the proven in vitro inhibition of periodontitis-related bacteria, future in vivo research using relevant animal models is needed to confirm the effectiveness of these drug delivery systems.Item c-FLIP, a Novel Biomarker for Cancer Prognosis, Immunosuppression, Alzheimer’s Disease, Chronic Obstructive Pulmonary Disease (COPD), and a Rationale Therapeutic Target(Taylor & Francis, 2019) Safa, Ahmad R.; Kamocki, Krzysztof; Saadatzadeh, M. Reza; Bijangi-Vishehsaraei, Khadijeh; Pharmacology and Toxicology, School of MedicineDysregulation of c-FLIP (cellular FADD-like IL-1β-converting enzyme inhibitory protein) has been shown in several diseases including cancer, Alzheimer’s disease, and chronic obstructive pulmonary disease (COPD). c-FLIP is a critical anti-cell death protein often overexpressed in tumors and hematological malignancies and its increased expression is often associated with a poor prognosis. c-FLIP frequently exists as long (c-FLIPL) and short (c-FLIPS) isoforms, regulates its anti-cell death functions through binding to FADD (FAS associated death domain protein), an adaptor protein known to activate caspases-8 and -10 and links c-FLIP to several cell death regulating complexes including the death-inducing signaling complex (DISC) formed by various death receptors. c-FLIP also plays a critical role in necroptosis and autophagy. Furthermore, c-FLIP is able to activate several pathways involved in cytoprotection, proliferation, and survival of cancer cells through various critical signaling proteins. Additionally, c-FLIP can inhibit cell death induced by several chemotherapeutics, anti-cancer small molecule inhibitors, and ionizing radiation. Moreover, c-FLIP plays major roles in aiding the survival of immunosuppressive tumor-promoting immune cells and functions in inflammation, Alzheimer’s disease (AD), and chronic obstructive pulmonary disease (COPD). Therefore, c-FLIP can serve as a versatile biomarker for cancer prognosis, a diagnostic marker for several diseases, and an effective therapeutic target. In this article, we review the functions of c-FLIP as an anti-apoptotic protein and negative prognostic factor in human cancers, and its roles in resistance to anticancer drugs, necroptosis and autophagy, immunosuppression, Alzheimer’s disease, and COPD.Item Development and characterization of novel ZnO-loaded electrospun membranes for periodontal regeneration(Elsevier, 2015-09) Münchow, Eliseu A.; Albuquerque, Maria Tereza P.; Zero, Bianca; Kamocki, Krzysztof; Piva, Evandro; Gregory, Richard L.; Bottino, Marco C.; Department of Restorative Dentistry, IU School of DentistryOBJECTIVES: This study reports on the synthesis, materials characterization, antimicrobial capacity, and cytocompatibility of novel ZnO-loaded membranes for guided tissue/bone regeneration (GTR/GBR). METHODS: Poly(ɛ-caprolactone) (PCL) and PCL/gelatin (PCL/GEL) were dissolved in hexafluoropropanol and loaded with ZnO at distinct concentrations: 0 (control), 5, 15, and 30wt.%. Electrospinning was performed using optimized parameters and the fibers were characterized via scanning and transmission electron microscopies (SEM/TEM), energy dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), contact angle (CA), mechanical testing, antimicrobial activity against periodontopathogens, and cytotoxicity test using human dental pulp stem cells (hDPSCs). Data were analyzed using ANOVA and Tukey (α=5%). RESULTS: ZnO nanoparticles were successfully incorporated into the overall submicron fibers, which showed fairly good morphology and microstructure. Upon ZnO nanoparticles' incorporation, the PCL and PCL/GEL fibers became thicker and thinner, respectively. All GEL-containing membranes showed lower CA than the PCL-based membranes, which were highly hydrophobic. Overall, the mechanical properties of the membranes were reduced upon ZnO incorporation, except for PCL-based membranes containing ZnO at the 30wt.% concentration. The presence of GEL enhanced the stretching ability of membranes under wet conditions. All ZnO-containing membranes displayed antibacterial activity against the bacteria tested, which was generally more pronounced with increased ZnO content. All membranes synthesized in this study demonstrated satisfactory cytocompatibility, although the presence of 30wt.% ZnO led to decreased viability. SIGNIFICANCE: Collectively, this study suggests that PCL- and PCL/GEL-based membranes containing a low content of ZnO nanoparticles can potentially function as a biologically safe antimicrobial GTR/GBR membrane.Item The development and maintenance of paclitaxel-induced neuropathic pain require activation of the sphingosine 1-phosphate receptor subtype 1(ASBMB, 2014-07-25) Janes, Kali; Little, Joshua W.; Li, Chao; Bryant, Leesa; Chen, Collin; Chen, Zhoumou; Kamocki, Krzysztof; Doyle, Timothy; Snider, Ashley; Esposito, Emanuela; Cuzzocrea, Salvatore; Bieberich, Erhard; Obedi, Lina; Petrache, Irina; Nicol, Grant; Neumann, William L.; Salvemini, Daniela; Department of Pharmacology and Toxicology, IU School of MedicineThe ceramide-sphingosine 1-phosphate (S1P) rheostat is important in regulating cell fate. Several chemotherapeutic agents, including paclitaxel (Taxol), involve pro-apoptotic ceramide in their anticancer effects. The ceramide-to-S1P pathway is also implicated in the development of pain, raising the intriguing possibility that these sphingolipids may contribute to chemotherapy- induced painful peripheral neuropathy, which can be a critical dose-limiting side effect of many widely used chemotherapeutic agents.We demonstrate that the development of paclitaxel-induced neuropathic pain was associated with ceramide and S1P formation in the spinal dorsal horn that corresponded with the engagement of S1P receptor subtype 1 (S1PR(1))- dependent neuroinflammatory processes as follows: activation of redox-sensitive transcription factors (NFκB) and MAPKs (ERK and p38) as well as enhanced formation of pro-inflammatory and neuroexcitatory cytokines (TNF-α and IL-1β). Intrathecal delivery of the S1PR1 antagonist W146 reduced these neuroinflammatory processes but increased IL-10 and IL-4, potent anti-inflammatory/ neuroprotective cytokines. Additionally, spinal W146 reversed established neuropathic pain. Noteworthy, systemic administration of the S1PR1 modulator FTY720 (Food and Drug Administration- approved for multiple sclerosis) attenuated the activation of these neuroinflammatory processes and abrogated neuropathic pain without altering anticancer properties of paclitaxel and with beneficial effects extended to oxaliplatin. Similar effects were observed with other structurally and chemically unrelated S1PR1 modulators (ponesimod and CYM-5442) and S1PR1 antagonists (NIBR-14/15) but not S1PR1 agonists (SEW2871). Our findings identify for the first time the S1P/S1PR1 axis as a promising molecular and therapeutic target in chemotherapy-induced painful peripheral neuropathy, establish a mechanistic insight into the biomolecular signaling pathways, and provide the rationale for the clinical evaluation of FTY720 in chronic pain patients.Item Dimensionally stable and bioactive membrane for guided bone regeneration: An in vitro study(Wiley Blackwell (John Wiley & Sons), 2016-04) Rowe, Matthew J.; Kamocki, Krzysztof; Pankajakshan, Divya; Li, Ding; Bruzzaniti, Angela; Thomas, Vinoy; Blanchard, Steve B.; Bottino, Marco C.; Department of Biomedical and Applied Sciences, School of DentistryComposite fibrous electrospun membranes based on poly(dl-lactide) (PLA) and poly(ε-caprolactone) (PCL) were engineered to include borate bioactive glass (BBG) for the potential purposes of guided bone regeneration (GBR). The fibers were characterized using scanning and transmission electron microscopies, which respectively confirmed the submicron fibrous arrangement of the membranes and the successful incorporation of BBG particles. Selected mechanical properties of the membranes were evaluated using the suture pullout test. The addition of BBG at 10 wt % led to similar stiffness, but more importantly, it led to a significantly stronger (2.37 ± 0.51 N mm) membrane when compared with the commercially available Epiguide® (1.06 ± 0.24 N mm) under hydrated conditions. Stability (shrinkage) was determined after incubation in a phosphate buffer solution from 24 h up to 9 days. The dimensional stability of the PLA:PCL-based membranes with or without BBG incorporation (10.07-16.08%) was similar to that of Epiguide (14.28%). Cell proliferation assays demonstrated a higher rate of preosteoblasts proliferation on BBG-containing membranes (6.4-fold) over BBG-free membranes (4- to 5.8-fold) and EpiGuide (4.5-fold), following 7 days of in vitro culture. Collectively, our results demonstrated the ability to synthesize, via electrospinning, stable, polymer-based submicron fibrous BBG-containing membranes capable of sustaining osteoblastic attachment and proliferation-a promising attribute in GBR.Item Effects of ciprofloxacin-containing antimicrobial scaffolds on dental pulp stem cell viability-In vitro studies(Elsevier, 2015-08) Kamocki, Krzysztof; Nör, Jacques E.; Bottino, Marco C.; Department of Restorative Dentistry, School of DentistryOBJECTIVE: A combination of antibiotics, including but not limited to metronidazole (MET) and ciprofloxacin (CIP), has been indicated to eradicate bacteria in necrotic immature permanent teeth prior to regenerative procedures. It has been shown clinically that antibiotic pastes may lead to substantial stem cell death. The aim of this study was to synthesise scaffolds containing various concentrations of CIP to enhance cell viability while preserving antimicrobial properties. DESIGN: Polydioxanone (PDS)-based electrospun scaffolds were processed with decreasing CIP concentrations (25-1 wt.%) and morphologically evaluated using scanning electron microscopy (SEM). Cytotoxicity assays were performed to determine whether the amount of CIP released from the scaffolds would lead to human dental pulp stem cell (hDPSC) toxicity. Similarly, WST-1 assays were performed to evaluate the impact of CIP release on hDPSC proliferation. Pure PDS scaffolds and saturated double antibiotic solution MET/CIP (DAP) served as both positive and negative controls, respectively. Antibacterial efficacy against E. faecalis (Ef) was tested. RESULTS: A significant decrease in hDPSC' viability at concentrations 5-25 wt.% was observed. However, concentrations below 5wt.% did not impair cell viability. Data from the WST-1 assays indicated no detrimental impact on cell proliferation for scaffolds containing 2.5 wt.% CIP or less. Significant antimicrobial properties were seen for CIP-scaffolds at lower concentrations (i.e., 1 and 2.5 wt.%). CONCLUSION: The obtained data demonstrated that a reduced concentration of CIP incorporated into PDS-based scaffolds maintains its antimicrobial properties while enhancing viability and proliferation of dental pulp stem cells.Item The role of ceramides in cigarette smoke-induced alveolar cell death(2012-11) Kamocki, Krzysztof; Petrache, Irina; Gunst, Susan J.; Quilliam, Laurence; Atkinson, SimonThe complex pathogenesis of emphysema involves disappearance of alveolar structures, in part attributed to alveolar cell apoptosis. The mechanism by which cigarette smoke (CS) induces alveolar cell apoptosis is not known. We hypothesized that ceramides are induced by CS via specific enzymatic pathways that can be manipulated to reduce lung cell apoptosis. CS increased ceramides in the whole lung and in cultured primary structural lung cells. Exposure to CS activated within minutes the acid sphingomyelinase, and within weeks the de novo- ceramide synthesis pathways. Pharmacological inhibition of acid sphingomyelinase significantly attenuated CS-induced apoptosis. To understand the mechanisms by which ceramides induce apoptosis, we investigated the cell types affected and the involvement of RTP801, a CS-induced pro-apoptotic and pro-inflammatory protein. Direct lung augmentation of ceramide caused apoptosis of both endothelial and epithelial type II cells. Ceramide upregulated RTP801 and the transgenic loss of RTP801 inhibited only epithelial, but not endothelial cell apoptosis induced by ceramide. In conclusion, CS induces acid sphingomyelinase-mediated ceramide upregulation and apoptosis in a cell-specific manner, which in epithelial cells involves induction of stress response proteins that may further amplify lung injury. Molecular targeting of amplification pathways may provide therapeutic opportunities to halt emphysema progression.Item Stimulation of sphingosine 1-phosphate signaling as an alveolar cell survival strategy in emphysema(American Thoracic Society, 2010-02-15) Diab, Khalil J.; Adamowicz, Jeremy J.; Kamocki, Krzysztof; Rush, Natalia I.; Garrison, Jana; Gu, Yuan; Schweitzer, Kelly S.; Skobeleva, Anastasia; Rajashekhar, Gangaraju; Hubbard, Walter C.; Berdyshev, Evgeny V.; Petrache, Irina; Medicine, School of MedicineRATIONALE: Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. OBJECTIVES: The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. METHODS: Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. MEASUREMENTS AND MAIN RESULTS: Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. CONCLUSIONS: Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development.Item Structural and functional characterization of endothelial microparticles released by cigarette smoke(SpringerNature, 2016-08-17) Serban, Karina A.; Rezania, Samin; Petrusca, Daniela N.; Poirier, Christophe; Cao, Danting; Justice, Matthew J.; Patel, Milan; Tsvetkova, Irina; Kamocki, Krzysztof; Mikosz, Andrew; Schweitzer, Kelly S.; Jacobson, Sean; Cardoso, Angelo; Carlesso, Nadia; Hubbard, Walter C.; Kechris, Katerina; Dragnea, Bogdan; Berdyshev, Evgeny V.; McClintock, Jeanette; Petrache, Irina; Department of Biochemistry & Molecular Biology, IU School of MedicineCirculating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.