James D. Lillich

Expression of pleiotrophin, an important regulator of cell migration, is inhibited in intestinal epithelial cells by treatment with non-steroidal anti-inflammatory drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs for the suppression of inflammation and pain. However, the analgesic properties of NSAIDs are also associated with significant negative side effects, most notably in the gastrointestinal (GI) tract. Increasingly, evidence indicates that the ulcerogenic properties of some NSAIDs are not exclusively the result of inhibition of cyclooxygenase isoforms in the GI tract, and other mechanisms, including inhibition of cell migration and epithelial restitution, are being explored. Recently, microarray analysis was used to identify potential novel targets of NSAID activity in intestinal epithelial cells. Treated cells exhibited significant reductions in the gene expression of pleiotrophin (PTN), a cytokine and growth factor known to participate in angiogenesis and bone growth. This report aimed to confirm the microarray results reported previously, and to measure protein expression of PTN in intestinal epithelial cells. Furthermore, we also examined the effects of exogenous PTN on cell migration in the presence and absence of either NSAIDs with variable ulcerogenic potential or PTN-specific siRNA. Our results demonstrated that indomethacin and NS-398, two NSAIDs with ulcerogenic potential significantly decrease both gene and protein expressions of PTN in IEC-6 cells and protein expression in IEC-6-Cdx2 cells. Additionally, cell migration experiments with PTN siRNA showed that PTN is an important mediator of IEC-6 cell migration, and addition of exogenous PTN partially restores the deficits in cell migration caused by treatment with indomethacin and NS-398. Finally, measurement of PTN protein expression in the GI tract of horses treated with phenylbutazone showed that PTN expression is reduced by NSAIDs in vivo. Our results show that PTN is an important mediator of cell migration in IEC-6 cells, and PTN is a potential target through which NSAIDs may inhibit cell migration, epithelial restitution, and wound healing in the GI tract.

Complications of dental surgery.

Both retrospective data and clinical experience indicate that complications of dental surgery are occasionally encountered and, to some extent, are inevitable. Many of the reported complications related to dental surgery such as incomplete removal of diseased teeth or removal of the wrong tooth can be avoided with sound preoperative planning and intraoperative technique. Diseased teeth should be properly identified prior to and during surgery. In addition, complete removal of the diseased tooth must be performed. Use of intraoperative radiographic examination to confirm the location of the diseased tooth and to document its removal cannot be overemphasized. Iatrogenic fracture of the maxillary or mandibular alveolar walls or palatine bone can be avoided by proper placement of the dental punch. The chances of developing incisional drainage or secondary sinusitis can be reduced by use of appropriate systemic antibiotics. These factors should guide the surgical approach to dental surgery to reduce the likelihood of developing common complications.

Drug-Induced Alterations to Gene and Protein Expression in Intestinal Epithelial Cell 6 Cells Suggest a Role for Calpains in the Gastrointestinal Toxicity of Nonsteroidal Anti-Inflammatory Agents

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used extensively as therapeutic agents, despite their well documented gastrointestinal (GI) toxicity. At this time, the mechanisms responsible for NSAID-associated GI damage are incompletely understood. In this study, we used microarray analysis to generate a novel hypothesis about cellular mechanisms that underlie the GI toxicity of NSAIDs. Monolayers of intestinal epithelial cells (IEC-6) were treated with NSAIDs that either exhibit (indomethacin, NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide]) or lack (SC-560 [5-(4-chlorphenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole]) inhibitory effects on IEC-6 migration. Bioinformatic analysis of array data identified the calpain cysteine proteases and their endogenous inhibitor calpastatin as potential targets of NSAIDs shown previously to retard IEC-6 migration. Accordingly, quantitative real-time reverse transcription polymerase chain reaction and immunoblotting were performed to assess the effects of NSAIDs on the expression of mRNA and protein for calpain 8, calpain 2, calpain 1, and calpastatin. In treated IEC-6 monolayers, NS-398 decreased the expression of mRNA for calpain 2 and calpain 8. Both NS-398 and indomethacin decreased the protein expression of calpains 8, 2, and 1. None of the NSAIDs affected expression of calpastatin mRNA or protein. The calpain inhibitors, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-Nle-CHO, retarded IEC-6 cell migration in a concentration-dependant fashion, and these inhibitory effects were additive with those of indomethacin and NS-398. Our experimental results suggest that the altered expression of calpain proteins may contribute to the adverse effects of NSAIDs on intestinal epithelial restitution.

Non-steroidal Anti-inflammatory Drugs Inhibit Calpain Activity and Membrane Localization of Calpain 2 Protease

Non-steroidal anti-inflammatory drugs (NSAIDs) are used frequently worldwide for the alleviation of pain despite their capacity to cause adverse gastrointestinal (GI) side effects. GI toxicity, once thought to be the result of non-specific inhibition of cyclooxegenase (COX) enzymes, is now hypothesized to have multiple other causes that are COX independent. In particular, NSAIDs inhibit intestinal epithelial restitution, the process by which barrier function in intestinal mucosa is restored at sites of epithelial wounds within hours through cell spreading and migration. Accordingly, recent evidence indicates that the expression of calpain proteases, which play a key role in cell migration, is decreased by NSAIDs that inhibit cell migration in intestinal epithelial cells (IEC). Here, we examine the effect of NSAIDs on calpain activity and membrane expression in IEC-6 cells. Indomethacin, NS-398, and SC-560 inhibited calpain activity and decreased expression of calpain 2 in total membrane fractions and in plasma membranes involved in cell attachment to the substrate. Additionally, we demonstrated that inhibition of calpain activity by NSAIDs or ALLM, a calpain inhibitor, limits cell migration and in vitro wound healing of IEC-6 cells. Our results indicate that NSAIDs may inhibit cell migration by decreasing calpain activity and membrane-associated expression of calpain 2. Our results provide valuable insight into the mechanisms behind NSAID-induced GI toxicity and provide a potential pathway through which these negative side effects can be avoided in future members of the NSAID class.

Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

In vivo assessment of a multicomponent and nanostructural polymeric matrix as a delivery system for antimicrobials and bone morphogenetic protein-2 in a unicortical tibial defect in goats

OBJECTIVE To determine the response of cortical bone to a multicomponent and nanostructural polymeric matrix as a drug delivery system for enhancing bone healing. ANIMALS 20 healthy adult crossbred goats. PROCEDURES A 3.5-mm-diameter unicortical defect was created in each tibia (day 0), and goats (4 goats/group) were treated as follows: not treated (control group), grafted with the matrix, grafted with antimicrobial (tigecycline and tobramycin)-impregnated matrix, grafted with recombinant human bone morphogenetic protein type 2 (rhBMP-2)-impregnated matrix, or grafted with antimicrobial- and rhBMP-2-impregnated matrix. Elution kinetics of antimicrobials was monitored through plasma concentrations. Bone response was assessed with radiographic scoring (days 1 and 30) and dual-energy x-ray absorptiometry (days 1, 14, and 30). Goats were euthanized on day 30, and histomorphologic analysis was performed. Categorical variables were analyzed with a generalized linear model, and continuous variables were analyzed with an ANOVA. RESULTS Plasma antimicrobial concentrations indicated continued release throughout the study. Radiography and dual-energy x-ray absorptiometry did not reveal significant differences among treatments on day 30. Periosteal reactions were significantly greater surrounding bone defects grafted with rhBMP-2-impregnated matrix than those not treated or grafted with matrix or with antimicrobial-impregnated matrix; periosteal reactions were similar in bone defects grafted with rhBMP-2-impregnated matrix and antimicrobial- and rhBMP-2-impregnated matrix. CONCLUSIONS AND CLINICAL RELEVANCE The matrix served as an antimicrobial delivery system and stimulated bone proliferation when rhBMP-2 was present. Antimicrobial and rhBMP-2 can be used concurrently, but the presence of antimicrobials may affect the performance of rhBMP-2.

Advanced Diagnostic Imaging Modalities Available at the Referral Center

While many equine diagnostic imaging procedures can be done in field, some procedures require specialized facilities, equipment or expertise which are generally only available in referral or specialty practices. As client awareness of the availability and advantages of these diagnostic procedures increases, veterinarians are faced with the increasing opportunity to utilize these services to provide optimal patient care. A working knowledge of the value and limitations of these methods is required to help guide veterinarians and clients in the selection of additional, and sometimes costly, diagnostic tests.

Diagnostic Approach to Exercise Intolerance in Racehorses

The diagnostic approach to the exercise intolerant racehorse depends on sound, fundamental steps to allow complete examination of the entire horse in a logical, time-efficient fashion. Availability of referral diagnostic modalities should not overshadow the need for collecting a clinical history or performing a complete physical examination. The diagnostic approach to exercise intolerance can be divided into a few basic steps. The first step, conducted without the horse, is a thorough discussion about the horse with the trainer and/or owner. Many clinical histories are complex and therefore following a predetermined pattern is helpful. Racing ability, general health history, historic problems with the major body systems including the musculoskeletal, respiratory, cardiovascular, and neurologic systems should be reviewed. The next step of the examination is conducted in the stall. A complete physical examination is required. The practitioner should pay close attention to the body systems that most frequently harbor causes of exercise intolerance. Before evaluating the horse for lameness, endoscopy of the upper airway should be performed. The final step is conducted outside the stall: completion of the lameness and neurologic examination or an exercise trial followed by further diagnostic tests such as a serum chemistry profile, bronchoalveolar lavage, or imaging studies. Decision for referral is best made after the physical examination is completed and capability for further diagnostics is exhausted.

Isolation and Culture of Juvenile Pig Thyroid Follicular Epithelia

Epithelial tissues are defined by their polarity and their ability to transport directionally. Thyroid is a tissue comprising functional epithelial units organized as enclosed follicles, with their luminal spaces defined by thyrocyte apices. Thus, the native arrangement of thyroid epithelia limits accessibility to the follicular space, presenting a challenge in studying transepithelial movements. This limitation can be overcome by studying thyrocytes grown as two-dimensional cultures. Herein we present methods for isolation of thyroid follicles from juvenile pigs and preparation of high-resistance, polarized cultures.

Modeling gold nanoparticle biodistribution after arterial infusion into perfused tissue: effects of surface coating, size and protein corona

Abstract A detailed understanding of the factors governing nanomaterial biodistribution is needed to rationally design safe nanomedicines. This research details the pharmacokinetics of gold nanoparticle (AuNP) biodistribution after arterial infusion of 40 or 80 nm AuNP (1 μg/ml) into the isolated perfused porcine skin flap (IPPSF). AuNP had surface coatings consisting of neutral polyethylene glycol (PEG), anionic lipoic acid (LA), or cationic branched polyethylenimine (BPEI). Effect of a porcine plasma corona (PPC) on 40 nm BPEI and PEG-AuNP were assessed in the IPPSF. Au concentrations were determined by ICP/MS and arterial to venous concentration-time profiles were analyzed over 8 hr (4 hr infusion, 4 hr washout) using a two-compartment pharmacokinetic model. IPPSF viability and vascular function were assessed by change in glucose utilization, vascular resistance, or weight gain after perfusion. All AuNP demonstrated some degree of AuNP arterial extraction and skin flap retention, as well as enhanced kinetic parameters of tissue uptake; with BPEI-AuNP consistently having the greatest biodistribution even with a PPC. Toxicological effects were not detected. Transmission electron microscopy confirmed intracellular uptake of AuNP. These studies paralleled previous in vitro cell culture studies using the same AuNP in human endothelial and renal proximal tubule cells, hepatocytes, keratinocytes, showing BPEI-AuNP having the greatest uptake, although the presence of a PPC did not reduce IPPSF biodistribution as in the cell culture studies. These findings clearly indicate arterial to the venous extraction of AuNP after infusion with the magnitude of extraction being greatest with the BPEI surface coating and provide data and model structure necessary to construct the whole body physiologically based pharmacokinetic models capable of utilizing available in vitro data.