Modern Intermittent Haemodialysis (IHD) is an Effective Method of Removing Salicylate in Chronic Topical Salicylate Toxicity

Introduction

There are limited data on modern intermittent hemodialysis (IHD) efficacy on salicylate elimination from topical poisoning.

Case report

A 54-year-old male sought treatment for dyspnea but was then diagnosed with salicylate toxicity from topical application of Dencorub Extra Strength Heat Gel® for 1 week. Each tube contained 100 g with 26 % methylsalicylate (26 g). Laboratory workup was remarkable for an elevated anion gap of 30 and salicylate concentration of 78.7 mg/dL [5.7 mmol/L (N?<?0.4 mmol/L)]. Treatment with urinary alkalinization was followed by hemodialysis for 5 h. Extraction ratios were 0.44 with clearance rates of 78.5 mL/min. Salicylate concentrations fell rapidly following initiation of hemodialysis with no rebound observed.

Discussion

Modern high flux IHD is an effective method of removing salicylates in the treatment of chronic topical poisoning.

     

Antinociceptive interaction of gabapentin with minocycline in murine diabetic neuropathy

Objective

Diabetic neuropathy (DN) is the most common complication of diabetes and pain is one of the main symptoms of diabetic neuropathy, however, currently available drugs are often ineffective and complicated by adverse events. The purpose of this research was to evaluate the antinociceptive interaction between gabapentin and minocycline in a mice experimental model of DN by streptozocin (STZ).

Methods

The interaction of gabapentin with minocycline was evaluated by the writhing and hot plate tests at 3 and 7 days after STZ injection or vehicle in male CF1 mice.

Results

STZ (150 mg/kg, i.p.) produced a marked increase in plasma glucose levels on day 7 (397.46 ± 29.65 mg/dL) than on day 3 (341.12 ± 35.50 mg/dL) and also developed neuropathic pain measured by algesiometric assays. Gabapentin produced similar antinociceptive activity in both writhing and hot plate tests in mice pretreated with STZ. However, minocycline was more potent in the writhing than in the hot plate test in the same type of mice. The combination of gabapentin with minocycline produced synergistic interaction in both test.

Conclusion

The combination of gabapentin with minocycline in a 1:1 proportion fulfills all the criteria of multimodal analgesia and this finding suggests that the combination provide a therapeutic alternative that could be used for human neuropathic pain management.

     

Survival After Severe Rhabdomyolysis Following Monensin Ingestion

Introduction

Monensin is a veterinary antibiotic with a narrow therapeutic window that has led to lethal intoxication in many animal species. Only two prior cases of human toxicity have been reported, both fatal. We present the first case of survival from severe toxicity following monensin ingestion.

Case

A 58-year-old man presented with 8 days of vomiting and abdominal pain. Due to delusions of central nervous system toxoplasmosis, he ingested 300 mg of monensin. His laboratory studies revealed severe rhabdomyolysis without renal dysfunction. Total creatine kinase (CK) peaked above 100,000 U/L. His CK decreased to 5192 U/L after 15 days of aggressive hydration and sodium bicarbonate therapy. His ejection fraction on echocardiogram decreased from 69 to 56%.

Discussion

Reports on acute clinical effects after human exposure to monensin are limited. Ingestion is known to cause skeletal and cardiac muscle rhabdomyolysis and necrosis. Animal studies demonstrate that monensin’s toxicity is due to increases in intracellular sodium concentrations and Ca²⁺ release. To date, no effective antidotal treatment has been described.

Conclusions

Monensin is a veterinary medication not approved for human use by the US Food and Drug Administration. Though poorly studied in humans, this case demonstrates the severe harm that may occur following ingestion.

     

Adjunctive Use of Ketamine for Benzodiazepine-Resistant Severe Alcohol Withdrawal: a Retrospective Evaluation

Introduction

Benzodiazepine (BZD)-resistant alcohol withdrawal remains a challenge for most institutions due to limited evidence with available agents. One published study currently exists utilizing the N-methyl-d-aspartate antagonist, ketamine, for alcohol withdrawal.

Objective

The purpose of our study was to evaluate the effect of adjunctive ketamine continuous infusion on symptom control and lorazepam infusion requirements for BZD-resistant alcohol withdrawal patients in the intensive care unit.

Methods

A retrospective review was conducted of patients receiving ketamine adjunctively with a lorazepam infusion for severe alcohol withdrawal between August 2012 and August 2014. Outcomes included time to symptom control, lorazepam infusion requirements, ketamine initial and maximum daily infusion rates, and adverse effects of ketamine.

Results

Thirty patients were included in the analysis. Mean time to initiation of ketamine after the initiation of a lorazepam infusion was 41.4 h. All patients achieved initial symptom control within 1 h of ketamine initiation. Median initial ketamine infusion rate was 0.75 mg/kg/h and the average maximum daily rate was 1.6 mg/kg/h. Significant decreases in lorazepam infusion rates from baseline were observed at 24 h (??4 mg/h; p?=?0.01) after ketamine initiation. No patients experienced documented CNS adverse effects. Two patients experienced hypertension and no patients experienced tachycardia related to ketamine.

Conclusion

Adjunctive ketamine could provide symptom control for BZD-refractory patients and may potentially reduce lorazepam infusion requirements. Future studies to determine optimal dosing, timing of initiation, and place in therapy for BZD-resistant alcohol withdrawal are needed. The mechanism of action via the NMDA receptor with ketamine may provide benefit for BZD-resistant alcohol withdrawal.

     

Estimating Maximal <Emphasis Type="BoldItalic">In Vitro</Emphasis> Skin Permeation Flux from Studies Using Non-sink Receptor Phase Conditions

Purpose

This study explored the impact of non-sink receptor conditions on the in vitro skin permeation test (IVPT) and sought to estimate equivalent sink condition IVPT data.

Methods

Simulated diffusion model and experimental IVPT data were generated for ethyl salicylate across human epidermal membranes in Franz diffusion cells using six different receptor phases, with a 10 fold variation in ethyl salicylate solubility.

Results

Both simulated and experimental IVPT – time profiles were markedly affected by receptor phase solubility and receptor sampling rates. Similar sink condition equivalent estimated maximum fluxes were obtained by nonlinear regression and adjustment of linear regression estimates of steady state flux for relative saturation of the receptor phase over time for the four receptor phases in which the ethyl salicylate was relatively soluble. The markedly lower steady - state fluxes found for the other two phases in which ethyl salicylate was less soluble was attributed to an aqueous solution boundary layer effect.

Conclusions

Non-sink receptor phase IVPT data can be used to derive equivalent sink receptor phase IVPT data provided the receptor phase solubility and hydrodynamics are sufficient to minimise the impact of aqueous diffusion layers on IVPT data.

     

Antioxidant effects of curcuminoids in patients with type 2 diabetes mellitus: a randomized controlled trial

Background

Oxidative stress has a key role in the pathogenesis of type II diabetes mellitus (T2DM) and its vascular complications. Antioxidant therapy has been suggested as a potential approach to blunt T2DM development and progression. The aim of this study was to assess the effects of supplementation with curcuminoids, which are natural polyphenolics from turmeric, on oxidative indices in diabetic individuals.

Methods

In this randomized double-blind placebo-controlled trial, 118 subjects with T2DM were randomized to curcuminoids (1000 mg/day co-administered with piperine 10 mg/day) or matching placebo for a period of 8 weeks. Serum total antioxidant capacity, superoxide dismutase (SOD) activities and malondialdehyde (MDA) concentrations were measured at baseline and after the supplementation period.

Results

Curcuminoids supplementation caused a significant elevation in serum total antioxidant capacity (TAC) (p < 0.001) and SOD activities (p < 0.001), while serum MDA levels were significantly reduced compared with the placebo group (p < 0.001). These results remained statistically significant after adjustment for potential confounders (baseline differences in body mass index and fasting serum insulin).

Conclusion

The present results support an antioxidant effect of curcuminoids supplementation in patients with T2DM, and call for future studies to assess the impact of these antioxidant effects on the occurrence of diabetic complications and cardiovascular endpoints.

     

Intranasal Delivery of Topically-Acting Levofloxacin to Rats: a Proof-of-Concept Pharmacokinetic Study

Purpose

To evaluate the potential of levofloxacin intranasal administration as a promising alternative approach to treat local infections such as chronic rhinosinusitis, by delivering drug concentrations directly to the site of infection.

Methods

Drug concentrations were measured in plasma, olfactory bulb and nasal mucosa of anterior (ANM) and posterior regions after intranasal (0.24 mg/kg) and intravenous (10 mg/kg) administration to rats, and pharmacokinetic parameters were compared between routes. For intranasal administration a thermoreversible in-situ gel was used.

Results

Plasma and olfactory bulb exposure to levofloxacin was minimal following intranasal dose, preventing systemic and central nervous system adverse effects. Levofloxacin concentration-time profile in ANM revealed higher concentrations during the first 60 min of the study following intranasal administration than the corresponding ones obtained after intravenous administration. A rapid and continuous decay of levofloxacin concentration in this nasal region was observed after intranasal delivery, resulting in much lower values at the last sampling time-points.

Conclusion

The higher dose-normalized concentrations and pharmacokinetic exposure parameters of levofloxacin in ANM after intranasal administration, demonstrates that intranasal delivery of the formulated gel is, by itself, advantageous for delivering levofloxacin to biophase and thus an attractive approach in management of chronic rhinosinusitis.

     

Enhanced Stability of Inactivated Influenza Vaccine Encapsulated in Dissolving Microneedle Patches

Purpose

This study tested the hypothesis that encapsulation of influenza vaccine in microneedle patches increases vaccine stability during storage at elevated temperature.

Methods

Whole inactivated influenza virus vaccine (A/Puerto Rico/8/34) was formulated into dissolving microneedle patches and vaccine stability was evaluated by in vitro and in vivo assays of antigenicity and immunogenicity after storage for up to 3 months at 4, 25, 37 and 45°C.

Results

While liquid vaccine completely lost potency as determined by hemagglutination (HA) activity within 1–2 weeks outside of refrigeration, vaccine in microneedle patches lost 40–50% HA activity during or shortly after fabrication, but then had no significant additional loss of activity over 3 months of storage, independent of temperature. This level of stability required reduced humidity by packaging with desiccant, but was not affected by presence of oxygen. This finding was consistent with additional stability assays, including antigenicity of the vaccine measured by ELISA, virus particle morphological structure captured by transmission electron microscopy and protective immune responses by immunization of mice in vivo.

Conclusions

These data show that inactivated influenza vaccine encapsulated in dissolving microneedle patches has enhanced stability during extended storage at elevated temperatures.

     

Metal-on-Metal Hip Joint Prostheses: a Retrospective Case Series Investigating the Association of Systemic Toxicity with Serum Cobalt and Chromium Concentrations

Introduction

There have been concerns about prosthesis failure and the potential for systemic toxicity due to release of cobalt and chromium from metal-on-metal hip joint prostheses (MoM-HP). There is conflicting evidence on whether there is a correlation between higher cobalt and chromium concentrations and systemic toxicity.

Methods

We undertook a retrospective review of consecutive patients with MoM-HP referred for outpatient review in toxicology clinics in London, UK, and in the USA recorded in the Toxicology Investigators Consortium (ToxIC) Registry from June 2011 to June 2015.

Results

Thirty-one cases were identified; the median (IQR) serum cobalt concentration was 10.0 (3.8–32.8) mcg/L, and the median (IQR) serum chromium concentration was 6.9 (3.7–18.7) mcg/L. Twenty-three (74.2%) had symptoms, most commonly lethargy, hearing loss, and tinnitus. The odds ratios of symptomatic/asymptomatic patients for metal ion concentrations above/below 7 mcg/L were 1.87 (95% CI 0.37–9.57, p = 0.45) and 0.60 (95% CI 0.10–3.50, p = 0.57) for cobalt and chromium, respectively. Two (6.5%) patients with systemic cobalt toxicity had median (IQR) serum cobalt concentrations significantly higher than those without systemic features (630.4 [397.6–863.2] mcg/L versus 9.8 [2.9–16.4] mcg/L; p = 0.017). However, overall, there were no differences between cobalt (p = 0.38) or chromium (p = 0.92) concentrations between symptomatic and asymptomatic patients and no clinical features or investigation results correlated with cobalt or chromium concentration.

Conclusion

Two (6.5%) of 31 individuals referred for assessment of MoM-HP were diagnosed with systemic cobalt toxicity. However, despite a high prevalence of reported symptoms, neither symptoms nor investigation results correlated with serum cobalt or chromium concentrations.

     

Efficacious Cefazolin Prophylactic Dose for Morbidly Obese Women Undergoing Bariatric Surgery Based on Evidence from Subcutaneous Microdialysis and Populational Pharmacokinetic Modeling

Purpose

To determine the efficacious cefazolin prophylactic dose for bariatric surgery using free subcutaneous concentrations accessed by microdialysis after 2 g or 3 g i.v. bolus dosing to morbidly obese women and POPPK modeling.

Methods

A POPPK model with variable plasma and subcutaneous tissue protein binding was developed to simultaneously describe plasma and tissue data sets. The outcomes was predicted for common surgical site infection (SSI) bacteria over 3, 4, 5 and 6 h periods postdose, as probability of target attainment (PTA) using Monte Carlo simulation.

Results

CFZ 2 g warrant up to 5 h SSI prophylaxis for bacteria with MICs ≤1 mg/L such as Escherichia coli and Staphylococcus aureus. For species such as Klebsiella pneumoniae, which present MIC distribution frequency of 2 mg/L, the maintenance of PTA?≥?90% occurs with a 3 g dose for surgeries lasting up to 5 h, and 2 g dose provide an adequate response up to 4 h (PTA of 89%).

Conclusions

Effectiveness of CFZ 2 g is similar to 3 g against bacteria with a MIC up to 2 mg/L, especially if the surgery does not last for more than 4 h.

     

Poly-ADP-ribose polymerase inhibition provides protection against lung injury in a rat paraquat toxicity model

Objectives

Paraquat (PQ) is a widely used herbicide. Exposure to PQ at toxic doses can result in fatal acute lung injury. Inhibition of the poly-(ADP-ribose) polymerase (PARP) enzyme alleviates inflammation and necrosis in various pathologies. Here we aimed to evaluate the effects of PARP inhibition on PQ-induced lung damage in a rat experimental model.

Methods

Female Sprague-Dawley rats (n = 24) were allocated into three groups: sham, PQ and PQ + 3-aminobenzamide (3-AB) that is a PARP inhibitor, groups. Experimental lung injury was induced by administration of 15 mg/kg PQ intraperitoneally in PQ and PQ + 3-AB groups. 3-AB (10 mg/kg twice per day) was administered to the PQ + 3-AB group for four consecutive days. The animals were killed on the fifth day following PQ administration. Lung tissue and blood samples were collected and stored until analysis.

Results

Serum lactate dehydrogenase (LDH) and neopterin levels, tissue oxidative stress parameters, transforming growth factor-beta1 (TGF-β) levels and histological injury scores in the PQ + 3-AB group were significantly lower than in the PQ group (P < 0.05, PQ vs. PQ + 3-AB). Total antioxidant capacity in the PQ + 3-AB group was significantly higher than in the PQ group (P < 0.05, PQ + 3-AB vs. PQ).

Conclusion

Our results suggested that the use of PARP inhibitors following PQ toxicity might be useful for minimizing lung injury due to paraquat toxicity.

     

Atorvastatin attenuates TNBS-induced rat colitis: the involvement of the TLR4/NF-kB signaling pathway

Aim

The aim of the present study is to explore whether atorvastatin improves intestinal inflammation through the inhibition of the TLR4/NFkB signaling pathway in TNBS-induced rat colitis.

Methods

Acute colitis was induced by intra-rectal administration of 100 mg/kg TNBS dissolved in 0.25 ml of 50 % ethanol. Twenty four hours after colitis induction, saline, atorvastatin (20 and 40 mg/kg) and sulfasalazine (100 mg/kg) were given to the animals by oral route. This was repeated daily for 1 week. Body weight changes, macroscopic and microscopic lesions were assessed. MPO and TNF-α activities were detected by immunohistochemistry (IHC) and the expression level of TLR4, MyD88 and NF-κB p65 proteins were measured by western blotting analysis.

Results

Atorvastatin and sulfasalazine reduced the body weight loss, macroscopic and microscopic lesions. Additionally, both drugs decreased the expression of MPO and TNF-α positive cells in the colon tissue. Furthermore, they inhibited the TNBS-induced expression of TLR4, MyD88 and NF-κB p65 proteins.

Conclusions

It is suggested that the anti-inflammatory effect of atorvastatin on TNBS-induced rat colitis may involve the inhibition of the TLR4/NFkB signaling pathway.

     

Differences between serum polar lipid profiles of male and female rheumatoid arthritis patients in response to glucocorticoid treatment

Objective

As there are pharmacological differences between males and females, and glucocorticoid (GC) treatment is associated with increased cardiovascular mortality rate in rheumatoid arthritis (RA) patients, it is important to study serum polar lipid profiles of male and female patients in response to GC therapy. Gender differences may require an adjustment to the treatment strategy for a selection of patients.

Methods

Serum samples from 281 RA patients were analysed using a targeted lipidomics platform. The differences in GC use and gender on polar lipid profiles were cross sectionally examined by multiple linear regressions, while correcting for confounding factors.

Results

Differences in polar lipids between GC users and non-GC users in females and males were merely restricted to lysophospholipids (lysophosphatidylcholines and lysophosphatidylethanolamines). Lysophospholipids in female patients treated with GCs were significantly higher than female patients not treated with GCs (p = 6.0 E?6), whereas no significant difference was observed in male GC users versus non-users (p = 0.397).

Conclusion

The lysophospholipid profiles in response to GCs were significantly different between male and female RA patients, which may have implications for the cardiovascular risk of GC treatment.

     

Reversal of Opioid-Induced Ventilatory Depression Using Low-Dose Naloxone (0.04 mg): a Case Series

Introduction

Naloxone is commonly administered in emergency department (ED) to reverse opioid intoxication. Several naloxone dose recommendations exist for acute management of opioid intoxication based on limited published clinical data. A case series of ED patients with opioid-induced ventilatory depression that was reversed using a low-dose naloxone (0.04 mg with titration) is presented.

Methods

ED patients with opioid-induced ventilatory depression requiring naloxone administration were identified through medical toxicology consultation. Retrospective review of medical records was performed. Collected data included history, and pre- and post-naloxone data, including respiratory rate (RR), pulse oximetry (pulse ox), end-tidal CO₂ level (ET-CO₂), and Richmond Agitation Sedation Scale (RASS).

Results

Fifteen ED patients with moderate to severe opioid-induced ventilatory depression (median RR, 6 breaths/min) who were managed using low-dose naloxone strategy were identified. Twelve of 15 patients reported ingestion of methadone (range, 30 to 180 mg). The median naloxone dose of 0.08 mg (range, 0.04 to 0.12 mg) reversed opioid-induced ventilatory and CNS depression. Two patients experienced acute opioid withdrawal after receiving 0.08 mg.

Conclusion

ED patients with moderate to severe opioid-induced ventilatory depression can be reversed using 0.04 mg IV naloxone with appropriate dose titration.

     

Pharmacokinetic-Pharmacodynamic Modeling of the Anti-Tumor Effect of Sunitinib Combined with Dopamine in the Human Non-Small Cell Lung Cancer Xenograft

Purpose

To investigate the anti-tumor effect of sunitinib in combination with dopamine in the treatment of nu/nu nude mice bearing non-small cell lung cancer (NSCLC) A549 cells and to develop the combination PK/PD model. Further, simulations were conducted to optimize the administration regimens.

Methods

A PK/PD model was developed based on our preclinical experiment to explore the relationship between plasma concentration and drug effect quantitatively. Further, the model was evaluated and validated. By fixing the parameters obtained from the PK/PD model, simulations were built to predict the tumor suppression under various regimens.

Results

The synergistic effect was observed between sunitinib and dopamine in the study, which was confirmed by the effect constant (GAMA, estimated as 2.49). The enhanced potency of dopamine on sunitinib was exerted by on/off effect in the PK/PD model. The optimal dose regimen was selected as sunitinib (120 mg/kg, q3d) in combination with dopamine (2 mg/kg, q3d) based on the simulation study.

Conclusions

The synergistic effect of sunitinib and dopamine was demonstrated by the preclinical experiment and confirmed by the developed PK/PD model. In addition, the regimens were optimized by means of modeling as well as simulation, which may be conducive to clinical study.

     

A Random Forest Approach for Counting Silicone Oil Droplets and Protein Particles in Antibody Formulations Using Flow Microscopy

Purpose

To evaluate a random forest model that counts silicone oil droplets and non-silicone oil particles in protein formulations with large class imbalance.

Methods

In this work, we present a novel approach for automated image analysis of flow microscopy data based on random forest classification enabling rapid analysis of large data sets. The random forest approach overcomes many of the limitations of traditional classification schemes derived from simple filters or regression models. In particular, the approach does not require a priori selection of important morphology parameters.

Results

We analyzed silicone oil droplets and non-silicone oil particles observed in four model systems with protein concentrations of 20, 50 and 125 mg/mL. Filters based on random forests achieve higher classification accuracies when compared to regression based filters. Additionally, we showcase a procedure that allows for accurate counting of particles ≥1 μm.

Conclusions

Our method is generally applicable for classification and counting of different classes of particles as long as class morphologies are differentially expressed.

     

Blood Urea Nitrogen/Creatinine Ratio and Response to Tolvaptan in Patients with Decompensated Heart Failure: A Retrospective Analysis

Introduction

Arginine vasopressin-stimulated reabsorption of urea occurs in the collecting duct via increased expression of the urea transporter.

Objective

The aim of this study was to evaluate whether the blood urea nitrogen/creatinine (BUN/Cr) ratio is useful for predicting tolvaptan response in patients with decompensated heart failure (HF).

Methods

Among 71 consecutive patients with HF who received oral tolvaptan between 2010 and 2014, we retrospectively studied 33 patients with decompensated HF without any mechanical circulatory assistance or inotropic support who had already been treated with loop diuretics. A responder to tolvaptan was defined as an individual who experienced a ≥30 % increase in their respective 24-h urine volume.

Results

Among the 33 patients, 21 met the criteria of a responder. The area under the receiver operating characteristic curves of BUN/Cr and BUN were 0.790 and 0.714, respectively, and the respective cut-off values for responders to tolvaptan were 23.8 and 49.0. BUN/Cr and BUN retained their significant relationships with the responder status (odds ratio for BUN/Cr >23.8: 20.9; 95 % confidence interval [CI] 2.7–531.1; p = 0.002; odds ratio for BUN ≥49: 7.7; 95 % CI 1.4–65.8; p = 0.02).

Conclusion

Our results suggest that high BUN/Cr may be a predictor of response to tolvaptan in decompensated HF patients. A prospective study with a large sample size is required to confirm this preliminary finding.

     

Anti-Tuberculosis Bacteriophage D29 Delivery with a Vibrating Mesh Nebulizer,Jet Nebulizer,and Soft Mist Inhaler

Purpose

To compare titer reduction and delivery rate of active anti-tuberculosis bacteriophage (phage) D29 with three inhalation devices.

Methods

Phage D29 lysate was amplified to a titer of 11.8 ± 0.3 log₁₀(pfu/mL) and diluted 1:100 in isotonic saline. Filters captured the aerosolized saline D29 preparation emitted from three types of inhalation devices: 1) vibrating mesh nebulizer; 2) jet nebulizer; 3) soft mist inhaler. Full-plate plaque assays, performed in triplicate at multiple dilution levels with the surrogate host Mycobacterium smegmatis, were used to quantify phage titer.

Results

Respective titer reductions for the vibrating mesh nebulizer, jet nebulizer, and soft mist inhaler were 0.4 ± 0.1, 3.7 ± 0.1, and 0.6 ± 0.3 log₁₀(pfu/mL). Active phage delivery rate was significantly greater (p < 0.01) for the vibrating mesh nebulizer (3.3x10⁸ ± 0.8x10⁸ pfu/min) than for the jet nebulizer (5.4x10⁴ ± 1.3x10⁴ pfu/min). The soft mist inhaler delivered 4.6x10⁶ ± 2.0x10⁶ pfu per 11.6 ± 1.6 μL ex-actuator dose.

Conclusions

Delivering active phage requires a prudent choice of inhalation device. The jet nebulizer was not a good choice for aerosolizing phage D29 under the tested conditions, due to substantial titer reduction likely occurring during droplet production. The vibrating mesh nebulizer is recommended for animal inhalation studies requiring large amounts of D29 aerosol, whereas the soft mist inhaler may be useful for self-administration of D29 aerosol.

     

Three-Dimensional Printing of Cell Exclusion Spacers (CES) for Use in Motility Assays

Purpose

Cell migration/invasion assays are widely used in commercial drug discovery screening. 3D printing enables the creation of diverse geometric restrictive barrier designs for use in cell motility studies, permitting on-demand assays. Here, the utility of 3D printed cell exclusion spacers (CES) was validated as a cell motility assay.

Methods

A novel CES fit was fabricated using 3D printing and customized to the size and contour of 12 cell culture plates including 6 well plates of basal human brain vascular endothelial (D3) cell migration cells compared with 6 well plates with D3 cells challenged with 1uM cytochalasin D (Cyto-D), an F-actin anti-motility drug. Control and Cyto-D treated cells were monitored over 3 days under optical microscopy.

Results

Day 3 cell migration distance for untreated D3 cells was 1515.943μm?±?10.346μm compared to 356.909μm?±?38.562μm for the Cyt-D treated D3 cells (p?<?0.0001). By day 3, untreated D3 cells reached confluency and completely filled the original voided spacer regions, while the Cyt-D treated D3 cells remained significantly less motile.

Conclusions

Cell migration distances were significantly reduced by Cyto-D, supporting the use of 3D printing for cell exclusion assays. 3D printed CES have great potential for studying cell motility, migration/invasion, and complex multi-cell interactions.

     

Human Immunodeficiency Virus (HIV) Separation and Enrichment via the Combination of Antiviral Lectin Recognition and a Thermoresponsive Reagent System

Purpose

In order to improve the detection limit of existing HIV diagnostic assays, we explored the use of a temperature-responsive magnetic nanoparticle reagent system in conjunction with cyanovirin-N for HIV recognition to rapidly and efficiently concentrate viral particles from larger sample volumes, ~ 1 ml.

Methods

Cyanovirin-N (CVN) mutant, Q62C, was expressed, biotinylated, and then complexed with a thermally responsive polymer-streptavidin conjugate. Confirmation of protein expression/activity was performed using matrix assisted laser desorption/ionization (MALDI) and a TZM-bl HIV inhibition assay. Biotinylated CVN mutant recognition with gp120 was characterized using surface plasmon resonance (SPR). Virus separation and enrichment using a thermoresponsive magnetic nanoparticle reagent system were measured using RT-PCR.

Results

Biotinylated Q62C exhibited a K_D of 0.6 nM to gp120. The temperature-responsive binary reagent system achieved a maximum viral capture of nearly 100% HIV, 1?×?10⁵ virus copies in 100 μl, using pNIPAAm-Q62C within 30 minutes. Additionally, the same reagent system achieved nearly 9-fold enrichment by processing a 10-times larger sample of 1000 μl (Fig. 3).

Conclusion

This work demonstrated a temperature-responsive reagent system that provides enrichment of HIV using antiviral lectin CVN for recognition, which is potentially amenable for use in point-of-care settings.