Denise Wilkes

Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein.

Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multispanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP-NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.

Pulsed radiofrequency treatment of lower extremity phantom limb pain.

BackgroundPhantom limb pain can be challenging to treat. We present a patient who developed severe phantom limb pain after revision of her lower extremity amputation due to the continued progression of peripheral vascular disease. Multiple treatment modalities had been tried without success. Pulsed radiofrequency has been successfully used to manage a number of pain syndromes. ObjectiveThe present case report describes the use of pulsed radiofrequency treatment for phantom limb pain. MethodsThe authors initially preformed regional blocks of femoral and sciatic nerve with 0.375% bupivicaine 15 cc and 50 μg clonidine to control the patients pain. The blocks provided good pain relief but with limited duration. Based on reports of prolonged pain relief provided by pulsed radiofrequency treatment for other chronic pain conditions such as lumbrosacral spondylosis, we decided to apply this treatment to the patients sciatic nerve. The patient underwent pulsed radiofrequency treatment with 2 cycles of 120 seconds at 42 degrees, pulse rate of 2 pulse/second, and pulse duration of 20 milliseconds. ResultsOur report shows that the sciatic nerve block with bupivicaine and clonidine, initiated approximately 3 years after amputation, produced modest short-term relief. The pulsed radiofrequency treatment resulted in long-term relief of phantom limb pain. The patient was able to wean herself off all oral medications and has been pain free for 4 months.

Pulsed Radiofrequency V2 Treatment and Intranasal Sphenopalatine Ganglion Block: A Combination Therapy for Atypical Trigeminal Neuralgia

Trigeminal neuralgia (TN) is a chronic condition affecting the fifth cranial nerve and resulting in sporadic intense burning and shock‐like pain lasting for seconds to minutes that can be incapacitating to patients. Atypical TN includes additional features such as continuous pain and sensory disturbances in the area innervated by one or more branches of the trigeminal nerve. Documented cases of TN have dated back to the 18th century. Today, there are roughly 140,000 people suffering with this condition in the U.S.A. Conventional treatments for this disorder include medical management with nonconvulsants such as carbamazepine, which decrease the nerves response to peripheral stimulation. These agents have good initial pain relief, but relief rates fall off dramatically over the long‐term. Recently, methadone has shown promise as a pharmacologic adjunct to patients with intractable neuropathic noncancer pain, including patients suffering from TN. Cases refractory to medical management can be treated with surgical microdecompression or minimally invasive procedures such as radiofrequency (RF) treatment. Pulsed RF (PRF) is a method gaining interest as it is delivered in pulses, allowing adequate time for dissipation of heat and energy resulting in less damage to surrounding structures. This case report describes the successful treatment of atypical V2 TN refractive to medical management requiring PRF treatment, a sphenopalatine block series, and low‐dose methadone.

Alcohol neurolysis of the sciatic and femoral nerves to improve pressure ulcer healing.

Successful pressure ulcer treatment is challenging and is often plagued with prolonged hospitalizations, multiple surgeries, and high recurrence rates. Pressure ulcer secondary to spinal cord injury is further complicated by spasticity, which contributes to both ulcer continuance and healing. This report illustrates the use of neurolytic regional techniques for spasticity control and pressure ulcer healing. Case report: We present our experience with a paraplegic man who suffered from chronic right trochanteric and ischial pressure ulcers that failed to heal despite surgical and conservative treatment. We report the successful treatment of knee and hip flexor spasticity with a femoral and sciatic alcohol neuroablation technique. It was not until the successful control of his lower extremity spasticity that the pressure ulcers showed signs of healing. Neuroablation nay be considered for spasticity control when more conservative approaches fail or are not feasible.

Programmable intrathecal pumps for the management of chronic pain: recommendations for improved efficiency.

The management of chronic pain can be very challenging. Often, physicians employ intrathecal (IT) drug delivery systems as a last resort to relieve intractable pain. The system consists of an implantable pump that stores and delivers medication through a catheter to the IT space. Programmability is achieved by positioning an external devise over the implanted pump to change the mode of drug delivery. The innovations in programmable IT drug delivery systems are expanding more rapidly than ever before. Unfortunately, the rapid expansion is accompanied by a lack of prospective randomized trials examining these new options. In an effort to improve results and reduce side effects, publications by experts or expert consensus panels provide guidance for the community. The purpose of this article is to provide a summary of high interest topics in recent publications.

Nucleotide triphosphatase activity of the N-terminal nucleotide-binding domains of the multidrug resistance proteins P-glycoprotein and MRP1.

The multidrug resistance proteins P-glycoprotein (Pgp) and MRP1 are drug-efflux pumps. In this study, we compared the nucleotide triphosphatase activities of the isolated N-terminal nucleotide binding domains (NBD1) of Pgp and MRP1, and explored the potential role of the phosphorylation target domain of Pgp on the regulation of Pgp NBD1 ATPase activity. We found that: (1) the NBD1s of Pgp and MRP1 have ATPase and GTPase activities, (2) the K(m)s of Pgp NBD1 for ATP and GTP hydrolysis are identical, while the K(m) of MRP1 NBD1 for ATP is lower than that for GTP, and (3) phosphorylation of MLD by PKA or PKC produces a marginal increase of V(max) for ATP hydrolysis, without affecting the affinity for ATP. These results show efficient GTP hydrolysis by the NBD1s of Pgp and MRP1, and a minor role of phosphorylation in the control of Pgp NBD1 ATPase activity.

A large animal neuropathic pain model in sheep: a strategy for improving the predictability of preclinical models for therapeutic development.

Background Evaluation of analgesics in large animals is a necessary step in the development of better pain medications or gene therapy prior to clinical trials. However, chronic neuropathic pain models in large animals are limited. To address this deficiency, we developed a neuropathic pain model in sheep, which shares many anatomical similarities in spine dimensions and cerebrospinal fluid volume as humans. Methods A neuropathic pain state was induced in sheep by tight ligation and axotomy of the common peroneal nerve. The analgesic effect of intrathecal (IT) morphine was investigated. Interspecies comparison was conducted by analyzing the ceiling doses of IT morphine for humans, sheep, and rats. Results Peroneal nerve injury (PNI) produced an 86% decrease in von-Frey filament-evoked withdrawal threshold on postsurgery day 3 and the decrease lasted for the 8-week test period. Compared to the pre-injury, sham, and contralateral hindlimb, the IT morphine dose that produces 50% of maximum analgesia (ED50) for injured PNI hindlimb was 1.8-fold larger and Emax, the dose that produces maximal analgesia, was 6.1-fold lower. The sheep model closely predicts human IT morphine ceiling dose by allometric scaling. This is in contrast to the approximately 10-fold lower morphine ceiling dose predicted by the rat spinal nerve ligated or spared nerve injury models. Conclusion PNI sheep model has a fast onset and shows stable and long-lasting pain behavioral characteristics. Since the antinociceptive properties of IT morphine are similar to those observed in humans, the PNI sheep model will be a useful tool for the development of analgesics. Its large size and consistent chronic pain behavior will facilitate the development and evaluation of surgical intervention and gene therapy. The PNI sheep pain model provides us with the opportunity for multi-species testing, which will improve the success of clinical trials.

Pulsed Radiofrequency V2 Treatment and Intranasal Sphenopalatine Ganglion Block: A Combination Therapy for Atypical Trigeminal Neuralgia: Combined Therapy for Atypical Trigeminal Neuralgia

Trigeminal neuralgia (TN) is a chronic condition affecting the fifth cranial nerve and resulting in sporadic intense burning and shock‐like pain lasting for seconds to minutes that can be incapacitating to patients. Atypical TN includes additional features such as continuous pain and sensory disturbances in the area innervated by one or more branches of the trigeminal nerve. Documented cases of TN have dated back to the 18th century. Today, there are roughly 140,000 people suffering with this condition in the U.S.A. Conventional treatments for this disorder include medical management with nonconvulsants such as carbamazepine, which decrease the nerves response to peripheral stimulation. These agents have good initial pain relief, but relief rates fall off dramatically over the long‐term. Recently, methadone has shown promise as a pharmacologic adjunct to patients with intractable neuropathic noncancer pain, including patients suffering from TN. Cases refractory to medical management can be treated with surgical microdecompression or minimally invasive procedures such as radiofrequency (RF) treatment. Pulsed RF (PRF) is a method gaining interest as it is delivered in pulses, allowing adequate time for dissipation of heat and energy resulting in less damage to surrounding structures. This case report describes the successful treatment of atypical V2 TN refractive to medical management requiring PRF treatment, a sphenopalatine block series, and low‐dose methadone.

Efficacy, Safety, and Feasibility of the Morphine Microdose Method in Community-Based Clinics

Objectives The goal of this study was to assess the success of the morphine microdose method in a community pain clinic setting by monitoring follow-up frequency, dose escalation, and monotherapy/polytherapy ratio. The morphine microdose method involves a pretrial reduction or elimination of systemic opioids followed by a period of abstinence. Intrathecal (IT) morphine is then started at doses of less than 0.2 mg per day. Systemic opioid abstinence is then continued after pump implant and IT morphine monotherapy. Design Retrospective review of medical records. Setting Private and academic pain clinic practices. Subjects Chronic noncancer pain patients. Methods We reviewed the charts of 60 patients who had completed a microdose regimen and had an IT pump implanted between June 11, 2008, and October 11, 2014. During IT therapy, dose change over time, pain scores, side effects, max dose, and duration were recorded. Results The majority of patients (35/60, 58%) were successfully managed solely on morphine microdose monotherapy. These patients did not require additional oral therapy. There was a significant reduction in mean pain scores, from 7.4 ± 0.32 before microdose therapy to 4.8 ± 0.3 after microdose therapy. Conclusions Microdose therapy achieved analgesia, improved safety, and avoided systemic side effects. The safety of IT therapy was increased by using a lower concentration (2 mg/mL) and lower daily doses (<3 mg/d) of morphine. Furthermore, microdose therapy was feasible, safe, and cost-effective in the outpatient setting.

Ultrasound-determined landmarks decrease pressure pain at epidural insertion site in immediate post-partum period

BACKGROUND Women have blamed epidurals for their post-partum back pain for decades. Survey-based studies have shown similar incidence of chronic back pain between women who delivered with epidurals compared to those who did not. However, epidural insertion site pain has yet to be evaluated by a quantitative measure: pressure pain threshold (PPT). Algometer measured PPT has been shown to be accurate and reproducible in acute, chronic, and postoperative pain studies. This study determines the effect of ultrasound-based landmarks on the PPT at the epidural insertion site in the post-partum period. METHODS Participants were randomized into either the ultrasound or sham groups. In addition, a non-randomized control group (no epidural) participated. Ultrasound of the lumbar region was used to mark mid intervertebral levels in the US group but not in the sham group. Epidural were placed using the marks in the US group or palpated bony landmarks in the sham group. PPT at each intervertebral space measured before and after the use of epidural. RESULTS Epidural placement did significantly decreased PPT in US (68%) and US sham (79%) groups and less in the control group (21%). US group showed decreased PPT only at insertion site whereas US sham group also showed decreased PPT at insertion site and adjacent levels. CONCLUSIONS We showed that epidural placed with ultrasound-determined landmarks not only improves the success of epidural placement but also minimizes the number of intervertebral levels with decreased PPT.