Protocol for isolating human BCAM-positive corneal progenitor cells by flow cytometry and cell sorting.

BCAM-positive basal limbal epithelial cells are an early transit-amplifying cell population (TAC) capable of holoclone formation and corneal epithelial differentiation. Here, we present a protocol for isolating BCAM-positive cells from human donor corneas by flow cytometry and cell sorting. We describe steps for cell dissection and dissociation, antibody staining, and flow cytometry. We then detail procedures for culturing the purified BCAM-positive and BCAM-negative cells for holoclone and cell sheet formation assays to study the factors that regulate corneal regeneration. For complete details on the use and execution of this protocol, please refer to Sasamoto et al.1.

Continuous Ketamine Infusion as a Treatment for Refractory Facial Pain.

Complex orofacial pain disorders, such as trigeminal neuralgia (TN) and atypical facial pain (AFP), can be excruciating and debilitating during attacks. Ketamine, an N-methyl-D-aspartate (NMDA) antagonist, is a powerful analgesic that has been used to treat various chronic pain conditions, but its role in treating complex facial pain has only been recently explored. In this retrospective case series, we reviewed the efficacy of continuous ketamine infusion for 12 patients with facial pain refractory to medical treatment. Patients who presented with a diagnosis of TN were more likely to have significant and sustained pain relief after receiving ketamine infusion. By contrast, those who did not respond to the treatment were more likely to have a diagnosis of AFP. The current report suggests a fundamental difference between these two facial pain disorders in their respective underlying pathophysiology and supports the use of continuous ketamine infusion for refractory TN, but not AFP.

Limbal BCAM expression identifies a proliferative progenitor population capable of holoclone formation and corneal differentiation.

The corneal epithelium is renowned for high regenerative potential, which is dependent on the coordinated function of its diverse progenitor subpopulations. However, the molecular pathways governing corneal epithelial progenitor differentiation are incompletely understood. Here, we identify a highly proliferative limbal epithelial progenitor subpopulation characterized by expression of basal cell adhesion molecule (BCAM) that is capable of holocone formation and corneal epithelial sheet generation. BCAM-positive cells can be found among ABCB5-positive limbal stem cells (LSCs) as well as among ABCB5-negative limbal epithelial cell populations. Mechanistically, we show that BCAM is functionally required for cellular migration and differentiation and that its expression is regulated by the transcription factor p63. In aggregate, our study identifies limbal BCAM expression as a marker of highly proliferative corneal epithelial progenitor cells and defines the role of BCAM as a critical molecular mediator of corneal epithelial differentiation.

Peripheral Nerve Stimulation for Pudendal Neuralgia: A Technical Note.

BACKGROUND: Pudendal neuropathy is a chronic, disabling form of perineal pain that involves the pudendal nerve, a mixed somatic and autonomic nerve that originates from sacral nerve roots. Peripheral nerve stimulation of the pudendal nerve can be useful to decrease symptom burden in patients who have failed initial conservative treatment modalities. METHODS: In this manuscript, we describe an approach to the placement of a peripheral nerve stimulator for the treatment of pudendal neuralgia. We present a case of complex pelvic neuropathy and review the factors that lead to successful placement. Technical aspects of stimulator placement and ultrasound landmarks are reviewed. RESULTS: A lateral to medial approach with ultrasound guidance at the level of the ischial spine is likely to facilitate proper lead placement along the course of the pudendal nerve. Aftercare and adherence to postimplant activity restrictions-particularly avoiding use of the extremes of hip flexion and extension for four weeks-lead to the absence of lead migration. CONCLUSIONS: Pudendal nerve stimulation is an emerging technique for neuromodulation of refractory pudendal neuralgia. Ultrasound-guided pudendal nerve stimulation is a viable technique for neuromodulation of pudendal neuralgia. Optimization of patient selection, ultrasound guidance, and proper adherence to postimplant activity restrictions may be helpful for long-term therapeutic success.

Targeting the ABC transporter ABCB5 sensitizes glioblastoma to temozolomide-induced apoptosis through a cell-cycle checkpoint regulation mechanism.

Glioblastoma multiforme (GBM) is a malignant brain tumor with a poor prognosis resulting from tumor resistance to anticancer therapy and a high recurrence rate. Compelling evidence suggests that this is driven by subpopulations of cancer stem cells (CSCs) with tumor-initiating potential. ABC subfamily B member 5 (ABCB5) has been identified as a molecular marker for distinct subsets of chemoresistant tumor-initiating cell populations in diverse human malignancies. In the current study, we examined the potential role of ABCB5 in growth and chemoresistance of GBM. We found that ABCB5 is expressed in primary GBM tumors, in which its expression was significantly correlated with the CSC marker protein CD133 and with overall poor survival. Moreover, ABCB5 was also expressed by CD133-positive CSCs in the established human U-87 MG, LN-18, and LN-229 GBM cell lines. Antibody- or shRNA-mediated functional ABCB5 blockade inhibited proliferation and survival of GBM cells and sensitized them to temozolomide (TMZ)-induced apoptosis in vitro Likewise, in in vivo human GBM xenograft experiments with immunodeficient mice, mAb treatment inhibited growth of mutant TP53, WT PTEN LN-229 tumors, and sensitized LN-229 tumors to TMZ therapy. Mechanistically, we demonstrate that ABCB5 blockade inhibits TMZ-induced G2/M arrest and augments TMZ-mediated cell death. Our results identify ABCB5 as a GBM chemoresistance marker and point to the potential utility of targeting ABCB5 to improve current GBM therapies.

Investigation of factors associated with ABCB5-positive limbal stem cell isolation yields from human donors.

PURPOSE: To identify factors associated with isolation yields of ATP-binding cassette (ABC) superfamily member B5 (ABCB5)-positive limbal stem cells (LSCs) from human cadaveric donor eyes. METHODS: Whole eye globes were obtained from the Saving Sight eye bank, Kansas City, MO and the CorneaGen eye bank, Seattle, WA. ABCB5-positive LSCs were sorted by flow cytometry upon anti-ABCB5 monoclonal antibody staining within one week after donor death. The yields of live limbal epithelial cells in their entirety and of isolated pure ABCB5-positive LSC subsets were correlated with variables contained in the eye donors' medical information. RESULTS: The mean isolation yield of live limbal epithelial cells and ABCB5-positive LSCs per donor eye was (340,000 ±â€¯160,000 and 2,608 ±â€¯1,842 respectively, mean ±â€¯SD). Stepwise regression analysis showed that cardiac disease-related death was the strongest negative predictor of the ABCB5-positive LSC isolation yield (p = 0.01). While we observed a trend for an age-related decline in the yield of ABCB5-positive LSCs, a statistically significant association could not be established (2% decrease/year, p = 0.11). Additionally, despite a trend for decreased isolation yields of total live limbal epithelial cells isolated from single donors with a longer time between death and tissue processing (p = 0.04), this did not affect the yields of purified ABCB5-positive LSC, which was independent of increasing time between death and tissue processing (p = 0.50). CONCLUSIONS: Our study identifies cardiac disease-related death as a donor variable significantly associated with lower ABCB5-positive LSC isolation yields.

Characteristics and advantages of adeno-associated virus vector-mediated gene therapy for neurodegenerative diseases.

Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.

Continuous Transversus Abdominis Plane Nerve Blocks: Does Varying Local Anesthetic Delivery Method-Automatic Repeated Bolus Versus Continuous Basal Infusion-Influence the Extent of Sensation to Cold?: A Randomized, Triple-Masked, Crossover Study in Volunteers.

BACKGROUND: It remains unknown whether continuous or scheduled intermittent bolus local anesthetic administration is preferable for transversus abdominis plane (TAP) catheters. We therefore tested the hypothesis that when using TAP catheters, providing local anesthetic in repeated bolus doses increases the cephalad-caudad cutaneous effects compared with a basal-only infusion. METHODS: Bilateral TAP catheters (posterior approach) were inserted in 24 healthy volunteers followed by ropivacaine 2 mg/mL administration for a total of 6 hours. The right side was randomly assigned to either a basal infusion (8 mL/h) or bolus doses (24 mL administered every 3 hours for a total of 2 bolus doses) in a double-masked manner. The left side received the alternate treatment. The primary end point was the extent of sensory deficit as measured by cool roller along the axillary line at hour 6 (6 hours after the local anesthetic administration was initiated). Secondary end points included the extent of sensory deficit as measured by cool roller and Von Frey filaments along the axillary line and along a transverse line at the level of the anterior superior iliac spine at hours 0 to 6. RESULTS: Although there were statistically significant differences between treatments within the earlier part of the administration period, by hour 6 the difference in extent of sensory deficit to cold failed to reach statistical significance along the axillary line (mean = 0.9 cm; SD = 6.8; 95% confidence interval -2.0 to 3.8; P = .515) and transverse line (mean = 2.5 cm; SD = 10.1; 95% confidence interval -1.8 to 6.8; P = .244). Although the difference between treatments was statistically significant at various early time points for the horizontal, vertical, and estimated area measurements of both cold and mechanical pressure sensory deficits, no comparison remained statistically significant by hour 6. CONCLUSIONS: No evidence was found in this study involving healthy volunteers to support the hypothesis that changing the local anesthetic administration technique (continuous basal versus hourly bolus) when using ropivacaine 0.2% and TAP catheters at 8 mL/h and 24 mL every 3 hours significantly influences the cutaneous effects after 6 hours of administration. Additional research is required to determine whether changing variables (eg, local anesthetic concentration, basal infusion rate, bolus dose volume, and/or interval) would provide different results.

Silencing Nociceptor Neurons Reduces Allergic Airway Inflammation.

Lung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation, we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8(+) sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large-pore ion channels to specifically block nociceptors, substantially reduced ovalbumin- or house-dust-mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce the release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4(+) and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma.

Evaluation of a portable markerless finger position capture device: accuracy of the Leap Motion controller in healthy adults.

Although motion analysis is frequently employed in upper limb motor assessment (e.g. visually-guided reaching), they are resource-intensive and limited to laboratory settings. This study evaluated the reliability and accuracy of a new markerless motion capture device, the Leap Motion controller, to measure finger position. Testing conditions that influence reliability and agreement between the Leap and a research-grade motion capture system were examined. Nine healthy young adults pointed to 15 targets on a computer screen under two conditions: (1) touching the target (touch) and (2) 4 cm away from the target (no-touch). Leap data was compared to an Optotrak marker attached to the index finger. Across all trials, root mean square (RMS) error of the Leap system was 17.30  ±  9.56 mm (mean ± SD), sampled at 65.47  ±  21.53 Hz. The % viable trials and mean sampling rate were significantly lower in the touch condition (44% versus 64%, p < 0.001; 52.02  ±  2.93 versus 73.98  ±  4.48 Hz, p = 0.003). While linear correlations were high (horizontal: r(2) = 0.995, vertical r(2) = 0.945), the limits of agreement were large (horizontal: -22.02 to +26.80 mm, vertical: -29.41 to +30.14 mm). While not as precise as more sophisticated optical motion capture systems, the Leap Motion controller is sufficiently reliable for measuring motor performance in pointing tasks that do not require high positional accuracy (e.g. reaction time, Fitt's, trails, bimanual coordination).

Bacteria activate sensory neurons that modulate pain and inflammation.

Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils and monocytes is not necessary for Staphylococcus aureus-induced pain in mice. Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin α-haemolysin, through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host-pathogen interactions.