Electrophysiology-based stratification of pancreatic tumorigenicity by label-free single-cell impedance cytometry.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer lacking specific biomarkers that can be correlated to disease onset, promotion and progression. To assess whether tumor cell electrophysiology may serve as a marker for PDAC tumorigenicity, we use multi-frequency impedance cytometry at high throughput (∼350 cells/s) to measure the electrical phenotype of single PDAC tumor cells from xenografts, which are derived from primary pancreatic tumors versus those from liver metastases of different patients. A novel phase contrast metric based on variations in the high and low frequency impedance phase responses that is related to electrophysiology of the cell interior is found to be systematically altered as a function of tumorigenicity. PDAC cells of higher tumorigenicity exhibited lowered interior conductivity and enhanced permittivity, which is validated by the dielectrophoresis on the respective cell types. Using genetic analysis, we suggest the role of dysregulated Na+ transport and removal of Ca2+ ions from the cytoplasm on key oncogenic KRAS-driven processes that may be responsible for lowering of the interior cell conductivity. We envision that impedance cytometry can serve as a tool to quantify phenotypic heterogeneity for rapidly stratifying tumorigenicity. It can also aid in protocols for dielectrophoretic isolation of cells with a particular phenotype for prognostic studies on patient survival and to tailor therapy selection to specific patients.

Dielectric characterization of Plasmodium falciparum-infected red blood cells using microfluidic impedance cytometry.

Although malaria is the world's most life-threatening parasitic disease, there is no clear understanding of how certain biophysical properties of infected cells change during the malaria infection cycle. In this article, we use microfluidic impedance cytometry to measure the dielectric properties of Plasmodium falciparum-infected red blood cells (i-RBCs) at specific time points during the infection cycle. Individual parasites were identified within i-RBCs using green fluorescent protein (GFP) emission. The dielectric properties of cell sub-populations were determined using the multi-shell model. Analysis showed that the membrane capacitance and cytoplasmic conductivity of i-RBCs increased along the infection time course, due to membrane alterations caused by parasite infection. The volume ratio occupied by the parasite was estimated to vary from less than 10% at earlier stages, to approximately 90% at later stages. This knowledge could be used to develop new label-free cell sorting techniques for sample pre-enrichment, improving diagnosis.

Analysis of Parasitic Protozoa at the Single-cell Level using Microfluidic Impedance Cytometry.

At present, there are few technologies which enable the detection, identification and viability analysis of protozoan pathogens including Cryptosporidium and/or Giardia at the single (oo)cyst level. We report the use of Microfluidic Impedance Cytometry (MIC) to characterise the AC electrical (impedance) properties of single parasites and demonstrate rapid discrimination based on viability and species. Specifically, MIC was used to identify live and inactive C. parvum oocysts with over 90% certainty, whilst also detecting damaged and/or excysted oocysts. Furthermore, discrimination of Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia, with over 92% certainty was achieved. Enumeration and identification of (oo)cysts can be achieved in a few minutes, which offers a reduction in identification time and labour demands when compared to existing detection methods.

Bilateral submandibular gland infection presenting as Ludwig's angina: first report of a case.

We diagnosed and treated a case of Ludwig's angina in a 45-year-old man who had edema of the floor of mouth and the tongue along with bilateral submandibular sialadenitis and sialolithiasis. We secured the patient's airway via nasal fiberoptic intubation in the surgical intensive care unit and administered intravenous antibiotics. The edema subsided, and the patient was extubated on the third postoperative day and discharged shortly thereafter. To our knowledge, this is the first reported case of a patient with bilateral submandibular sialadenitis and sialolithiasis presenting as Ludwig's angina. Despite the decreasing incidence of this disease, Ludwig's angina remains an important disease process because a failure to control the airway can have disastrous consequences. Proper diagnosis, airway control, antibiotic therapy, and occasionally surgical management are essential to ensure the safety of the patient.

Tongue reduction in Beckwith-Weidemann syndrome.

OBJECTIVE: To review our experience with patients with macroglossia as a component of Beckwith-Weidemann Syndrome (BWS). DESIGN: Chart review of six patients treated with BWS. SETTING: Tertiary care teaching hospital. PATIENTS: Six patients diagnosed with BWS and macroglossia. INTERVENTIONS: Four patients underwent at least one surgical procedure to address their macroglossia. The surgical options and potential complications are discussed. RESULTS: Three patients who have undergone tongue reduction have a functioning tongue with normal mobility. Two patients have required tracheotomy as apart of their management and still have significant tongue enlargement. CONCLUSIONS: Macroglossia as a part of BWS may present a difficult management problem. Various methods of tongue reductions have been reported with mixed results.

Pediatric temporal bone fractures.

OBJECTIVE: To examine the etiology, presentation, and management of temporal bone fractures in children. STUDY DESIGN: Case control. METHOD: Retrospective review of a level I pediatric trauma center from July 1, 1990 to November 1, 1996 identified 680 patients. Inclusion criteria of age less than 14 years and only blunt temporal bone trauma identified 122 patients, with 97 charts available for review. The criteria for temporal bone fracture consisted of both clinical and radiologic information. Only patients with temporal bone fractures confirmed by computed tomography, a complete otolaryngology examination, and audiometric evaluations were included in the study. The data were analyzed with the Kruskal-Wallis analysis of variance (ANOVA) for examining the three separate age groups of fractures. Chi-squared analysis was used to compare these data with previously published adult and pediatric temporal bone fracture series and to examine the three separate age groups of fractures. RESULTS: The review identified 72 children with 79 temporal bone fractures: 47 boys and 25 girls. The patients ranged from 6 months to 14 years of age, with a bimodal distribution of patients with peaks at 3 years and 12 years of age. The most common causes of fractures were motor vehicle accidents (47%), falls (40%), biking accidents (8%), and blows to the head (7%). Common presenting signs and symptoms included hearing loss (82%), hemotympanum (81%), loss of consciousness (63%), intracranial injuries (58%), bloody otorrhea (58%), extremity fractures (8%), and facial nerve weakness (3%). The most common causes of temporal bone fractures were falls and motor vehicle accidents. Forty-two patients were noted to have bloody otorrhea and possible cerebrospinal fluid leak. Twenty-four received intravenous antibiotics. No patient developed prolonged otorrhea or meningitis during hospitalization and the follow-up period. The classification of fracture patterns was longitudinal, 54%; transverse, 6%; oblique, 10%; squamous, 27%; and other, 3%. Hearing loss was found in 59 patients, with conductive hearing loss being the most common finding in 56% of the patients, followed by sensorineural hearing loss in 17% and mixed hearing loss in 10%. CONCLUSIONS: Pediatric temporal bone fractures are associated with falls and motor vehicle accidents. There is a high incidence of associated intracranial injuries and hearing loss, but facial nerve injuries are uncommon. Timely management minimizes complications.

Biochemical and pharmacological characterization of mu, delta and kappa 3 opioid receptors expressed in BE(2)-C neuroblastoma cells.

Total opioid binding in the human neuroblastoma cell line BE(2)-C has a density similar to that found in brain, with a Bmax value of 383 +/- 60 fmol/mg protein and a KD of 0.4 +/- 0.07 nM for the nonselective opioid antagonist 3H-diprenorphine. Selective assays reveal a binding distribution of mu (38%), delta (16%) and kappa 3 (43%) opioid receptors. There is no observable kappa 1 or kappa 2 binding. The sum of the Bmax values in the selective binding assays (370 +/- 39 fmol/mg protein) approximates closely that observed with 3H-diprenorphine, suggesting that mu, delta and kappa 3 sites account for most of the binding. The binding selectivities of various opiates and opioid peptides in the BE(2)-C cells are similar to those in rat brain. Delta and mu binding are defined easily by traditional selective ligands. The binding profiles also distinguish clearly mu from kappa 3 binding. The selective mu ligand DAMGO competes with mu binding over 35-fold more potently than kappa 3 binding, whereas morphine shows a 10-fold selectivity. Functionally, selective mu, delta and kappa 3 agonists inhibit forskolin-stimulated cAMP accumulation through distinct receptor mechanisms that are pertussis toxin-sensitive. In addition to demonstrating that BE(2)-C cells provide a useful model system for studying mu, kappa 3 and delta receptors, these studies confirm that kappa 3 receptors represent a pharmacologically distinct receptor class in this cell line.