Horizontal and vertical microchamber platforms for evaluation of the paracellular permeability of an epithelial cell monolayer.

Epithelial cells serve as a barrier by tightly adhering to each other and contribute to the homeostasis of living organisms by controlling substance permeation. Therefore, evaluation of the barrier function is important in pharmaceutical development processes. However, the widely used Transwell-based assays require the development of the defect-free epithelial cell monolayer above several tens of mm2, often resulting in low reproducibility and requiring a long incubation time. In addition, the culture surface of cells is far from the bottom of the well plate, making it difficult to observe the cell morphology using an optical microscope. Herein, we propose simple polydimethylsiloxane microfluidic devices for evaluating the barrier function of an epithelial monolayer using a microchamber array. After the formation of the epithelial monolayer over microchambers, the permeation of the marker molecules introduced above resulted in increased fluorescence intensity in microchambers, which was monitored using confocal laser scanning microscopy. We show that using this technique, alteration of the paracellular permeability induced by sodium caprate (C10) and cytochalasin-D, permeation enhancing factors, can be elucidated. Furthermore, by tilting the microchamber device 90 degrees, the vertical cell section and microchambers were imaged in the same focal plane, allowing for live visualization of the passage of fluorescent substances across the cell monolayer. This technique is expected to be useful for investigating the relationship between paracellular permeability and cell morphology, which is unattainable through conventional methods.

A simple microfluidic device for live-imaging of the vertical section of epithelial cells.

We investigated the capability of simple microfluidic devices with trenches having vertical sidewalls for live-cell fluorescence imaging of adherent cells. An epithelial cell line that forms a two-dimensional (2D) sheet was cultured to adhere to the vertical sidewall so that its vertical section can be imaged directly using ordinal inverted-type laser-scanning microscopy. The material and the structure of the device were characterized. We show that the detailed distribution of intracellular organelles, such as microtubules and mitochondria, and of intercellular apparatus, such as claudin and zonula occludens, can be imaged with high spatio-temporal resolution with a single scan.

A New Approach for Determining Short-Term, Objective Prognostic Predictive Methods for Terminal Cancer Patients Based on the Change Point of Laboratory Test Values.

BACKGROUND: In terminal phase cancer, predicting a prognosis precisely plays an important role for patients and their families to live meaningful lives. However, there are no established short-term, objective prognostic predictive methods. OBJECTIVE: To develop simple, short-term, objective prognostic predictive methods through detecting a change point for laboratory test values. DESIGN: A retrospective chart review. SETTING/SUBJECTS: Subjects were cancer patients aged ≥16 years and discharged dead from Osaka University Hospital in 2008. MEASUREMENTS: Using different laboratory test values, new prognostic predictive methods were determined based on either six laboratory test values (white blood cell [WBC], platelet [PLT], C-reactive protein, blood urea nitrogen [BUN], aspartate aminotransferase [AST], and lactase dehydrogenase [LDH]): the WPCBAL score, or five test values (WBC, PLT, BUN, AST, and LDH): the WPBAL score. Their utility, including sensitivity and specificity, was compared with that of Glasgow prognostic scores (GPSs). RESULTS: In total, 121 cancer patients were enrolled. WPCBAL and WPBAL scores showed higher sensitivity (0.88 and 0.91 vs. 0.68), specificity (0.79 and 0.70 vs. 0.53), negative predictive value (0.98 and 0.97 vs. 0.76), and a much larger relative risk (16.5 and 14.2 vs. 1.78) as prognostic predictors within two weeks of death than GPS as a prognostic predictor within three weeks of death. CONCLUSION: This is the first study that suggests that the objective prognostic predictive methods, through detecting the change point of laboratory test values, are useful for predicting short-term prognosis. The WPCBAL score and WPBAL score could objectively predict the remaining lifetime within two weeks of mortality.

Microchamber device for detection of transporter activity of adherent cells.

We present a method to detect the transporter activity of intact adherent cells using a microchamber device. When adherent cells are seeded onto the poly-di-methyl siloxane substrate having microchambers with openings smaller than the size of a cell, the cells form a confluent layer that covers the microchambers, creating minute, confined spaces. As substances exported across the cell membrane accumulate, transporter activity can be detected by observing the fluorescence intensity increase in the microchamber. We tested the microchamber device with HeLa cells over-expressing MDR1, an ATP-binding cassette transporter, and succeeded in detecting the transport of fluorescence-conjugated paclitaxel, the anti-cancer drug, at the single-cell level.

L-glutamine decreases the severity of mucositis induced by chemoradiotherapy in patients with locally advanced head and neck cancer: a double-blind, randomized, placebo-controlled trial.

The incidence of severe mucositis in the oral cavity, pharynx and larynx is high among patients with head and neck cancer (HNC) receiving chemoradiotherapy (CRT), resulting in significant pain and impairment of quality of life. The present study investigated whether L-glutamine (glutamine) decreases the severity of mucositis in the oral cavity, pharynx and larynx induced by CRT. This double-blind, randomized, placebo-controlled trial included 40 untreated patients with squamous cell carcinoma of the nasopharynx, oropharynx, hypopharynx or larynx. Patients received 66 or 70 Gy of total radiation at the rate of 2 Gy/fraction daily and 5 fractions/week. Cisplatin (20 mg/m2) and docetaxel (10 mg/m2) were intravenously co-administered once a week for 6 weeks. Patients were randomized to orally receive either glutamine (group G) or placebo (group P) at a dose of 10 g 3 times a day throughout the CRT course. Mucositis was assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0. The primary end point was mucositis severity. Mucositis developed in all patients. A maximal mucositis grade of G4 was observed in 0 and 25% group G and P patients, respectively, while that of G2 was observed in 10 and 0% group G and P patients, respectively (p=0.023). Glutamine significantly decreased the maximal mucositis grade (group G, 2.9±0.3; group P, 3.3±0.4; p=0.005) and pain score at weeks 4, 5 and 6. Glutamine significantly decreased mucositis severity in the oral cavity, pharynx and larynx induced by CRT in patients with HNC.

[Effect of the administration of zoledronic acid on life expectancy in patients with multiple myeloma with or without renal impairment].

Zoledronic acid(ZA)is believed to exert anticancer effects in patients with multiple myeloma(MM). For patients with impaired renal function, its dosage should be determined according to creatinine clearance(Ccr). However, there is no reported difference in life expectancy improvement between those with and without renal impairment. Therefore, we conducted a retrospective study to investigate this clinical question. Seventy-eight MM patients receiving ZA injections were selected and divided into 2 groups: (1)normal group(n=39), baseline Ccr≥60mL/min, and(2)impaired group(n=39), baseline Ccr<60mL/min. Patients in the normal group received a significantly higher initial dose(p<0.001), were of a younger age(p<0.001), had lower b2-microglobulin(b2-M)levels(p<0.001), and had higher rates of prior hematopoietic stem cell transplantation(p<0.001)than those in the impaired group. We then compared the survival rate between 31 patients in the normal group and 27 patients in the impaired group whose treatment outcome data were available and found no significant difference(p=0.251). Therefore, our results suggest that the survival rate on ZA administration may not differ between MM patients with and without renal impairment.

Polysulfides are possible H2S-derived signaling molecules in rat brain.

Accumulating evidence shows that hydrogen sulfide (H2S) has a variety of physiological functions. H2S is produced from cysteine by 3 sulfurtransferases. H2S, in turn, generates polysulfides, the functions of which are not well understood. H2S induces Ca(2+) influx in astrocytes, a type of glia. However, the receptor that mediates the response has not been identified. Here, we have shown that polysulfides induce Ca(2+) influx by activating transient receptor potential (TRP)A1 channels in rat astrocytes (EC50 91 nM, Hill coefficient value 1.77±0.26) and that the maximum response was induced at 0.5 µM, which is 1/320 of the concentration of H2S required to achieve a response of similar magnitude (160 µM, EC50 116 µM). TRPA1-selective agonists, allyl isothiocyanate and cinnamaldehyde, induced Ca(2+) influx, and responses to polysulfides were suppressed by TRPA1-selective inhibitors, HC-030031 and AP-18, as well as by siRNAs selective to TRPA1. The present study suggests that polysulfides are possible H2S-derived signaling molecules that stimulate TRP channels in the brain.

Can gradual dose titration of ketamine for management of neuropathic pain prevent psychotomimetic effects in patients with advanced cancer?

BACKGROUND: Ketamine is often used to manage neuropathic pain in patients with cancer. However, it occasionally causes psychotomimetic effects such as vivid dreams, nightmares, illusions, hallucinations, and altered body image. OBJECTIVE: To examine whether gradual dose titration of ketamine for management of neuropathic pain prevents psychotomimetic effects in patients with advanced cancer. METHODS: This was a retrospective chart review. We administered ketamine when neuropathic pain in patients with advanced cancer became refractory to opioids and oral adjuvant analgesics. The starting dose of ketamine was 10 mg/d by continuous intravenous infusion. The dose was gradually increased by 10 mg/d every 4 to 6 hours to 50 mg/d or until the pain was relieved. It was subsequently increased by 25 mg/d every 12 to 24 hours until the pain was relieved. RESULTS: For this study, we enrolled 46 patients with advanced cancer. The mean age was 52.2 ± 16.9 years. The mean dose at onset of action and maximum dose of ketamine were 56 ± 58 and 272 ± 214 mg/d, respectively. The mean pain intensity (numerical rating scale) decreased significantly from 7.3 ± 2.0 to 3.5 ± 2.2 after the administration of ketamine (P < .01). The effectiveness was 69.5%. No psychotomimetic effect of less than 300 mg/d was observed during the introduction phase even though psychotropic drugs were not prescribed. Mild sedation was observed in 3 patients (7%) as the only adverse effect during the introduction phase. CONCLUSION: Gradual dose titration of ketamine for management of neuropathic pain can prevent psychotomimetic effects in patients with advanced cancer.

A retrospective chart review of terminal patients with cancer with agitation and their risk factors.

BACKGROUND: Agitated delirium is often observed in terminal patients with cancer. To clarify the risk factors for agitated delirium in terminal patients with cancer, we conducted a retrospective chart review of 126 patients with cancer who died at a university hospital in 2008. METHOD: As a working definition, we define agitated delirium as a score of 2 or more in item 9 of the Memorial Delirium Assessment Scale with diurnal variation. RESULTS: The results were as follows: agitated delirium was observed in 49 (42%) of the 115 patients, and it occurred within the last week before death in 49% of the patients. Univariate analysis revealed older age, male gender, smoking history, lung cancer, diabetes, and high C-reactive protein (CRP) value as major risk factors, while dendritic analysis revealed lung cancer, high CRP value, diabetes, older age, and smoking history as key factors for predicting agitation. CONCLUSION: It is necessary to consider risk factors in order to categorize terminal patients with cancer into high- and low-risk groups and undertake possible counter-measures.

Can milnacipran used for neuropathic pain in patients with advanced cancer cause neuromuscular and somatosensory disorders?

BACKGROUND: Milnacipran is one of the classes of drugs that are serotonin and norepinephrine reuptake inhibitors (SNRIs). It is a promising drug for the treatment of neuropathic pain in patients with advanced cancer. However, we found that neuromuscular and somatosensory disorders occurred when milnacipran was used as an adjuvant analgesic. CASE REPORT: A 66-year-old woman with a history of neuropathic pain was given 15 mg of milnacipran after dinner. The next morning she developed stiffness of the fingers, numbness in the mandible, and the soles of her feet felt swollen. Milnacipran was discontinued and her symptoms disappeared immediately. We managed this case, which was becoming severe, by discontinuing milnacipran on early detection of symptoms. DISCUSSION: This is the first report that demonstrates an adverse reaction of milnacipran when used as an analgesic adjuvant, and not as an antidepressant drug, for neuropathic pain in patients with advanced cancer. The analgesic effect of SNRIs will likely be used in the management of neuropathic pain in the future; however, clinicians should be aware of the early adverse reactions to these agents.

Light-induced deterioration test of carboplatin under clinical settings.

Chemotherapeutic drug dosages are calculated precisely based on the patient's height, body weight, and renal function, etc. To ensure safe and favorable outcomes of treatment, dosing solutions are prepared by appropriate mixing of the drug solutions based on such calculations. The package inserts for many injectable preparations include a warning for storing the product "shielded from light." However, there are no reports of stability assessment of a mixed product against light exposure or the residual amount of active ingredient in the dosing solution during or at the end of treatment. We evaluated the stability of carboplatin from the time of mixing of the dosing solution until the end of drug infusion in a clinical-like setting. With 4-hour exposure to outdoor scattered light, the dosing solution began to show discoloration by 1 hour, becoming dark yellow by 4 hours, with reduction of the percent residual carboplatin to about 23%. To identify the optimal light-shielding shade, the dosing solution was shielded from outdoor scattered light with 1 of 3 protective covers: aluminum foil, yellow plastic shade, and brown plastic shade. The yellow plastic shade prevented any changes of the appearance of the dosing solution during the 4-hour exposure period. The percent residual carboplatin, determined by HPLC, in the dosing solution shielded with a yellow plastic shade was about 85.2% at 2 hours and 78.6% at 4 hours. Thus carboplatin dosing solution should be completely shielded from light until infusion is completed.

Can anti-infective drugs improve the infection-related symptoms of patients with cancer during the terminal stages of their lives?

BACKGROUND: Appropriate use of anti-infective drugs is essential in clinical practice. No evidence-based guidelines or protocols have been published on the appropriate use of anti-infective drugs in patients receiving palliative care as yet. METHODS: The medical records, which included the demographic data of patients, anti-infective drug use, bacteriologic findings, symptoms, and hematologic findings were reviewed retrospectively to determine the potential factors that contribute to symptom improvement of patients in terminal phase. RESULTS: Seventy-one patients (64%) who received anti-infective drugs and had a total of 326 episodes of infection were assessed. Symptom improvement was seen in 33.1%. A total of 22.6% of episodes were started on anti-infective drugs during the last week of life and the symptom improvement in these episodes was 9.2%. Symptom improvement was hardly observed when the anti-infective drug was administered during the last week of life. The association between the decrease in the C-reactive protein (CRP) levels, the decrease of the leukocyte count, reduction of fever, and symptom improvement was determined. The decrease of CRP levels was 42.4%; leukocyte, 56.7%; and reduction of fever was 28.4%. The symptom improvement of individual treatment history was also investigated. The symptom improvement of the group who took positive treatment such as chemotherapy, radiotherapy, surgery, and catheter placement was significantly lower than that of no-treatment group. CONCLUSIONS: Active cancer treatment probably induces the symptoms related to infection and the use of anti-infective drugs. Unnecessary and excessive treatment should be avoided, and the symptoms should be managed with consideration of the patient's state of mind in order to improve the quality of life of terminally ill patients.

A retrospective chart review of opioid-induced nausea and somnolence on commencement for cancer pain treatment.

Morphine, oxycodone, and fentanyl are major opioids available as controlled-release morphine (CRM), controlled-release oxycodone (CRO), and transdermal fentanyl (TDF), respectively, in Japan. The authors conducted a retrospective chart review to examine (1) nausea and somnolence on commencement of CRM, CRO, and TDF for cancer pain treatment, (2) the antiemetic effectiveness of prochlorperazine to prevent opioid-induced nausea, and (3) the side effect of prochlorperazine on somnolence in patients with cancer pain. Four hundred thirteen patients with cancer were prescribed with CRM (N = 66), CRO (N = 196), and TDF (N = 151). The incidence of nausea on commencement of the TDF group (6.8 percent) was significantly lower than that of both the CRM group (22.6 percent) and the CRO group (35.4 percent; p < 0.001). There was no significant difference in the incidence of nausea on commencement of all groups combined with prochlorperazine at dosage of 15 mg/d. The incidence of somnolence on commencement of the TDF group (9.0 percent) was significantly lower than that of both the CRM group (31.3 percent) and the CRO group (41.5 percent; p < 0.001). The incidence of somnolence on commencement of the CRO group combined with prochlorperazine was significantly higher than that of the CRO combined without prochlorperazine (p < 0.05). In conclusion, the incidence of nausea and somnolence on commencement of TDF are significantly lower than that of both CRM and CRO for cancer pain treatment. Prochlorperazine at a dosage of 15 mg/d may not be effective in preventing opioid-induced nausea and may cause somnolence in patients with cancer pain.

Differentiated astrocytes acquire sensitivity to hydrogen sulfide that is diminished by the transformation into reactive astrocytes.

Hydrogen sulfide (H2S) enhances the induction of hippocampal long-term potentiation (LTP) and induces calcium waves in astrocytes. Based on these observations, H2S has been proposed to be a synaptic modulator in the brain. Here we show that differentiated astrocytes acquire sensitivity to H2S that is diminished by their transformation into reactive astrocytes. Although sodium hydrosulfide hydrate (NaHS), a donor of H2S, did not increase the intracellular concentration of Ca2+ in progenitors, exposure of progenitors to leukemia inhibitory factor (LIF), which induces differentiation into glial fibrillary acidic protein (GFAP)-positive astrocytes, greatly increased the sensitivity to NaHS. In contrast, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), dibutyryl cyclic AMP (db cAMP) and interleukin-1beta (IL-1beta) induced the conversion to reactive astrocytes with diminished sensitivity to NaHS. This suppressive effect of EGF on the sensitivity to NaHS was inhibited by cycloheximide, indicating that de novo protein synthesis was required for the suppression of H2S sensitivity.