Novel method to analyze cell kinetics for the rapid diagnosis and determination of the causative agent in allergy.

Drug-induced allergy (DIA), an unexpectedly triggered side effect of drugs used for therapeutic purposes, is a serious clinical issue that needs to be resolved because it interrupts the treatment of the primary disease. Since conventional allergy testing is insufficient to accurately predict the occurrence of DIA or to determine the drugs causing it, the development of diagnostic and predictive tools for allergic reactions is important. We demonstrated a novel method, termed high-sensitive allergy test (HiSAT), for the rapid diagnosis of allergy (within 1 hr; with true-positive diagnosis rates of 89% and 9% for patients with and without allergy-like symptoms, respectively). HiSAT analyzes the cell kinetics as an index against chemotactic factors in a patient's serum, as different from the diagnosis using conventional methods. Once allergy has occurred, HiSAT can be used to determine the causative medicine using culture supernatants incubated with the subject's lymphocytes and the test allergen. This test is more efficient (60%) than the lymphocyte transformation test (20%). Furthermore, in HiSAT, cell mobility significantly increases in a dose-dependent manner against supernatant incubated with lymphocytes from a subject with pollinosis collected at a time when the subject is without allergic symptoms and the antigen. The result demonstraed that HiSAT might be a promising method to rapidly diagnose DIA or to determine with high accuracy the antigen causing allergy.

Influence of Memantine on Continuous Treatment with Rivastigmine Patches-Retrospective Study Using the Logistic Regression Analysis.

Rivastigmine patches exhibit stable effects when attached once a day, and may reduce Alzheimer's disease (AD) patient's or caregiver's burden. On the other hand, it was reported that adverse events, such as dermal disorder, frequently appeared after the start of rivastigmine administration. We retrospectively investigated medical records in 120 patients with moderate or mild AD in whom rivastigmine administration was started in the Department of Neurology, Fukuoka University Hospital between July 2011 and June 2014 (43 males, 77 females, mean age: 76.9±8.0 years). In 72 patients (60.0%), rivastigmine administration was discontinued within 52 weeks after its start. In 45 of these, it was discontinued before reaching a dose of 18 mg/d which was proven to be effective for AD patients. A primary reason for discontinuation was the appearance or deterioration of adverse events in 64 patients. Of these, 43 complained of dermal disorder, accounting for the highest percentage. To clarify factors influencing the continuous administration of rivastigmine, multivariate analysis was performed in 114 patients meeting criteria. Combination therapy with memantine was extracted as a factor (p=0.008). The results of this study suggest that adherence to combination therapy with rivastigmine and memantine is more favorable than that to monotherapy with rivastigmine.

[Continued Use of Rotigotine Transdermal Patches for Parkinson Disease].

Transdermal patches containing rotigotine, a dopamine agonist (DA) for treatment of Parkinson disease, continuously exert stable effects when applied once daily. Therefore, they are expected to reduce the patient burdens due to complications such as wearing-off and dysphagia. However, dosing is occasionally reduced or discontinued after application because of several reasons such as skin reactions or unsatisfactory efficacy. To identify the risk factors involved in the reduced or discontinued use of rotigotine patches, a retrospective study was conducted with reference to the medical records of patients with Parkinson disease who received rotigotine patches in our hospital. 85 patients were involved in this study. Dosing of rotigotine was reduced or discontinued in 53 patients during the study period. The factors associated with charges in treatment included combination therapy with clonazepam and oral administration of another DA before the application of rotigotine. The reduction or discontinuation rate of rotigotine patches in patients who reduced the equivalent dose of DA on the introduction of rotigotine patches was 94.7%, showing a significantly higher rate compared with 61.3% in the increased dose group. To improve adherence to rotigotine patch therapy, physicians need to carefully consider concomitant drugs and total dose of DAs. (Received December 7, 2015; Accepted February 22, 2016; Published June 1, 2016).

Change of teicoplanin loading dose requirement for incremental increases of systemic inflammatory response syndrome score in the setting of sepsis.

Background Target trough concentrations are recommended for teicoplanin (TEIC) to minimize its adverse effects and to maximize efficacy in sepsis caused by grampositive cocci, including methicillin-resistant Staphylococcus aureus infection. However, optimal doses to attain proper trough values in patients with sepsis have not yet been well established for TEIC. Objective This study investigated whether the systemic inflammatory response syndrome (SIRS) score could predict the pharmacokinetics of TEIC in patients with sepsis. Setting This study was conducted at Fukuoka University Hospital in Japan. Methods We retrospectively reviewed the records of patients using TEIC between April 2012 and March 2015. SIRS positive was defined as infection with a SIRS score ≥2. Estimates of pharmacokinetic parameters were calculated using a Bayesian method. Creatinine clearance rates were estimated by the Cockcroft-Gault formula (eCcr). Main outcome measure Change of TEIC loading dose requirement for incremental increases of SIRS score. Results In total, 133 patients were enrolled: 50 non-SIRS patients and 83 patients with SIRS. The TEIC plasma trough concentration was significantly lower in SIRS than non-SIRS patients (15.7 ± 7.1 vs. 20.1 ± 8.6 µg/mL; P < 0.01), although there was no significant difference in the loading dose administered. Moreover, SIRS scores were increasingly predictive of eCcr and TEIC clearance in a stepwise manner. To achieve the target trough concentration (15-30 µg/mL), the optimal doses required in non-SIRS versus SIRS patients were 12-24 versus 18-30 mg/kg/day, respectively, during the first 48 h. Conclusions These findings suggest that the pharmacokinetics of TEIC are altered in SIRS patients, who required higher doses than non-SIRS patients to achieve the target trough concentration. We suggest that the SIRS score can become a new modality to determine the initial TEIC loading dose.

Elevated permeability of the blood-brain barrier in mice intratracheally administered porcine pancreatic elastase.

Chronic obstructive pulmonary disease (COPD) shows progressive, irreversible airflow limitation induced by emphysema and lung inflammation. The aim of the present study was to determine if COPD conditions induce blood-brain barrier (BBB) dysfunction. We found that the intratracheal administration of porcine pancreatic elastase (PPE; 3 U) induced alveolar enlargement, increased neutrophil number in bronchoalveolar lavage fluid, and decreased blood oxygen saturation in mice at 21 days after inhalation. In parallel with these lung damages, BBB permeability to sodium fluorescein and Evans blue albumin was markedly increased. Our findings demonstrate that COPD conditions are associated with risk for BBB impairment.

Tumor necrosis factor-α reduces beta-amyloid accumulation primarily by lowering cellular prion protein levels in a brain endothelial cell line.

Disruption of beta-amyloid (Aß) transport across the blood-brain barrier is thought to cause Aß accumulation in the brain, thus leading to the development of Alzheimer's disease (AD). As AD patients show increased serum tumor necrosis factor-α (TNFα) levels, we examined the effect of TNFα on the function and expression of Aß transport-related proteins including cellular prion protein (PrP(C)) in the mouse brain microvascular endothelial cell line MBEC4. TNFα decreased PrP(C) levels and intracellular radiolabeled Aß. Similarly, anti-prion protein antibody also decreased radiolabeled Aß. These results suggest that TNFα lowers PrP(C) levels, which in turn, reduces Aß in the brain endothelium.

[Necessity of personalized initial loading dose calculation of teicoplanin by clinical pharmacist-examination of the utility of using systemic inflammatory response syndrome score-].

When teicoplanin (TEIC) is injected at the maintenance dose, a long period is required for achievement of the target plasma trough concentration because of its long elimination half-life. An initial loading dose is necessary for rapid achievement of an effective plasma trough concentration. Thus, we proposed that it is necessary for a pharmacist determine the initial loading dose of TEIC to reach an effective plasma trough concentration rapidly after its administration to a patient. In the present study, we evaluated the effectiveness of initial loading dose determination by pharmacists and physicians by comparing the achievement rate of target plasma trough concentrations (>15 µg/mL) and expression of adverse effects. Among 61 patients, 34 were treated according to an initial loading dose determined by a pharmacist (pharmacist intervention) and 27 were treated according to the treating physician's discretion (non-pharmacist intervention). The achievement rate of target concentrations was 91.2% (plasma trough concentration 23.3±5.3 µg/mL) in the pharmacist intervention group and 25.9% (plasma trough concentration 14.0±5.9 µg/mL) in the non-pharmacist intervention group. There was no difference in the incidence of adverse effects between the two groups. Also, we found that systemic inflammatory response syndrome (SIRS) may have a correlation with plasma trough concentrations of TEIC. We suggest that the SIRS score could become a means way of determining initial loading dose. These findings suggest that it is potentially effective for a pharmacist to determine this initial dose in order to rapidly achieve the target plasma trough concentration of TEIC.

Tumor necrosis factor-α-stimulated brain pericytes possess a unique cytokine and chemokine release profile and enhance microglial activation.

Brain pericytes are involved in neurovascular dysfunction, neurodegeneration and/or neuroinflammation. In the present study, we focused on the proinflammatory properties of brain pericytes to understand their participation in the induction of inflammation at the neurovascular unit (NVU). The NVU comprises different cell types, namely, brain microvascular endothelial cells, pericytes, astrocytes and microglia. Among these, we found pericytes to be the most sensitive to tumor necrosis factor (TNF)-α, possessing a unique cytokine and chemokine release profile. This was characterized by marked release of interleukin (IL)-6 and macrophage inflammatory protein-1α. Furthermore, TNF-α-stimulated pericytes induced expression of inducible nitric oxide synthase and IL-1ß mRNAs, as an index of BV-2 microglial cell activation state, to the highest levels. Based on these findings, the possibility that brain pericytes act specifically as TNF-α-sensitive cells and as effectors of TNF-α through the release of proinflammatory factors, and that, as such, they have a role in inducing brain inflammation, should be considered.

Involvement of CXCL14 in osteolytic bone metastasis from lung cancer.

To investigate the molecular mechanisms of lung cancer-induced bone metastasis, we established a bone-seeking subclone (HARA-B4) from a human squamous lung cancer cell line (HARA) using an in vivo selection method. We compared comprehensive gene expression profiles between HARA and HARA-B4, and identified the critical factors for the formation of bone metastasis using in vitro and in vivo assays. The number of bone metastatic colonies in the hind legs was significantly higher in HARA-B4-inoculated mice than in HARA-inoculated mice at 4 weeks after inoculation. In addition, visceral (adrenal) metastases were not found in HARA-B4-inoculated mice at autopsy, suggesting an increase in cancer cell tropism to bone in HARA-B4. Based on a comprehensive gene expression analysis, the expression level of CXC chemokine ligand 14 (CXCL14) was 5-fold greater in HARA-B4 than in HARA. Results of a soft agar colony formation assay showed that anchorage-independent growth ability was 4.5-fold higher with HARA-B4 than with HARA. The murine pre-osteoblast cell line MC3T3-E1 and the pre-osteoclast/macrophage cell line RAW264.7 migrated faster toward cultured HARA-B4 cells than toward HARA cells in a transwell cell migration assay. Interestingly, CXCL14 was shown to be involved in all events (enhancement of cancer cell tropism to the bone, anchorage-independent growth and/or recruitment of bone marrow cells) based on siRNA experiments in HARA-B4 cells. Furthermore, in clinical specimens of lung cancer-induced bone metastasis, expression of CXCL14 was observed in the tumor cells infiltrated in bone marrow in all specimens examined. CXCL14 was able to promote bone metastasis through enhancement of cancer cell tropism to the bone and/or recruitment of bone marrow cells around metastatic cancer cells.

[The timing of the appearance of oral mucositis induced by concurrent chemoradiotherapy with S-1 for head and neck cancer].

Oral mucositis is a frequent adverse event in patients receiving concurrent chemoradiotherapy for head and neck cancer. Although the management of oral mucositis is essential to improve treatment completion rates, no detailed studies on the time of oral mucositis appearance have been reported. We conducted a retrospective study on the timing of the appearance of oral mucositis induced by concurrent chemoradiotherapy with S-1 for head and neck cancer. A total of 11 patients with head and neck cancer who received concurrent chemoradiotherapy with S-1 were examined. All patients developed oral mucositis within 13.8 ± 5.6 days after the initiation of radiotherapy (20.4 ± 8.1 Gy). In addition, the effects of pain-associated symptoms caused by oral mucositis on the patients' nutritional status, including reduction in caloric intake (24.4% ± 31.1%), weight loss (5.2% ± 5.2%), and duration of a regular diet (24.5 ± 17.1 days), were observed and lasted until the completion of radiation therapy. The delineation of the timing of oral mucositis appearance has become a key motivator for the patients to perform oral care proactively to limit severity and serves as a necessary index for monitoring oral health and managing pain and nutrition.

[Evaluation of the resident pharmacist program of Fukuoka University Hospital].

Fukuoka University Hospital began to employ a resident pharmacist system in 2006. In the present study, to evaluate our resident program, we conducted a questionnaire survey of graduates who completed program as well as staff of the Pharmaceutical Department of this hospital. In addition, based on the results of this survey, we examined the current state and future of postgraduate training programs that can be offered to those who have completed a 6-year pharmacy course. The proportion of residents and staff who responded to the survey was 76.5% (13/17) and 100% (42/42), respectively. Of these two groups, the program was rated as beneficial by 92% and 72%, respectively. Regarding the contents of the training program, both residents and staff highly evaluated guidance on drug management on wards and the preparation of drugs (including anti-cancer agents) because these were useful for actual work. The necessity and usefulness of a resident program under the 6-year course system were also suggested. According to those graduating from a 6-year pharmacy course, a training program should include training instructions for students in a long-term internship during the 1st year, and specialty pharmacist education during the 2nd year. The results of our study suggest that it is advisable to begin to provide graduates on a 6-year course who participate in a resident program with specialized education focused on ward duties from the latter half of the 1st year based on their undergraduate education.

Paracellular barrier and tight junction protein expression in the immortalized brain endothelial cell lines bEND.3, bEND.5 and mouse brain endothelial cell 4.

The blood-brain barrier (BBB) is formed by brain endothelial cells. Many immortalized brain endothelial cell lines have been established; these have been used as in vitro BBB models. The aim of the present study was to assess the paracellular barrier properties of the immortalized mouse brain endothelial cell lines bEND.3, bEND.5 cells, and mouse brain endothelial cell 4 (MBEC4), and those of the primary mouse brain endothelial cells pMBECs. bEND.3 cells showed low permeability to sodium fluorescein and obvious staining of tight junction proteins (claudin-5, occludin and ZO-1) similar to pMBECs; these barrier properties of MBEC4 and bEND.5 cells were low. In addition, bEND.3 cells expressed the highest level of claudin-5 among all cells. These results suggest that bEND.3 cells are a convenient and useful model for evaluating BBB function, especially the paracellular barrier.

Atorvastatin stimulates neuroblastoma cells to induce neurite outgrowth by increasing cellular prion protein expression.

Recently, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors were reported to induce neurite outgrowth in vitro. However, the mechanism underlying this effect remains unclear. Cellular prion protein (PrP(C)) is a ubiquitous glycoprotein present on the surfaces of various cells, including neurons, and is suggested to be involved in neurite outgrowth. Therefore, the present study aimed to determine whether PrP(C) mediates neurite outgrowth induced by HMG-CoA reductase inhibitors. Atorvastatin, a strong HMG-CoA reductase inhibitor, induced neurite outgrowth and increased PrP(C) levels in Neuro2a cells in a time- and dose-dependent manner. PrP(C) mRNA expression was also increased by atorvastatin. Farnesol, a non-sterol mevalonate derivative, attenuated the atorvastatin-induced neurite outgrowth and increase in PrP(C). Neuro2a cells overexpressing PrP(C) showed a remarkable enhancement of atorvastatin-induced neurite outgrowth compared with mock cells transfected with empty pCI-neo vector. These findings suggest that PrP(C) contributes, at least in part, to atorvastatin-induced neurite outgrowth. This phenomenon may be included among the mechanisms underlying decreased risk of Alzheimer's disease in patients treated with HMG-CoA reductase inhibitors.

[Establishment of an educational program concerning adverse drug reaction reporting-system in long-term practical training at a hospital].

The model core curriculum for pharmaceutical education specifies the specific behavioral objectives (SBOs) concerning adverse drug reactions, which aims to train pharmacy students to manage adverse drug reactions. Fukuoka University Hospital has developed a problem-based learning (PBL) program concerning adverse drug reactions as long-term practical training to collect adverse event information, identify adverse effects, and acquire management skills. Students' level of satisfaction with the program was high (approximately 90%), and the mean self-evaluation score for the SBOs concerning adverse reaction was 4.4 (5-grade scale), showing a high level of understanding. In addition, students' will of participation to the adverse drug reaction-reporting system was significantly improved after the PBL program, showing the usefulness of this program (p=0.02). However, the results of the PBL program revealed students' insufficient knowledge of adverse reactions and lack of reviewing skills, suggesting the need to improve the education system whereby students can learn adverse drug reactions in clinical settings.

Evaluation of a practical training program for drug information services for fifth-year pharmacy students in a hospital.

Drug information (DI) services is an essential resource for pharmacists to provide counseling to patients and guide appropriate medication use. We devised a DI practical training course that incorporated an inquiry-based practical training program and evaluated its effectiveness. A total of 91 fifth-year students in Pharmaceutical Sciences at Fukuoka University took part in the following DI sessions based on specific behavioral objectives (SBOs) for DI in the Model Core Curriculum for Practical Training: inquiry practice, simulated pharmacy and therapeutics committee, DI newsletter, use of emergency and safety information, off-label use in clinical trials, PRE-AVOID (Be prepared to avoid the adverse drug reactions), adverse drug reactions, and small group discussions about drug poisoning. The level of understanding of the SBOs for DI training was >4.2 for each item assessed, and the level of satisfaction for each practice was >3.9. This DI practical training successfully facilitated students' ability to provide DI. The number of students interested in DI services significantly increased (p<0.01). After the DI practical training, many students made statements such as "I realized that DI services is a very important job" and "I feel that pharmacists have much to contribute to DI services by evaluating the most appropriate information from a pharmacist's standpoint." It appears that students recognized the pharmacist's role and importance of DI services in clinical practice through the DI training. These results suggest that this DI practical training program was effective.

Involvement of the cellular prion protein in the migration of brain microvascular endothelial cells.

The conversion of cellular prion protein (PrP(C)) to its protease-resistant isoform is involved in the pathogenesis of prion disease. Although PrP(C) is a ubiquitous glycoprotein that is present in various cell types, the physiological role of PrP(C) remains obscure. The present study aimed to determine whether PrP(C) mediates migration of brain microvascular endothelial cells. Small interfering RNAs (siRNAs) targeting PrP(C) were transfected into a mouse brain microvascular endothelial cell line (bEND.3 cells). siPrP1, selected among three siRNAs, reduced mRNA and protein levels of PrP(C) in bEND.3 cells. Cellular migration was evaluated with a scratch-wound assay. siPrP1 suppressed migration without significantly affecting cellular proliferation. This study provides the first evidence that PrP(C) may be necessary for brain microvascular endothelial cells to migrate into damaged regions in the brain. This function of PrP(C) in the brain endothelium may be a mechanism by which the neurovascular unit recovers from an injury such as an ischemic insult.

Measurement and assessment of cytomegalovirus of immunoglobulin (Ig) g titer in preparations.

Cytomegalovirus (CMV) remains the most important pathogen following solid organ transplantation and is the major cause of recipient morbidity and mortality during the first 6 months posttransplantation. To prevent CMV infection and/or to prevent symptomatic CMV disease, immunoglobulin (Ig) G including hyperimmune CMV IgG are used alone or in combination with antiviral medications. The CMV IgG titer, however, has a wide range and frequently depends on the company supplying the Ig preparation even if the preparations come from the plasma pool of a national blood donation agency. In the present study, we therefore simultaneously measured and evaluated the CMV IgG titers in various Ig preparations using two common methods: the neutralizing antibody (NT) and enzyme immunoassay (EIA). The CMV IgG titer in the present study indicated different values using both methods among Ig preparations that were made from the plasma pool of a national blood donation agency (about 3.5- or about 1.7-fold difference using the NT or EIA methods, respectively). Furthermore, there were no correlations in the CMV IgG titer between our findings and published data from the manufacturers, or between the two methods tested here. These findings suggest the importance and necessity of a standard method and/or sample for the measurement and assessment of CMV IgG in Ig preparations.

[Establishment of a system for early exposure provided by new pharmacists in a hospital].

It is very important for students to undergo early exposure in 6-year pharmaceutical education; through this experience they will understand roles of pharmacists, map out their future career, and increase their motivation for learning. Therefore we had newly recruited pharmacists provide an early exposure program in a hospital. According to the results of a questionnaire survey involving students, educational staff of the Faculty of Pharmaceutical Sciences and the new pharmacists, 99% of the students were satisfied with the program, and their motivation for learning was enhanced. Interestingly, they were more careful regarding their grooming and appearance after completing the program. Educational staff of the Faculty of Pharmaceutical Sciences evaluated the new pharmacists and their teaching very positively, and, in turn, the pharmacists assessed an early exposure program in a hospital as significant. Therefore we conclude that the system for early exposure was useful for both students and new pharmacists.

[Pharmaceutical services for patients receiving AC chemotherapy].

General treatments for breast cancer patients, such as surgery, chemotherapy, radiotherapy and lymphatic edema drainage, are performed at the Department of Breast Surgery in Kyushu Central Hospital. In those treatments, pharmacists provide the pharmaceutical treatment. Combination chemotherapy of doxorubicin and cyclophosphamide (AC therapy) is one of the standard regimens for breast cancer. In breast cancer patients who received AC therapy, we carried out investigations on side effects, and prepared pamphlets to support patients' self-management in their daily lives. In the process of preparing pamphlets, we made check sheets to monitor the severity and incidence of side effects. Based on the results of analysis and patients' opinions as well as staff remarks, we prepared pamphlets. According to the evaluation survey, pamphlets are regarded as useful. To meet the needs of patients, we intend to periodically revise pamphlets by continuing investigations on side effects, and provide up-to-date information.

Rapid and sensitive HPLC method for the simultaneous determination of paraquat and diquat in human serum.

A rapid and sensitive HPLC method for the simultaneous determination of paraquat and diquat in human serum has been developed. After deproteinization of the serum with 10% trichloroacetic acid, the samples were separated on a reversed-phase column, and subsequently reduced to their radicals with alkaline sodium hydrosulfite solution. These radicals were monitored with a UV detector at 391 nm. This method permitted the reliable quantification of paraquat over linear ranges of 50 ng - 10 microg/ml and 100 ng - 10 microg/ml for diquat in human serum. The within- and between-day variations are lower than 2.3 and 2.2%, respectively. This technique was also utilized to determine the paraquat and diquat serum levels in a patient who had ingested herbicide (Prigrox L) containing paraquat and diquat.