Bortezomib enhances G-CSF-induced hematopoietic stem cell mobilization by decreasing CXCL12 levels and increasing vascular permeability.

Bortezomib (BTZ) is known to enhance the mobilization of hematopoietic stem and progenitor cells (HSPCs) induced by granulocyte colony-stimulating factor (G-CSF). However, the most effective time at which to administer BTZ to produce this enhancing effect remains debatable, and the precise mechanism underlying the effect of BTZ is poorly understood. We addressed these questions in this article by performing animal experiments. First, in agreement with previous studies, BTZ administration 12 hours before blood collection was most effective for HSPC mobilization; in contrast, BTZ administration 3 days before blood collection negatively affected HSPC harvesting. Next, in terms of the mechanism of action, G-CSF, but not BTZ, downregulated the expression of very late antigen-4 on HSPCs and vascular cell adhesion molecule-1 on bone marrow (BM) stromal cells; however, intriguingly, both G-CSF and BTZ downregulated CXCL12 chemokine expression in BM. Notably, BTZ treatment also increased BM vascular permeability. These results suggest that the pro-mobilization effect of BTZ could involve the dissociation of HSPCs from BM stromal cells triggered by G-CSF, vascular hyperpermeability elicited by BTZ, and downregulation of CXCL12 concomitantly induced by G-CSF and BTZ.

The Effects of Silver Sulfadiazine on Methicillin-Resistant Staphylococcus aureus Biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA), the most commonly detected drug-resistant microbe in hospitals, adheres to substrates and forms biofilms that are resistant to immunological responses and antimicrobial drugs. Currently, there is a need to develop alternative approaches for treating infections caused by biofilms to prevent delays in wound healing. Silver has long been used as a disinfectant, which is non-specific and has relatively low cytotoxicity. Silver sulfadiazine (SSD) is a chemical complex clinically used for the prevention of wound infections after injury. However, its effects on biofilms are still unclear. In this study, we aimed to analyze the mechanisms underlying SSD action on biofilms formed by MRSA. The antibacterial effects of SSD were a result of silver ions and not sulfadiazine. Ionized silver from SSD in culture media was lower than that from silver nitrate; however, SSD, rather than silver nitrate, eradicated mature biofilms by bacterial killing. In SSD, sulfadiazine selectively bound to biofilms, and silver ions were then liberated. Consequently, the addition of an ion-chelator reduced the bactericidal effects of SSD on biofilms. These results indicate that SSD is an effective compound for the eradication of biofilms; thus, SSD should be used for the removal of biofilms formed on wounds.

FF-10501 induces caspase-8-mediated apoptotic and endoplasmic reticulum stress-mediated necrotic cell death in hematological malignant cells.

FF-10501 is a novel inhibitor of inosine monophosphate dehydrogenase (IMPDH). Clinical trials of FF-10501 for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are currently being conducted in the United States. Although it has been shown that FF-10501 induces apoptosis in hematological malignant cells, the intracellular mechanisms of this effect have not been characterized. We conducted an in vitro study to elucidate the mechanisms of FF-10501-induced cell death using 12 hematological malignant cell lines derived from myeloid and lymphoid malignancies. FF-10501 suppressed the growth of each cell line in a dose-dependent manner. However, the clinically relevant dose (40 µM) of FF-10501 induced cell death in three cell lines (MOLM-13, OCI-AML3, and MOLT-3). Investigation of the cell death mechanism suggested that FF-10501 induces both apoptotic and necrotic cell death. FF-10501-induced apoptosis was mediated by caspase-8 activation followed by activation of the mitochondrial pathway in MOLM-13 and MOLT-3 cells. FF-10501 induced necrotic cell death via endoplasmic reticulum stress in OCI-AML3 cells. The present study is the first to identify intracellular pathways involved in FF-10501-induced cell death.

Inhibitory effects of polysorbate 80 on MRSA biofilm formed on different substrates including dermal tissue.

Methicillin-resistant Staphylococcus aureus (MRSA) forms biofilms on necrotic tissues and medical devices, and causes persistent infections. Surfactants act on biofilms, but their mode of action is still unknown. If used in the clinic, cytotoxicity in tissues should be minimized. In this study, we investigated the inhibitory effect of four different surfactants on MRSA biofilm formation, and found that a nonionic surfactant, polysorbate 80 (PS80), was the most suitable. The biofilm inhibitory effects resulted from the inhibition of bacterial adhesion to substrates rather than biofilm disruption, and the effective dose was less cytotoxic for 3T3 fibroblasts. However, the effects were substrate-dependent: positive for plastic, silicon, and dermal tissues, but negative for stainless-steel. These results indicate that PS80 is effective for prevention of biofilms formed by MRSA on tissues and foreign bodies. Therefore, PS80 could be used in medical practice as a washing solution for wounds and/or pretreatment of indwelling catheters.

Adsorption of Nafamostat Mesilate on AN69ST Membranes: A Single-Center Retrospective and In Vitro Study.

We examined whether AN69ST (acrylonitrile and methallyl sulfonate copolymer) membranes adsorb nafamostat mesilate. This study retrospectively analyzed 87 continuous hemodiafiltration sessions in vivo. We divided the continuous hemodiafiltration sessions into AN69ST and non-AN69ST groups using the nafamostat mesilate dose and activated clotting time as indicators of nafamostat mesilate adsorption onto the membrane. Furthermore, we studied the in vitro adsorption of nafamostat mesilate from nafamostat mesilate solutions onto four different hemodialysis membranes. This in vivo study shows that nafamostat mesilate doses were significantly higher, but activated clotting times were shorter (P < 0.001) in the AN69ST group than in the non-AN69ST group. These results suggest that AN69ST adsorbs nafamostat mesilate. Further, the in vitro experiments show that nafamostat mesilate adsorbs AN69ST on membranes significantly more than the other membranes tested. These in vitro and clinical findings provide evidence that AN69ST may adsorb nafamostat mesilate.

Acceleration Mechanisms of Skin Wound Healing by Autologous Micrograft in Mice.

A micrograft technique, which minces tissue into micro-fragments >50 µm, has been recently developed. However, its pathophysiological mechanisms in wound healing are unclear yet. We thus performed a wound healing study using normal mice. A humanized mouse model of a skin wound with a splint was used. After total skin excision, tissue micro-fragments obtained by the Rigenera protocol were infused onto the wounds. In the cell tracing study, GFP-expressing green mice and SCID mice were used. Collagen stains including Picrosirius red (PSR) and immunohistological stains for α-smooth muscle actin (αSMA), CD31, transforming growth factor-ß1 (TGF-ß1) and neutrophils were evaluated for granulation tissue development. GFP-positive cells remained in granulation tissue seven days after infusion, but vanished after 13 days. Following the infusion of the tissue micrograft solution onto the wound, TGF-ß1 expression was transiently upregulated in granulation tissue in the early phase. Subsequently, αSMA-expressing myofibroblasts increased in number in thickened granulation tissue with acceleration of neovascularization and collagen matrix maturation. On such granulation tissue, regenerative epithelial healing progressed, resulting in wound area reduction. Alternative alteration after the micrograft may have increased αSMA-expressing myofibroblasts in granulation tissue, which may act on collagen accumulation, neovascularization and wound contraction. All of these changes are favorable for epithelial regeneration on wound.

Increased drug resistance of meticillin-resistant Staphylococcus aureus biofilms formed on a mouse dermal chip model.

PURPOSE: Meticillin-resistant Staphylococcus aureus (MRSA) biofilm formation in humans is of serious clinical concern. Previous in vitro studies have been performed with biofilms grown only on inorganic substrates; therefore, we investigated the vancomycin (VCM) resistance of MRSA biofilms grown on skin tissue. METHODOLOGY: We established a novel tissue substrate model, namely MRSA grown on segments of mouse skin tissue (dermal chips, DCs), and compared its resistance capacity against VCM with that of MRSA biofilms grown on plastic chips (PCs).Results/Key findings. For one MRSA isolate, we found that the VCM MIC was identical (1.56 µg ml-1) for planktonic cultures and for biofilms-formed on PCs (PC-BF), although the minimum bactericidal concentration (MBC) increased to 6.25 µg ml-1 in PC-BF. On the contrary, the MIC and MBC for biofilms formed on DCs (DC-BF) significantly increased (25 and 50 µg ml-1, respectively). Furthermore, the minimum biofilm-eradicating concentration was higher for DC-BF (100 µg ml-1) than for PC-BF (25 µg ml-1). Using six MRSA strains, we found that in PC-BF, the c.f.u. number decreased with increasing VCM concentration, whereas in DC-BF, it greatly increased until the MIC was reached, accompanied by the formation of large colonies, thicker bacterial walls and the presence of many mitotic cells. CONCLUSION: Our results indicate that the VCM resistance of MRSA was greater in DC-BF. We conclude that DCs may provide a specific environment for MRSA that enhances bacterial growth under cytotoxic VCM concentrations, and might be useful for the study of skin wound infections and the effects of antimicrobial drugs.

A Novel Skin Splint for Accurately Mapping Dermal Remodeling and Epithelialization During Wound Healing.

The mouse excisional dorsal full-thickness wound model with a silicon splint fixed on the skin has been widely used to mimic human wound healing. However, the method cannot accurately quantify dermal remodeling, since the initial point of epithelialization on the wound surface is unclear. To overcome this limitation, we have developed a novel mouse excisional wound model to assess the degree of epithelial extension and regeneration, using a plastic ring-shaped splint fixed beneath the surrounding epidermal tissue. At the end of the experiment, tissue samples were fixed in formalin, the splint was excised, and paraffin sections were prepared. Splint holes, corresponding to the prior location of the splint, were evident on the tissue cross-sections, and the epidermis above the holes was considered the initial excision site. The epidermal contraction and epithelial regeneration, as independent essential tissue alterations in wound healing, could be distinguishable and quantified. Compared with previous splint models, this method provides an accurate evaluation of epidermal processes in wound healing, and can be a platform to assess the effects of various wound healing factors. J. Cell. Physiol. 232: 1225-1232, 2017. © 2016 Wiley Periodicals, Inc.

Inhibition of glucose transporter 1 induces apoptosis and sensitizes multiple myeloma cells to conventional chemotherapeutic agents.

Despite the recent development of anti-myeloma drugs, the prognosis of high-risk multiple myeloma remains poor. Therefore, new effective treatment strategies for this disease are needed. It has been reported that high intensity of 18-fluorodeoxyglucose positron emission tomography is high-risk factor in myeloma, suggesting that glucose uptake can be therapeutic target in high-risk myeloma. In this study, we addressed the utility of glucose transporter 1 (GLUT1) as a therapeutic target for myeloma with increased glucose uptake. We found myeloma cell lines with elevated glucose uptake activity via GLUT1 up-regulation. STF-31, a selective GLUT1 inhibitor, completely suppressed the glucose uptake activity and induced apoptosis in GLUT1 expressing myeloma cells. On the other hand, this agent little shows the cytotoxicity in normal peripheral blood mononuclear cells. Moreover, STF-31 synergistically enhanced the cell death induced by melphalan, doxorubicin, and bortezomib. GLUT1 may be promising therapeutic target in myeloma with elevated glucose uptake.

Therapeutic Dose Monitoring for Linezolid in a Patient with MRSA Pneumonia with Bacteremia in Diabetes Insipidus.

INTRODUCTION: Few studies have investigated the effect of increased creatinine clearance (CrCl) on linezolid (LZD) concentration. Herein, we report the pharmacokinetic/pharmacodynamic (PK/PD) profile of LZD used in the management of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia with concomitant bacteremia in a patient with high CrCl caused by diabetes insipidus (DI). CASE REPORT: A 19-year-old man was admitted to the intensive care unit following a traumatic brain injury. After admission, he underwent a craniotomy for the severe brain injury. However, he developed DI after the operation. Despite treatment with vasopressin, his urine output reached 5-6 L/day as a result of the DI, and his CrCl increased to 180-278 mL/min. We were required to administer 6-7 L of fluid a day to compensate for the high urinary fluid output. On day 55, MRSA pneumonia with sepsis was suspected and, consequently, LZD was administrated intravenously (600 mg every 12 h). He was treated with LZD for 14 days. The patient has since successfully recovered from MRSA pneumonia with concomitant bacteremia, and was transferred to the general ward on day 82. Blood LZD levels from days 60-68, which were measured after the patient's transfer to the general ward, showed that the trough levels were lower than the threshold level of detection. The blood 24-h area under the plasma LZD concentration-time curve (AUC)24/minimum inhibitory concentration (MIC) was 69.3. CONCLUSION: In spite of the low level of LZD AUC24/MIC caused by the high CrCl with DI, MRSA pneumonia with concomitant bacteremia was successfully treated with LZD.

5-Fluorouracil Chemotherapy for Dihydropyrimidine Dehydrogenase-deficient Patients: Potential of the Dose-escalation Method.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) degrades approximately 85% of administered 5-fluorouracil (5-FU). With a reported high mortality rate, chemotherapy is generally contraindicated for patients with DPD deficiency. PATIENTS AND METHODS: Chemotherapy was initiated for a 73-year-old man with DPD deficiency. Capecitabine was administered in incrementally increasing doses, beginning with a single pill while monitoring plasma 5-FU concentration, and neutrophil and platelet counts. RESULTS: DPD protein level was 2.35 U/mg. After increasing the capecitabine dose to 1,800 mg, oxaliplatin and bevacizumab were added. Subsequent DPD protein measurement showed that the level had increased to approximately 12-fold the one before chemotherapy. Sequencing of all 23 exons of DPYD gene revealed a mutation of guanine to thymine in exon 11 (1156 G>T). CONCLUSION: This is the first report to indicate that DPD activity can be induced. These findings may provide early indications of a new method for chemotherapy for DPD-deficient patients.

A simple high-performance liquid chromatography for the determination of linezolid in human plasma and saliva.

Linezolid is an antimicrobial agent for the treatment of multiresistant Gram-positive infections. A practical high-performance liquid chromatography method was developed for the determination of linezolid in human plasma and saliva. Linezolid and an internal standard (o-ethoxybenzamide) were extracted from plasma and saliva with ethyl acetate and analyzed on a Capcell Pak C18 MG column with UV detection at 254 nm. The calibration curve was linear through the range 0.5-50 µg/mL using a 200 µL sample volume. The intra- and interday precisions were all <6.44% for plasma and 5.60% for saliva. The accuracies ranged from 98.8 to 110% for both matrices. The mean recoveries of linezolid were 80.8% for plasma and 79.0% for saliva. This method was used to determine the plasma and saliva concentrations of linezolid in healthy volunteers who were orally administered a 600 mg dose of linezolid. Our liquid-liquid extraction procedure is easy and requires a small volume of plasma or saliva (200 µL). This small volume can be advantageous in clinical pharmacokinetic studies, especially if children participate.

Infection mechanism of biofilm-forming Staphylococcus aureus on indwelling foreign materials in mice.

Indwelling foreign-body infections are a critical medical problem, especially in immunocompromised patients. To examine the pathogenicity of biofilm-forming bacteria settling on foreign materials, mice implanted with plastic discs were infected with Staphylococcus aureus. After opening a wide subcutaneous pocket on the dorsal side of mice with or without temporal leukocytopenia, a plastic sheet was placed in the left subcutaneous space; subsequently, bacteria in a planktonic state were dispersed over the subcutaneous space. Bacterial numbers were examined 7 days after inoculation. In subcutaneous tissue on the right, S. aureus was found only in leukocytopenic mice. Meanwhile, bacteria were detected on the plastic and neighbouring tissue in both leukocytopenic and normal mice; however, colony-forming analysis indicated that leukocytopenic mice possessed significantly more bacteria. Tissue reaction against bacteria was pathologically examined. Invading S. aureus induced severe inflammation. In transient leukocytopenic mice, bacterial microcolonies formed on the plastic as well as in the developed necrotic tissue - both of which were shielded from inflammatory cell infiltration - result in bacteraemia. These results indicate that biofilm-forming S. aureus settling on indwelling foreign material are tolerant against host immunity and assault neighbouring tissue, which may lead to chronic wound infection.

Safety and pharmacokinetic evaluation of repeated intravenous administration of palonosetron 0.75 mg in patients receiving highly or moderately emetogenic chemotherapy.

PURPOSE: The aims of this study were to evaluate the safety, efficacy, and pharmacokinetics of repeated doses of palonosetron 0.75 mg on days 1 and 3 in Japanese patients who received highly or moderately emetogenic chemotherapy. METHODS: Twenty- six patients received palonosetron 0.75 mg intravenously before chemotherapy on days 1 and 3 plus dexamethasone (12-16 mg before chemotherapy on day 1 and 4-8 mg on days 2 and 3). The primary endpoints were safety and pharmacokinetics. Pharmacokinetics were evaluated in a subset of patients (n=6). Complete response and complete protection were evaluated as secondary endpoints. RESULTS: The accumulation ratios for C max and AUClast after the second dose on day 3 were 1.42 and 1.37, respectively. These values were consistent with the theoretical values expected from the half-life of palonosetron on day 1. Almost all of the patients had no nausea or vomiting in the acute phase (complete response (CR) rate, 96.2% [25/26]; CP rate, 92.3% [24/26]). In the delayed phase (24-192 h post-chemotherapy), the complete response and complete protection rates were 76.9% (20/26) and 61.5% (16/26), respectively. Treatment was well tolerated. CONCLUSIONS: This is the first study to report the pharmacokinetics of multiple doses of palonosetron 0.75 mg, given on days 1 and 3, in Japanese patients. Repeated treatment with palonosetron was safe and well tolerated by patients who received highly or moderately emetogenic anticancer chemotherapy.

[A study of the sialogogic effect of cevimeline gargle].

The purpose of this study was to clarify the sialogogic effect iveness of cevimeline gargle. Sequential comparison tests were conducted on healthy adult males and females who received both cevimeline and a placebo. The effect was evaluated by measuring the amount of saliva using the Saxon test and Mucus, and by recording the subjects' own perceptions of oral mucosal moisture reported according to 4 levels. First, ten subjects' measurements were taken before gargling, as well as 5 minutes, 90 minutes and 3 hours after gargling. For the cevimeline treatment, the amount of saliva reached the maximum level within 5 minutes after gargling, and a significant salivary secreting effect(p<0. 05-p<0. 001)was observed up to 90 minutes after gargling. The degree of oral moisture also reached the maximum level within 5 minutes after gargling with cevimeline, and a significant moisture effect(p<0. 05-p<0. 001)persisted up to 3 hours after gargling. After safety profiles were determined to be acceptable, an additional 19 subjects entered the study and the same results were observed. The present data, indicating that cevimeline gargle increases the saliva in healthy individuals for at least 3 hours, will be useful in clinical trials of cevimeline gargle for patients with salivary secretion disorder.

Simultaneous determination of cytosine arabinoside and its metabolite, uracil arabinoside, in human plasma using hydrophilic interaction liquid chromatography with UV detection.

A practical high-performance liquid chromatography using a Cosmosil HILIC column and UV detection was developed for determining the concentrations of cytosine arabinoside (Ara-C) and uracil arabinoside (Ara-U), which is a major metabolite of Ara-C, in human plasma. This method was used to determine the plasma concentrations of Ara-C and Ara-U in a patient treated with high-dose Ara-C therapy for end-stage renal failure.

Importance of inducible multidrug resistance 1 expression in HL-60 cells resistant to gemtuzumab ozogamicin.

Resistance to gemtuzumab ozogamicin (GO) hampers the effective treatment of refractory acute myeloid leukemia (AML). To clarify the mechanism of resistance to GO, HL-60 cells were persistently exposed to GO in order to establish GO-resistant HL-60 (HL-60/GOR) cells. Multidrug resistance 1 (MDR-1) was strongly expressed in HL-60/GOR cells, but not in HL-60 cells. Although withdrawal of GO after the chronic exposure of HL-60/GOR cells to this compound gradually decreased MDR-1 expression to trace levels, reintroducing GO restored high MDR-1 expression in HL-60/GOR cells, but not in HL-60 cells. These results indicate that HL-60/GOR cells acquired the ability to induce MDR-1 expression in response to GO. U0126, a MEK1/2 inhibitor, prevented GO-inducible MDR-1 expression and abrogated GO resistance in HL-60/GOR cells. These results suggest that in the clinical use of GO, inducible MDR-1 expression in tumor cells should be investigated before treatment with GO. If the cells are positive then MEK1/2 inhibitors may be effective in overcoming resistance to GO.

Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction.

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N(10-) methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7-OH-MTX, DAMPA and 2,4-diaminopteroic acid (DAPA) as an internal standard were separated on a reversed-phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7-OH-MTX and DAMPA from plasma at 100 pmol/mL were 81.5±5.4, 82.5±5.3 and 56.2±7.0%, respectively. The limits of quantification for MTX, 7-OH-MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within- and between-day variations for MTX, 7-OH-MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy.

Interferon-α and zidovudine for relapsed/refractory adult T cell leukemia/lymphoma: case reports of Japanese patients.

Combination therapy with interferon-α and zidovudine (IFN/AZT) has been regarded as standard care for acute and indolent (i.e., chronic and smouldering) ATL based on reports involving a limited number of patients. This treatment approach has not been evaluated in Japan, a major endemic area of this disease in the world. This is the first Japanese report of IFN/AZT for ATL. It is impossible to draw any definitive conclusion from this small study; however, IFN/AZT showed clear anti-ATL effects for refractory/relapsed ATL patients. This report would contribute for developing future ATL treatment in Japan.

Population pharmacokinetic study of a test dose oral busulfan in Japanese adult patients undergoing hematopoietic stem cell transplantation.

PURPOSE: The aim of this study is to determine the population pharmacokinetics of oral busulfan in Japanese adults. METHODS: We previously underwent a clinical trial involving the dose adjustment of oral busulfan depending on the individual pharmacokinetics using a test dose in hematopoietic stem cell transplantation recipients. Seventy-one Japanese patients aged from 16 to 67 years were enrolled. After taking oral busulfan 0.5 mg/kg as a test dose, blood samples were collected at five time points from each patient. Busulfan concentrations were measured by high-performance liquid chromatography, and the individual parameters were estimated by using the nonlinear mixed effects model computer program. A one-compartment model with first-order absorption was sufficient to describe the concentration-time profile. RESULTS: The final pharmacokinetic parameter were the clearance (CL/F) = 0.153 L/h/kg, distribution volume (Vd/F) = 0.695 L/kg, and absorption rate constant (ka) = 2.39 h(-1). The inter-individual variabilities in CL/F, Vd/F and ka were 25.9, 26.2, and 111.8%, respectively, and the residual variability was 12.1% as the coefficient of variation. CONCLUSION: We developed a population pharmacokinetic model of oral busulfan in Japanese adults. The final population model was implemented into the program excel, leading to an easy and proper therapeutic monitoring of oral BU by using small number of samples.