Exploration of optimal dosing regimens of vancomycin in patients infected with methicillin-resistant Staphylococcus aureus by modeling and simulation.

WHAT IS KNOWN AND OBJECTIVE: Vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infection and shows time-dependent bacterial killing. The current study evaluated the pharmacokinetics (PK) and pharmacodynamics (PD) of vancomycin and explored its optimal dosing regimens by modeling and simulation. METHODS: Pharmacokinetics study was performed for 20 patients who were treated with vancomycin intravenously, 1000 mg, every 12 h, and blood for PK was randomly drawn within prespecified time windows. PD study was in vitro time-kill experiment for vancomycin against 20 MRSA strains independent of the PK study, where bacterial titre was measured at 0, 2, 4, 8, 24 h after the beginning of vancomycin exposure at 0, 1, 2, 4, 8, 16, 32× minimum inhibitory concentrations. PK and PD models were built from each data set, and simulation for MRSA titre changes over time in human body was performed for various vancomycin dosing regimens using NONMEM(®) . RESULTS: Vancomycin followed a two-compartment PK model, and creatinine clearance was the significant covariate affecting the clearance of vancomycin. PD model described the in vitro time-kill data well. The PK/PD model predicted clear dose-response relationships of vancomycin. The therapeutic dosing regimens of vancomycin, suggested by the simulation studies, showed good agreement with the current clinical practice guidance, which indicates that this PK/PD modeling and simulation approach could prove useful for identifying optimal dosing regimens of other antibiotics and expediting novel antibiotic development. Using PD model from in vitro time-kill study and human PK model from phase 1 study, we could predict whether the drug is going to be efficacious or obtain insight into the optimal dosing regimens for a novel antibiotic agent in the early phases of drug development process.

Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes.

Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ10), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 µM H2O2)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ10 can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ10 not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ10 prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ10 preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ10 may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies.

[Recovery from parkinsonism with N-acetylcysteine-differentiated neurons].

The upregulation of dopaminergic neuronal differentiation is necessary for stem cell therapy in Parkinson's disease (PD). In this study, neuronal differentiation efficiency increased by more than 2 times in P19 embryonic stem cells (ESCs) induced by N-acetylcysteine (NAC) and retinoic acid (RA) as compared to RA alone, with suppressed glial differentiation. The majority of NAC-treated stem cells grafted into brains of PD mice differentiated into dopaminergic neurons and persisted well for 6 weeks. Parkinsonism was also greatly improved after grafting NAC-treated cells in comparison to cells treated with only RA. Our results strongly suggest that NAC treatment may be an effective strategy for generating stem cells fated to become dopaminergic neurons for PD clinical therapy.

N-acetylcysteine enhances neuronal differentiation of P19 embryonic stem cells via Akt and N-cadherin activation.

We examined whether N-acetylcysteine (NAC) enhanced embryonic body (EB) formation and neuronal differentiation in terms of EB formation, neuronal marker (microtubule-associated protein 2; MAP-2) expression, and neuron maturation using P19 embryonic stem cells. The size and numbers of EBs were greatly increased, together with the up-regulated N-cadherin expression. Also, MAP-2 expression and neurite outgrowth were much increased with activation of serine/threonine protein kinase (Akt) and blocked by addition of an Akt inhibitor (LY294002). Our results suggested that NAC increased EB formation by up-regulating the N-cadherin expression. Furthermore, NAC-enhanced neuronal differentiation was mediated by activation of Akt.

A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics.

Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1(enu/+) mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1(enu/+) mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.

Correlation between the immature characteristics of umbilical cord blood-derived mesenchymal stem cells and engraftment of hematopoietic stem cells in NOD/SCID mice.

Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSC) facilitate the engraftment of human (h) hematopoietic stem cells when transplanted simultaneously in animal and human studies. However, the type of MSCs that preferentially enhance the engraftment of HSCs is unknown. Recent studies have shown that MSCs derived from a single source are heterogeneous in terms of cell size, morphology, proliferation rate, and differentiation potential. This study was designed to investigate the properties of UCB-MSCs, which influence the engraftment of hHSCs in a NOD/SCID mouse model. We categorized MSCs as being the most effective (UCB-352 MSCs) or the least effective (UCB-156 MSCs) at promoting the homing and engraftment of HSCs, and compared the characteristics of these 2 MSC populations. We observed that the 2 populations showed differences in characteristics typical of immature MSCs, and related to proliferation potential. We showed that UCB-352 MSCs, which proliferate quickly, preferentially enhanced the engraftment of HSCs in NOD/SCID mice. In addition, we observed differences in the pattern of both PODXL and Oct4 expression, and in the levels of cytokines such as SDF-1 and SCF using flow cytometry and membrane arrays. The more effective UCB-352 MSCs expressed higher levels of PODXL and Oct4, which were associated with immaturity, than did the UCB-156 MSCs. Furthermore, UCB-352 cells secreted greater levels of SDF-1 and SCF, both of which are required for hematopoiesis. We propose that the proliferation potential of UCB-MSCs, coupled with their immature characteristics, may serve as a novel standard to promote the homing and engraftment of HSCs.

Expression of Ca2+ -activated K+ channels in human dermal fibroblasts and their roles in apoptosis.

BACKGROUNDS/AIMS: Cell proliferation and apoptosis are responsible for maintaining normal tissue homeostasis, and K(+) currents play important roles in regulating the physiological balance between them. This function of Ca(2+)-activated K(+) (K(Ca)) channels has been demonstrated in many types of tissues, but not in dermal fibroblasts. We investigated the expression of K(Ca) channels and their effects on proliferation and apoptosis in human dermal fibroblasts. METHODS: We used discoidin domain receptor 2 immunostaining to identify human dermal fibroblasts, and reverse transcription polymerase chain reaction, Western blot analysis and electrophysiological patch clamp recordings to evaluate the expression and characteristics of 3 members of the K(Ca) channel family, large-conductance K(Ca) (BK), intermediate-conductance K(Ca) (IK) and small-conductance K(Ca) channels. We also used the 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, Hoechst 33258 staining and Depsipher staining to investigate the effects of K(Ca) channels on cell proliferation and the mechanisms involved. RESULTS AND CONCLUSIONS: All 3 members of the K(Ca) channel family were found in fibroblasts. 1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619, a BK channel activator) or 1-ethyl-2-benzimidazolinone (EBIO, an IK channel activator) decreased the proliferation of fibroblasts and induced apoptotic changes by mitochondrial membrane potential disruption. However, a pan-caspase inhibitor (Z-VAD-fmk) failed to prevent the apoptotic changes. Our findings indicate that 3 types of functional K(Ca) channels are expressed in human dermal fibroblasts and are involved in apoptosis of the cells through the mitochondrial apoptotic pathway, but seemingly in a caspase-independent manner.

Detection of malignant cells in pleural fluid or ascites by CD44v8-10/CD44v10 competitive RT-PCR.

BACKGROUND: CD44 is a cell surface adhesion molecule which has been implicated in various biologic functions as lymphocyte homing and activation, cellular migration and extracellular matrix adhesion. Over-expression of CD44v8-10 has been found in several cancers and is considered to be associated with tumor progression and metastasis. Recently, a novel molecular method, CD44v8-10/CD44v10 competitive reverse transcription-polymerase chain reaction(RT-PCR) has been developed for detecting cancer cells over-expressing CD44v8-10. METHODS: We analyzed from benign and malignant pleural effusion and ascites by CD44 competitive RT-PCR and compared to the conventional cytology. RESULTS: The CD44 competitive RT-PCR analysis showed that all the 24 samples associated with benign disease presented a predominant expression of the CD44v10 transcript (v8-10/v10 ratio: 0.126-0.948), whereas 6 of 7 malignant pleural samples associated with cytology positive cancer expressed the CD44v8-10 transcript (v8-10/v10 ratio > 1.00). CONCLUSION: These results indicate that CD44 competitive RT-PCR assay is a useful and adjunct to cytological examination in cancer diagnosis, especially in detecting exfoliated cancer cells in pleural effusion.

Detection of circulating tumor cells in patients with gastrointestinal tract cancer using RT-PCR and its clinical implications.

To investigate the relationship between the presence of circulating tumor cells in different stages of gastrointestinal tract cancer and the subsequent relapse or distant metastasis, circulating levels of CEA mRNA was serially examined at an interval of 10.6+/-4.5 or 13.7+/-3.0 months in gastric or colorectal cancer patients, respectively. CEA mRNA was measured by means of RT-PCR amplification as an indicator for micrometastatic malignant cells. Seven of twenty-nine respectable gastric cancer patients (24.1%) [EGC: 2/9 (22.2%), AGC IIIa: 1/5 (20.0%), AGC IIIb: 4/15 (26.6%)] were positive for CEA mRNA on the initial test and 10 of 29 patients (34.4%) [EGC: 2/ 9 (22.2%), AGC IIIa: 1/5 (20.0%), AGC IIIb: 7/15 (46.7%)] were positive on a follow-up test. Only in AGC IIIb, the positive rate for CEA mRNA increased about twice and 6 of 7 positive cases (85.7%) relapsed within 2.6+/-2.4 months after the follow-up test. In colorectal cancer, 4 of 19 patients (21.1%) [B2: 1/6 (16.7%), C2: 3/13 (23.0%)] were positive on the initial test and 10 of 19 patients (52.6%) [B2: 4/6 (66.7%), C2: 6/13 (46.2%)] were positive on a follow-up test showing an increase in positive rates during a follow-up, however, no significant correlation between CEA mRNA positivity and subsequent relapse was demonstrated. These results suggest that an early tumor cell dissemination may occur in gastrointestinal tract cancer without subsequent relapse, however, the serial regular examination of CEA mRNA level may contribute to predicting a subsequent relapse in AGC IIIb in gastric cancer.

Clinical impacts of tumor cell contamination of hematopoietic stem cell products in metastatic breast cancer patients undergoing autologous peripheral blood stem cell transplantation: multicenter trial.

To determine whether the tumor cell contamination of peripheral blood stem cells influences clinical impacts on high-dose chemotherapy in patients with metastatic breast cancer, we analyzed carcinoembryonic antigen (CEA) mRNA in the apheresis products by nested RT-PCR (reverse transcriptase-polymerase chain reaction). A total of 38 metastatic breast cancer patients and ten normal healthy subjects as a negative control were included. Twenty out of 38 (51.3%) apheresis products from patients with metastatic breast cancer were positive for CEA mRNA. CEA mRNA was noted in 54.8% (17/31) of patients mobilized with chemotherapy plus G-CSF and 42.8% (3/7) of patients with G-CSF alone. There was no significant difference in age, estrogen receptor, menopausal status, mobilization method, disease free interval, or number of metastasis sites (1 vs > or = 2) between positive and negative groups. The presence of CEA mRNA in apheresis products did not influence the time to progression and overall survival in both groups. However, both the univariate and the multivariate analysis disclosed that the number of metastasis was associated with survival significantly. We suggest that the tumor cell contamination does not predict poor treatment outcome in patients with metastatic breast cancer.

Detection of tumor cell contamination in peripheral blood by RT-PCR in gastrointestinal cancer patients.

We analyzed the peripheral blood of patients with gastrointestinal tract cancer at different stages to assess the presence of carcinoembryonic antigen (CEA) mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR), which we used as an indicator for micrometastatic malignant cells. A total of 35 gastric, 24 colorectal, 4 esophageal and 4 biliary tract cancer patients and nine normal healthy subjects were studied. No CEA mRNA was detected in the nine normal healthy volunteers. CEA mRNA was detected in 100% (10/10) of metastatic, 33.3% (3/9) of early gastric cancer (EGC), and 18.8% (3/16) resectable gastric cancer patients, respectively. In colorectal cancer, 55.6% (5/9) of metastatic cancers were positive for CEA mRNA, and 26.7% (4/15) Duke stage B/C showed positive. One patient with stage III gastric cancer who was negative CEA mRNA initially and turned positive during follow-up, developed multiple bone metastasis one month later. Another stage III patient, who was positive for CEA mRNA, preoperatively revealed early relapse in two months. These results suggest that the identification of circulating tumor cells using RT-PCR for the detection of CEA mRNA is feasible and this analysis may be a promising tool for early detection of micrometastatic circulating malignant cells in patients with gastrointestinal tract cancer.

Telomerase activity and its clinicopathological significance in gastric cancer.

In order to assess the role of telomerase in development of malignant gastric cancer, we measured the telomerase activity in gastric cancer tissues and normal tissues obtained from 95 patients by employing recently developed sensitive PCR (polymerase chain reaction)-based telomerase assay (telomeric repeat amplification protocol, TRAP). We also investigated how telomerase activity related to other clinicopathological findings including DNA ploidy and K-RAS gene point mutation. The telomerase activity was present in 85 of the 95 gastric cancer tissues, whereas we detected no telomerase activity in any normal tissue. The incidence of telomerase activity in gastric cancer tissues was not correlated to age, sex, tumour stage, histological grade, DNA ploidy or K-RAS mutation. Disease-free or overall survival of patients having tumours with detectable telomerase activity was not significantly different from that of those without telomerase activity. These findings suggest that telomerase may play a key role in the establishment and progression of the gastric cancer and further studies will be needed to elucidate the biological role of telomerase in gastric cancer.