COL8A1 facilitates the growth of triple-negative breast cancer via FAK/Src activation.

PURPOSE: Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes, and treatment options are limited because of the lack of signature molecules and heterogeneous properties of cancer. COL8A1 expression is higher in breast cancer than in normal tissues and is strongly correlated with worse overall survival in patients with breast cancer. However, the biological function of COL8A1 on cancer progression is not fully understood. In this study, we investigated the biological function of COL8A1 on TNBC progression. METHODS: COL8A1-deficient cells were generated using the CRISPR-Cas9 system. The tumor growth and metastasis of TNBC cells were evaluated using three-dimensional culture (3D) methods and xenograft mouse models. The activation of focal adhesion kinase (FAK)/Src by COL8A1 in TNBC cells was evaluated by immunoblotting. RESULTS: COL8A1 expression was primarily distributed into TNBC cell lines. Further, relapse-free survival in TNBC patients with the MSL subtype was strongly associated with the COL8A1 expression. MDA-MB-231 and Hs578T cells, classified as the MSL subtype, strongly express COL8A1, and COL8A1 protein expression was induced by hypoxia in both cell lines. Loss of COL8A1 expression inhibited spheroid /tumor growth and metastasis in vitro and in vivo. Further, exogenous COL8A1 promoted TNBC growth via the FAK/Src activation. Finally, the spheroid growth of MDA-MB-231 and Hs578T cells was inhibited by defactinib, a FAK inhibitor, without cytotoxicity. CONCLUSION: These results indicate that COL8A1-mediated FAK/Src activation produces a more aggressive phenotype in TNBC, and its target inhibition may be an efficacious treatment for TNBC.

253 J at 0.2†Hz, LD pumped cryogenic helium gas cooled Yb:YAG ceramics laser.

A 253 J with 26 ns at 0.2 Hz laser performance was demonstrated using a LD pumped cryogenically cooled Yb:YAG ceramics laser amplifier. A high energy storage of 344 J was achieved with a stored energy density of 0.58 J/cm3 using a 1 kJ output multidirectional-pumping system. High energy-extraction efficiency of 56.5% was achieved with high energy fluence of 4.63 J /cm2. To the best of our knowledge, this is the highest output energy obtained with a repetitive nanosecond pulse by LD pumped solid-state laser. This paper presented a design of 1 kJ amplifier based on experimentally proven numerical data.

Effect of CYP3A5*3 genetic variant on the metabolism of direct-acting antivirals in vitro: a different effect on asunaprevir versus daclatasvir and beclabuvir.

Direct-acting antivirals, asunaprevir (ASV), daclatasvir (DCV), and beclabuvir (BCV) are known to be mainly metabolized by CYP3A enzymes; however, the differences in the detailed metabolic activities of CYP3A4 and CYP3A5 on these drugs are not well clarified. The aim of the present study was to elucidate the relative contributions of CYP3A4 and CYP3A5 to the metabolism of ASV, DCV, and BCV, as well as the effect of CYP3A5*3 genetic variant in vitro. The amount of each drug and their major metabolites were determined using LC-MS/MS. Recombinant CYP3As and CYP3A5*3-genotyped human liver microsomes (CYP3A5 expressers or non-expressers) were used for the determination of their metabolic activities. The contribution of CYP3A5 to ASV metabolism was considerable compared to that of CYP3A4. Consistently, ASV metabolic activity in CYP3A5 expressers was higher than those in CYP3A5 non-expresser. Moreover, CYP3A5 expression level was significantly correlated with ASV metabolism. In contrast, these observations were not found in DCV and BCV metabolism. To our knowledge, this is the first study to directly demonstrate the effect of CYP3A5*3 genetic variants on the metabolism of ASV. The findings of the present study may provide basic information on ASV, DCV, and BCV metabolisms.

Minor contribution of CYP3A5 to the metabolism of hepatitis C protease inhibitor paritaprevir in vitro.

Paritaprevir (PTV) is a non-structural protein 3/4A protease inhibitor developed for the treatment of hepatitis C disease as a fixed dose combination of ombitasvir (OBV) and ritonavir (RTV) with or without dasabuvir. The aim of this study was to evaluate the effects of cytochrome P450 (CYP) 3A5 on in vitro PTV metabolism using human recombinant CYP3A4, CYP3A5 (rCYP3A4, rCYP3A5) and human liver microsomes (HLMs) genotyped as either CYP3A5*1/*1, CYP3A5*1/*3 or CYP3A5*3/*3. The intrinsic clearance (CLint, Vmax/Km) for the production of a metabolite from PTV in rCYP3A4 was 1.5 times higher than that in rCYP3A5. The PTV metabolism in CYP3A5*1/*1 and CYP3A5*1/*3 HLMs expressing CYP3A5 was comparable to that in CYP3A5*3/*3 HLMs, which lack CYP3A5. CYP3A4 expression level was significantly correlated with PTV disappearance rate and metabolite formation. In contrast, there was no such correlation found for CYP3A5 expression level. This study represents that the major CYP isoform involved in PTV metabolism is CYP3A4, with CYP3A5 having a minor role in PTV metabolism. The findings of the present study may provide foundational information on PTV metabolism, and may further support dosing practices in HCV-infected patients prescribed PTV-based therapy.

Quantification and tracking of genetically engineered dendritic cells for studying immunotherapy.

PURPOSE: Genetically encoded reporters can assist in visualizing biological processes in live organisms and have been proposed for longitudinal and noninvasive tracking of therapeutic cells in deep tissue. Cells can be labeled in situ or ex vivo and followed in live subjects over time. Nevertheless, a major challenge for reporter systems is to identify the cell population that actually expresses an active reporter. METHODS: We have used a nucleoside analog, pyrrolo-2'-deoxycytidine, as an imaging probe for the putative reporter gene, Drosophila melanogaster 2'-deoxynucleoside kinase. Bioengineered cells were imaged in vivo in animal models of brain tumor and immunotherapy using chemical exchange saturation transfer MRI. The number of transduced cells was quantified by flow cytometry based on the optical properties of the probe. RESULTS: We performed a comparative analysis of six different cell lines and demonstrate utility in a mouse model of immunotherapy. The proposed technology can be used to quantify the number of labeled cells in a given region, and moreover is sensitive enough to detect less than 10,000 cells. CONCLUSION: This unique technology that enables efficient selection of labeled cells followed by in vivo monitoring with both optical and MRI. Magn Reson Med 79:1010-1019, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Endocan as a prognostic biomarker of triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) has aggressive characteristics and fewer treatment options than other subtypes. The purpose of this study was to explore prognostic biomarkers for TNBC that can be easily detected from the blood samples. METHODS: MDA-MB-231 and MDA-MB-231BR, a brain metastatic variant of the human TNBC cell line MDA-MB-231, were used as less and more aggressive models of TNBC, respectively. The extent to which the candidate gene/protein identified by RNA sequencing correlated well with aggressiveness of TNBC and how much protein was detected from the blood of tumor-bearing mice were evaluated. RESULTS: Both the in vitro proliferation and in vivo tumor growth of MDA-MB-231BR were more rapid than those of MDA-MB-231. RNA sequencing identified ESM1 as a gene that was expressed significantly more in MDA-MB-231BR than in MDA-MB-231, and qRT-PCR confirmed a significantly higher expression of ESM1 in MDA-MB-231BR xenograft in vivo. Furthermore, Kaplan-Meier analysis of relapse-free survival demonstrated that TNBC patients with high ESM1 expression had clearly worse relapse-free survival than those with low ESM1 expression, which was consistent with our preclinical findings. Endocan, a protein of ESM1 gene product, was successfully detected in both conditioned medium from MDA-MB-231BR and plasma samples from mice bearing MDA-MB-231BR xenograft, which showed a significantly distinct pattern from less aggressive MDA-MB-231. Moreover, bisulfite sequence analysis revealed that overexpression of ESM1 in MDA-MB-231BR might be attributed to DNA demethylation in an upstream region of the ESM1 gene. CONCLUSION: This study indicates that endocan could be used as a blood-based prognostic biomarker in TNBC patients.

Controlled Secretion of the Anticancer Protein MDA-7 from Engineered Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been explored as a "live" carrier of cytokines for targeted cancer therapy, but, in earlier reports in the literature, the secretion process of therapeutic cytokines was not regulated. The purpose of this study was to generate MSCs to conditionally secrete the melanoma differentiation-associated gene-7 (MDA-7) tumor-suppressor protein. To control the secretion of MDA-7 from MSCs, a well-established tetracycline-controlled transcriptional activation system was incorporated into MDA-7 plasmid. MDA-7 gene expression was induced in the engineered MSCs only in the presence of doxycycline, as characterized by quantitative reverse transcription (qRT)-PCR. Enzyme-linked immunosorbent assay (ELISA) also revealed that the MDA-7 protein was secreted from the engineered MSCs only after the cells had been exposed to doxycycline. Both recombinant human MDA-7 protein and the conditioned medium from the engineered MSCs in the presence of doxycycline significantly inhibited tube formation of human umbilical vascular endothelial cells (HUVECs), indicating that our system could be used for targeted, antiangiogenic therapy. Overall, this study provides useful information on the potential use of engineered MSCs for the controlled secretion of therapeutic proteins, in this case MDA-7, for targeted cancer therapy.

Role of apoptosis signal-regulating kinase 1 (ASK1) as an activator of the GAPDH-Siah1 stress-signaling cascade.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays roles in both energy maintenance, and stress signaling by forming a protein complex with seven in absentia homolog 1 (Siah1). Mechanisms to coordinate its glycolytic and stress cascades are likely to be very important for survival and homeostatic control of any living organism. Here we report that apoptosis signal-regulating kinase 1 (ASK1), a representative stress kinase, interacts with both GAPDH and Siah1 and is likely able to phosphorylate Siah1 at specific amino acid residues (Thr-70/Thr-74 and Thr-235/Thr-239). Phosphorylation of Siah1 by ASK1 triggers GAPDH-Siah1 stress signaling and activates a key downstream target, p300 acetyltransferase in the nucleus. This novel mechanism, together with the established S-nitrosylation/oxidation of GAPDH at Cys-150, provides evidence of how the stress signaling involving GAPDH is finely regulated. In addition, the present results imply crosstalk between the ASK1 and GAPDH-Siah1 stress cascades.

A preclinical murine model for the early detection of radiation-induced brain injury using magnetic resonance imaging and behavioral tests for learning and memory: with applications for the evaluation of possible stem cell imaging agents and therapies.

Stem cell therapies are being developed for radiotherapy-induced brain injuries (RIBI). Magnetic resonance imaging (MRI) offers advantages for imaging transplanted stem cells. However, most MRI cell-tracking techniques employ superparamagnetic iron oxide particles (SPIOs), which are difficult to distinguish from hemorrhage. In current preclinical RIBI models, hemorrhage occurs concurrently with other injury markers. This makes the evaluation of the recruitment of transplanted SPIO-labeled stem cells to injury sites difficult. Here, we developed a RIBI model, with early injury markers reflective of hippocampal dysfunction, which can be detected noninvasively with MRI and behavioral tests. Lesions were generated by sub-hemispheric irradiation of mouse hippocampi with single X-ray beams of 80 Gy. Lesion formation was monitored with anatomical and contrast-enhanced MRI and changes in memory and learning were assessed with fear-conditioning tests. Early injury markers were detected 2 weeks after irradiation. These included an increase in the permeability of the blood-brain barrier, demonstrated by a 92 ± 20 % contrast enhancement of the irradiated versus the non-irradiated brain hemispheres, within 15 min of the administration of an MRI contrast agent. A change in short-term memory was also detected, as demonstrated by a 40.88 ± 5.03 % decrease in the freezing time measured during the short-term memory context test at this time point, compared to that before irradiation. SPIO-labeled stem cells transplanted contralateral to the lesion migrated toward the lesion at this time point. No hemorrhage was detected up to 10 weeks after irradiation. This model can be used to evaluate SPIO-based stem cell-tracking agents, short-term.

Linezolid-Induced Thrombocytopenia Is Caused by Suppression of Platelet Production via Phosphorylation of Myosin Light Chain 2.

Linezolid (LZD) is an antimicrobial that is commonly used for treatment of vancomycin-resistant Enterococci and methicillin-resistant Staphylococcus aureus infections. However, the development of thrombocytopenia, one of the most frequent adverse side effects of this antimicrobial, can lead to discontinuation of LZD treatment. While clinical studies indicate that risk factors for the development of LZD-induced thrombocytopenia include treatment for >14 consecutive days, renal dysfunction, and chronic liver disease, the fundamental mechanism governing the pathogenesis of this disorder remains unclear. In this study, we aimed to elucidate the mechanism of LZD-induced thrombocytopenia by investigating the impact of LZD treatment on platelet destruction and production using rat platelet-rich plasma (PRP) and human immortalized cell lines, respectively. Compared to the control population, an increase in lactate dehydrogenase release was not detected upon exposure of rat PRP to varying concentrations of LZD, indicating that this compound is not cytotoxic towards platelets. Meanwhile, LZD treatment resulted in a significant dose-dependent increase in the proliferation of HEL human erythroleukemia and MEG-01 human megakaryoblast cells in vitro, but did not influence the differentiation of these cell lines. Lastly, LZD treatment yielded elevated levels of phosphorylation of myosin light chain 2 (MLC2), which regulates platelet release, in MEG-01 cells. Based on these results, we speculate that LZD induces thrombocytopenia by promoting MLC2 phosphorylation and thereby suppressing the release of platelets from mature megakaryocytes. These findings provide the first insight into the mechanism of LZD-mediated thrombocytopenia and may facilitate the development of strategies to treat and/or prevent this disease.

Dynamic glucose enhanced (DGE) MRI for combined imaging of blood-brain barrier break down and increased blood volume in brain cancer.

PURPOSE: Recently, natural d-glucose was suggested as a potential biodegradable contrast agent. The feasibility of using d-glucose for dynamic perfusion imaging was explored to detect malignant brain tumors based on blood brain barrier breakdown. METHODS: Mice were inoculated orthotopically with human U87-EGFRvIII glioma cells. Time-resolved glucose signal changes were detected using chemical exchange saturation transfer (glucoCEST) MRI. Dynamic glucose enhanced (DGE) MRI was used to measure tissue response to an intravenous bolus of d-glucose. RESULTS: DGE images of mouse brains bearing human glioma showed two times higher and persistent changes in tumor compared with contralateral brain. Area-under-curve (AUC) analysis of DGE delineated blood vessels and tumor and had contrast comparable to the AUC determined using dynamic contrast enhanced (DCE) MRI with GdDTPA, both showing a significantly higher AUC in tumor than in brain (P < 0.005). Both CEST and relaxation effects contribute to the signal change. CONCLUSION: DGE MRI is a feasible technique for studying brain tumor enhancement reflecting differences in tumor blood volume and permeability with respect to normal brain. We expect DGE will provide a low-risk and less expensive alternative to DCE MRI for imaging cancer in vulnerable populations, such as children and patients with renal impairment.

Noninvasive Imaging of Liposomal Delivery of Superparamagnetic Iron Oxide Nanoparticles to Orthotopic Human Breast Tumor in Mice.

PURPOSE: Magnetic resonance imaging (MRI) is widely used for diagnostic imaging in preclinical studies and in clinical settings. Considering the intrinsic low sensitivity and poor specificity of standard MRI contrast agents, the enhanced delivery of MRI tracers into tumors is an important challenge to be addressed. This study was intended to investigate whether delivery of superparamagnetic iron oxide nanoparticles (SPIONs) can be enhanced by liposomal SPION formulations for either "passive" delivery into tumor via the enhanced permeability and retention (EPR) effect or "active" targeted delivery to tumor endothelium via the receptors for vascular endothelial growth factor (VEGFRs). METHODS: In vivo MRI of orthotopic MDA-MB-231 tumors was performed on a preclinical 9.4 T MRI scanner following intravenous administration of either free/non-targeted or targeted liposomal SPIONs. RESULTS: In vivo MRI study revealed that only the non-targeted liposomal formulation provided a statistically significant accumulation of SPIONs in the tumor at four hours post-injection. The EPR effect contributes to improved accumulation of liposomal SPIONs in tumors compared to the presumably more transient retention during the targeting of the tumor vasculature via VEGFRs. CONCLUSIONS: A non-targeted liposomal formulation of SPIONs could be the optimal option for MRI detection of breast tumors and for the development of therapeutic liposomes for MRI-guided therapy.

Gosha-jinki-gan reduced oxaliplatin-induced hypersensitivity to cold sensation and its effect would be related to suppression of the expression of TRPM8 and TRPA1 in rats.

Peripheral neuropathy is a common side effect of the chemotherapeutic agent oxaliplatin (Oxp), and is associated with hypersensitivity to cold sensation in the acute stage. Recently, gosha-jinki-gan (GJG), a Japanese herbal medicine, was reported to improve Oxp-induced cold hypersensitivity. However, the mechanism for this effect was not elucidated. We hypothesized that the effect of GJG on Oxp-induced cold hypersensitivity may be associated with the expression of the transient receptor potential melastatin 8 (TRPM8) and transient receptor potential ankyrin 1 (TRPA1) channels, which are cold-gated ion channels. To assess this hypothesis, we examined alteration of the withdrawal response to cold stimulation following coadministration of GJG and Oxp in rats, and the relationship between this altered withdrawal response and the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG). Assessment of cold hypersensitivity was performed at 4 and 10°C using a cold plate. Compared with Oxp administration alone, coadministration of GJG (oral dose: 1 g/kg/day for 12 days) and Oxp (intraperitoneal dose: 4 mg/kg twice a week) significantly reduced the withdrawal response to cold stimulation. On the 12th day of drug administration, the L4-L6 DRG were removed and the expression of TRPM8 and TRPA1 mRNA was determined using RT-PCR. The expression of TRPM8 and TRPA1 in the DRG of rats that were coadministered GJG and Oxp decreased significantly compared with that in the rats administered Oxp alone. These results suggest that coadministration of GJG may improve Oxp-induced cold hypersensitivity by suppressing the overexpression of TRPM8 and TRPA1 mRNA.

Water exchange-minimizing DCE-MRI protocol to detect changes in tumor vascular parameters: effect of bevacizumab/paclitaxel combination therapy.

OBJECTIVE: The purpose of this study was to assess changes in the tumor microvasculature induced by combination antiangiogenic therapy in MCF-7 breast tumor mouse models, using a noninvasive DCE-MRI method that minimizes the effect of water exchange. MATERIALS AND METHODS: 3D quantitative DCE-MRI images were acquired with a heavily T1-weighted saturation recovery gradient echo sequence with a recovery delay of 20 ms. Tumor vascular volume (VV) and vascular permeability-surface area product (PS) were obtained through a linear regression of the albumin-Gd-DTPA-enhanced dynamic image intensity on MCF-7 breast tumor mouse models treated with combination bevacizumab/paclitaxel therapy. RESULTS: Measured tumor VV values were significantly higher than the values that have been reported previously using quantitative T1 mapping, and are in good agreement with micro-CT (computed tomography) results reported earlier from other tumor models. A trend of decreasing tumor PS was detected in the group of MCF-7 tumor bearing mice treated with the bevacizumab/paclitaxel combination regimen. CONCLUSION: VV and PS maps obtained by a heavily T1-weighted acquisition protocol revealed the large peripheral blood vessels as well as the permeable areas within the tumor. A 12-day/three-dose combination treatment of bevacizumab and paclitaxel resulted in delayed tumor growth and a trend of decreasing tumor vascular permeability surface area product.

Muscles used for chest compression under static and transportation conditions.

BACKGROUND: Unstable conditions during ambulance transportation are not conducive to the performance of high-quality cardiopulmonary resuscitation by emergency medical technicians. OBJECTIVE: The present study was conducted to clarify differences in the quality of chest compression and associated muscle activity between static and ambulance transportation conditions. METHODS: Nine paramedic students performed chest compression for 5 minutes on the floor and during ambulance transportation. Compression rate and depth and success and error rates of chest compression were determined using the Resusci Anne manikin with a PC SkillReporting System (Laerdal Medical). Integrated electromyography (i-EMG) values of eight different muscles were also recorded bilaterally during the first and last 30 seconds of compression. RESULTS: There was no significant difference in compression rate per minute (p = 0.232) and depth of chest compression (p = 0.174) between the two conditions. The success rate was significantly lower under the ambulance transportation condition than under the static condition (p = 0.0161). Compared with those under the static condition, the total i-EMG values were significantly lower for the multifidus (p = 0.0072) and biceps femoris (p < 0.0001) muscles and significantly higher for the deltoid (p = 0.0032), pectoralis major (p = 0.0037), triceps brachii (p = 0.0014), vastus lateralis (p < 0.0001), and gastrocnemius (p = 0.0004) muscles under the ambulance transportation condition. CONCLUSIONS: Chest compression is performed mainly through flexion and extension of the hip joint while kneeling on the floor and through the elbow and shoulder joints while standing in a moving ambulance. Therefore, the low quality of chest compression during ambulance transportation may be attributable to an altered technique of performing the procedure.

Natural D-glucose as a biodegradable MRI contrast agent for detecting cancer.

PURPOSE: Modern imaging technologies such as CT, PET, SPECT, and MRI employ contrast agents to visualize the tumor microenvironment, providing information on malignancy and response to treatment. Currently, all clinical imaging agents require chemical labeling, i.e. with iodine (CT), radioisotopes (PET/SPECT), or paramagnetic metals (MRI). The goal was to explore the possibility of using simple D-glucose as an infusable biodegradable MRI agent for cancer detection. METHODS: D-glucose signals were detected using chemical exchange saturation transfer (glucoCEST) MRI of its hydroxyl groups. Feasibility was established in phantoms as well as in vivo using two human breast cancer cell lines, MDA-MB-231 and MCF-7, implanted orthotopically in nude mice. PET and contrast-enhanced MRI were also acquired. RESULTS: Both tumor types exhibited significant glucoCEST signal enhancement during systemic sugar infusion (mild hyperglycemia), allowing their noninvasive visualization. GlucoCEST showed differences between types, while PET and CE-MRI did not. Data are discussed in terms of signal contributions from the increased vascular volume in tumors and especially from the acidic extracellular extravascular space (EES), where glucoCEST signal is expected to be enhanced due to a slow down of hydroxyl proton exchange. CONCLUSIONS: This observation opens up the possibility for using simple non-toxic sugars as contrast agents for cancer detection with MRI by employing hydroxyl protons as a natural label.

Toward in vivo imaging of heart disease using a radiolabeled single-chain Fv fragment targeting tenascin-C.

Antibodies specific to a particular target molecule can be used as analytical reagents, not only for in vitro immunoassays but also for noninvasive in vivo imaging, e.g., immunoscintigraphies. In the latter case, it is important to reduce the size of antibody molecules in order to achieve suitable in vivo "diagnostic kinetics" and generate higher-resolution images. For these purposes, single-chain Fv fragments (scFvs; M(r) < 30 kDa) have greater potential than intact immunoglobulins (~150 kDa) or Fab (or Fab') fragments (~50 kDa). Our recent observation of enhanced tenascin-C (Tnc) expression at sites of cardiac repair after myocardial infarction prompted us to develop a radiolabeled scFv against Tnc for in vivo imaging of heart disease. We cloned the genes encoding the heavy and light chain variable domains of the mouse anti-Tnc monoclonal antibody 4F10, and combined them to create a single gene. The resulting scFv-4F10 gene was expressed in E. coli cells to produce soluble scFv proteins. scFv-4F10 has an affinity for Tnc (K(a) = 3.5 × 10(7) M(-1)), similar to the Fab fragment of antibody 4F10 (K(a) = 1.3 × 10(7) M(-1)) and high enough to be of practical use. A cysteine residue was then added to the C-terminus to achieve site-specific (111)In labeling via a chelating group. The resulting (111)In-labeled scFv was administered to a rat model of acute myocardial infarction. Biodistribution and quantitative autoradiographic studies indicated higher uptake of the radioactivity at the infarcted myocardium than the noninfarcted one. Single photon emission computed tomography (SPECT) provided in vivo cardiac images that coincided with the ex vivo observations. Our results will promote advances in diagnostic strategies for heart disease.

Oligosaccharide-assisted direct immunosensing of small molecules.

"Sandwich-type" noncompetitive (immunometric) assays allow for high-sensitivity high-throughput macromolecule sensing and determination but cannot be used on small molecules (haptens). Here, we isolated single-chain Fv fragments from a phage-display library, which bound to complexes of particular haptens (vitamin D and A derivatives) with immobilized beta-cyclodextrin or beta-maltosyl residues, and formed ternary complexes. These scFvs enabled novel "semisandwich-type" immunometric assays of haptens with nanomole-range sensitivities.

Two-step in vitro antibody affinity maturation enables estradiol-17beta assays with more than 10-fold higher sensitivity.

Immunoassays for haptens depend on competitive hapten-anti-hapten reactions, and consequently their sensitivities are significantly influenced by the affinities of anti-hapten antibodies. Thus, genetically engineered antibodies, which have much higher affinities than native antibodies, should increase assay sensitivities. Here, we created a mutated single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) that allowed immunoassays with a much improved sensitivity. Two steps of affinity maturation were performed on a "wild-type" scFv (scFv#E4-4) composed of V(H) and V(L) domains from a mouse anti-E(2) antibody (Ab#E4-4). First, we conducted complementarity-determining region (CDR)-targeted mutagenesis by "CDR-shuffling". Gene fragments encoding CDRs H2, H3, L1, and L3, each of which contained random point mutations, were combined by "shuffling" into the gene encoding the scFv#E4-4 scaffold. After phage display and repeated panning, we isolated a mutated scFv clone [scFv#m1-e7; Ile(L29)Val] that had 5-fold higher affinity (K(a) = 2.6 x 10(8) M(-1)) compared to the Ab#E4-4 Fab fragment (Fab#E4-4). Next, the entire V(H) and V(L) of this clone were randomly mutated by error-prone polymerase chain reaction (PCR). From this library, we found an improved clone, scFv#m2-c4 (K(a) = 6.3 x 10(8) M(-1); Lys(H19)Arg, Tyr(H56)Phe, Ser(H84)Pro, Glu(H85)Gly, Gln(L27)Arg, Leu(L36)Met, Ser(L63)Gly, and Ser(L77)Gly). ScFv#m2-c4 had more than 10-fold higher sensitivity (the midpoint of its dose-response curve was 0.56 ng) than Fab#E4-4 (midpoint 9.0 ng/assay) in a competitive E(2) radioimmunoassay, and even higher sensitivity [midpoint 21 pg/assay, and a limit of detection of 0.47 pg (1.7 fmol)/assay] in a competitive enzyme-linked immunosorbent assay. Cross-reactivity with selected E(2)-related endogenous steroids strongly suggested that scFv#m2-c4 has improved specificity compared to conventional antibodies.

Monitoring of release of cargo from nanocarriers by MRI/MR spectroscopy (MRS): significance of T2/T2* effect of iron particles.

To monitor the release of cargo molecules from nanocarriers, a novel MRI/MRS technique was developed and tested. This novel approach uses a simultaneous encapsulation of superparamagnetic iron oxide (SPIO) nanoparticles and either a gadolinium (Gd)-based paramagnetic contrast agent, Gd-diethylenetriamine pentaacetic acid bismethylamide(GdDTPA-BMA), for MRI, or an anticancer agent, 5-fluorouracil (5-FU), for MRS. These agents have significantly different diffusion properties due to their different molecular sizes. Strong negative signal enhancement due to the T(2) effects of SPIO dominates the positive T(1) contrast generated by GdDTPA-BMA when SPIO and GdDTPA-BMA are in close proximity (intact form). Positive T(1) contrast becomes evident upon release of GdDTPA-BMA from the carrier once the distance between GdDTPA-BMA and SPIO molecules is beyond the T(2) enhancement range. Similarly, intact nanocarriers loaded with 5-FU and SPIO have a broad (19)F resonance line because line-width is inversely proportional to T*2, while free 5-FU appears as a narrow resonance line once it is released from the liposomes. This technique allowed monitoring of the release of cargo molecules from liposomes encapsulating both SPIO and either GdDTPA-BMA or 5-FU by MRI/MRS in vitro using 2% agarose gel phantoms. Experimental results demonstrate successful demarcation of the released cargo molecules vs. encapsulated molecules.