The interplay of T1- and T2-relaxation on T1-weighted MRI of hMSCs induced by Gd-DOTA-peptides

Three Gd-DOTA-peptide complexes with different peptide sequence are synthesized and used as T₁ contrast agent to label human mesenchymal stem cells (hMSCs) for magnetic resonance imaging study. The peptides include a universal cell penetrating peptide TAT, a linear MSC-specific peptide EM7, and a cyclic MSC-specific peptide CC9. A significant difference in labeling efficacy is observed between the Gd-DOTA-peptides as well as a control Dotarem. All Gd-DOTA-peptides as well as Dotarem induce significant increase in T₁ relaxation rate which is in favor of T₁-weighted MR imaging. Gd-DOTA-CC9 yields the maximum labeling efficacy but poor T₁ contrast enhancement. Gd-DOTA-EM7 yields the minimum labeling efficacy but better T₁ contrast enhancement. Gd-DOTA-TAT yields a similar labeling efficacy as Gd-DOTA-CC9 and similar T₁ contrast enhancement as Gd-DOTA-EM7. The underlying mechanism that governs T₁ contrast enhancement effect is discussed. Our results suggest that T₁ contrast enhancement induced by Gd-DOTA-peptides depends not only on the introduced cellular Gd content, but more importantly on the effect that Gd-DOTA-peptides exert on the T₁-relaxation and T₂-relaxation processes/rates. Both T₁ and particularly T₂ relaxation rate have to be taken into account to interpret T₁ contrast enhancement. In addition, the interpretation has to be based on cellular instead of aqueous longitudinal and transverse relaxivities of Gd-DOTA-peptides.     

Manipulating the surface coating of ultra-small Gd2O3 nanoparticles for improved T1-weighted MR imaging

In this report, monodispersed ultra-small Gd₂O₃ nanoparticles capped with hydrophobic oleic acid (OA) were synthesized with average particle size of 2.9 nm. Two methods were introduced to modify the surface coating to hydrophilic for bio-applications. With a hydrophilic coating, the polyvinyl pyrrolidone (PVP) coated Gd₂O₃ nanoparticles (Gd₂O₃-PVP) showed a reduced longitudinal T₁ relaxation time compared with OA and cetyltrimethylammonium bromide (CTAB) co-coated Gd₂O₃ (Gd₂O₃-OA-CTAB) in the relaxation study. The Gd₂O₃-PVP was thus chosen for its further application study in MRI with an improved longitudinal relaxivity r₁ of 12.1 mm⁻¹ s⁻¹ at 7 T, which is around 3 times as that of commercial contrast agent Magnevist^®. In vitro cell viability in HK-2 cell indicated negligible cytotoxicity of Gd₂O₃-PVP within preclinical dosage. In vivo MR imaging study of Gd₂O₃-PVP nanoparticles demonstrated considerable signal enhancement in the liver and kidney with a long blood circulation time. Notably, the OA capping agent was replaced by PVP through ligand exchange on the Gd₂O₃ nanoparticle surface. The hydrophilic PVP grants the Gd₂O₃ nanoparticles with a polar surface for bio-application, and the obtained Gd₂O₃-PVP could be used as an in vivo indicator of reticuloendothelial activity.     

Mn-doped silica nanoparticles for hepatocyte-targeted detection of liver cancer in T1-weighted MRI

With an aim to examine the possibility of developing a liver-specific MRI contrast agent that takes advantages of brightly enhanced MR images by Mn²⁺ whilst making up the limitations of the pre-developed contrast agent, the Mn²⁺-doped SiO₂ nanoparticles (Mn–SiO₂) were synthesized and their characteristics as MR contrast agents were investigated. The in vitro and in vivo investigations showed that Mn–SiO₂ has unique MR contrast-enhancing characteristics that activate positive contrast enhancement in T₁-weighted MR images only under low pH conditions by liberating Mn²⁺ ions from MR inactive nanoparticles. The administration of Mn–SiO₂ to an orthotopic xenograft model of human hepatocellular carcinoma (HCC) resulted in a differentiation of enhancement periods between HCC and normal parenchyma tissues on T₁-weighted MR images and consequently presented the duplicates of the highly contrast-enhanced liver image with an equal liver-to-HCC contrast ratio but opposite contrast. The Mn–SiO₂-enhanced MR imaging therefore allowed for the repetitive detection of the HCC within a single MR imaging session, which can help us to achieve more reliable diagnosis and characterization of liver lesions than is possible with any currently used Mn²⁺-based contrast agent. In addition, the in vivo biodistribution study also supported the effectiveness of Mn–SiO₂ nanoparticles as a liver-specific MRI contrast agent, which efficiently delivers and releases the T₁-contrasting Mn²⁺ ions to targeted hepatocytes.     

Functional differences between D1 and D5 revealed by high resolution imaging on live neurons

The interaction between the dopaminergic and glutamatergic systems governs normal behavior and is perturbed in many psychiatric disorders including schizophrenia. Hypofunction of the D1 family of receptors, to which the D₁ and D₅ subtypes belong, is a typical feature of schizophrenia. Here we have used confocal live cell imaging of neurons to examine the distinct roles of the D₁ and D₅ receptors in the intra-neuronal interaction with the glutamatergic system. Using fluorescently tagged D₁ or D₅ expressed in cultured striatal neurons, we show that both receptor subtypes are primarily transported via lateral diffusion in the dendritic tree. D₁ is to a much larger extent than D₅ expressed in spines. D₁ is primarily expressed in the head whereas D₅ is largely localized to the neck of the spine. Activation of N-methyl-d-aspartic acid (NMDA) receptors slowed the diffusion rate and increased the number of D₁ positive spines, while no effect on D₅ diffusion or spine localization could be observed. The observed differences between D₁ and D₅ can be attributed to structural differences in the C-terminus and its capacity to interact with NMDA receptors and PSD-95. Identification of a unique role of D₁ for the intra-neuronal interaction between the dopaminergic and glutamatergic systems will have implications for the development of more specific treatments in many neuropsychiatric disorders.     

Oral administration of a dominant T-cell determinant peptide inhibits allergen-specific TH1 and TH2 cell responses in Cry j 2–primed mice

Background: Oral immunotherapy with a peptide for allergic immune responses is theoretically a promising therapy but has not been established yet. Objective: To evaluate immune suppressive efficacy of oral administration of an immunodominant peptide, we investigated changes in T-cell proliferation, T_H1 - and T_H2 -cytokine production, and T_H1 - and T_H2 -mediated antibody production in mice after oral administration of a peptide. Methods: Peptide p246-259, containing a dominant T-cell determinant of Cry j 2, which is the major allergen in Japanese cedar pollen, was used in this study. Groups of mice received p246-259 or PBS alone before or after they were primed intranasally with Cry j 2 and cholera toxin. In another experiment mice were primed intraperitoneally with Cry j 2 and alum. Proliferative response and cytokine production by nasal-associated lymph node cells against Cry j 2 were investigated. Amounts of systemic anti-Cry j 2 IgE and IgG antibodies were also measured. Results: Oral administration of the peptide to mice before, or even after, the sensitization induced oral tolerance in T-cell responses against the allergen; the tolerance was associated with decreased production of T_H1 (IFN-γ and IL-2) and T_H2 (IL-4) cytokines. Allergen-specific T_H1 -mediated (IgG2a and IgG2b) and T_H2 -mediated (IgG1 and IgE) antibody responses were also inhibited. Conclusions: Oral administration of a dominant T-cell determinant peptide induces immunologic tolerance in both T_H1 and T_H2 cell responses against the whole protein allergen. Our study is the first, to our knowledge, to demonstrate the potential for peptide-based oral immunotherapy in order to treat allergic immune responses. (J Allergy Clin Immunol 1998;102:961-7.)     

Ultrastructural analysis of TiO2 nanotubes with photodecomposition of water into O2 and H2 implanted in the nude mouse

To analyze the biocompatibility and O2 generation of TiO2 nanotubes via photodecomposition of water into O2 and H2 in vivo, samples were implanted under the inguinal skin of the nude mouse. Venous oxygen saturation (SvO2) of the inguinal skin over the implanted region was measured with a tissue oximeter and the ultrastructures were examined with an electron microscope. Four weeks after the implantation, SvO2 of the inguinal skin of the groups with TiO2 nanotubes was 30-40% higher than that of the opposite control region (54%). SvO2 of the other groups, comprising splenic autografts, fetal cardiac tissue transplantation and surgical procedure without TiO2 nanotubes, was roughly the same as that of controls. Ultrastructurally, TiO2 nanotubes were phagocytized by the macrophage and promoted filament formation in its cytoplasm. Neither death of the cell nor destruction of the tissue was recognized. These findings indicate excellent biocompatibility and O2 generation of TiO2 nanotubes in vivo.     

Fates of Fe3O4 and Fe3O4@SiO2 nanoparticles in human mesenchymal stem cells assessed by synchrotron radiation-based techniques

Superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as the magnetic resonance imaging (MRI) contrast agent in biomedical studies and clinical applications, with special interest recently in in vivo stem cell tracking. However, a full understanding of the fate of SPIOs in cells has not been achieved yet, which is particularly important for stem cells since any change of the microenvironment may disturb their propagation and differentiation behaviors. Herein, synchrotron radiation-based X-ray fluorescence (XRF) in combination with X-ray absorption spectroscopy (XAS) were used to in situ reveal the fate of Fe₃O₄ and Fe₃O₄@SiO₂ NPs in human mesenchymal stem cells (hMSCs), in which the dynamic changes of their distribution and chemical speciation were precisely determined. The XAS analysis evidences that Fe₃O₄ NPs cultured with hMSCs are quite stable and almost keep their initial chemical form up to 14 days, which is contradictory to the previous report that Fe₃O₄ NPs were unstable in cell labeling assessed by using a simplified lysosomal model system. Coating with a SiO₂ shell, Fe₃O₄@SiO₂ NPs present higher stability in hMSCs without detectable changes of their chemical form. In addition, XRF analysis demonstrates that Fe₃O₄@SiO₂ NPs can label hMSCs in a high efficiency manner and are solely distributed in cytoplasm during cell proliferation, making it an ideal probe for in vivo stem cell tracking. These findings with the help of synchrotron radiation-based XAS and XRF improve our understanding of the fate of SPIOs administered to hMSCs and will help the future design of SPIOs for safe and efficient stem cells tracking.     

Development of ZrO2/SiO2 bioinert ceramic coatings for biomedical application

Gas tunnel-type plasma spraying (GTPS) was employed to deposit ZrO₂/SiO₂ bioinert ceramic composite coatings with an appropriate thickness on SUS 304 substrate. Zirconia and fused silica powders, with equal wt%, have been mixed together in ceramic pot for 30 min and internally fed in the plasma jet. The composite coatings were sprayed at two different gas flow rates (120 and 150 l/min) and constant vortex arc current of 450 A and gun current of 50 A. The microstructure of as-sprayed coatings was examined by scanning electron microscope. Elemental analysis was achieved for the composite coatings using EDS analysis unit which is attached to SEM. Phase structure was investigated by X-ray diffraction. The hardness and abrasive wear test of the coatings were investigated. The biological property of the coatings was examined by immersing the as-sprayed coatings in simulated body fluid (SBF) solution for 20 days at 36.5 C. The growth of apatite (HA) on the coatings surfaces was observed by SEM and EDX analysis.     

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate

At the rat motor nerve terminals, activation of muscarinic M₁ receptors negatively modulates the activity of inhibitory muscarinic M₂ receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involved endogenous adenosine tonically activating A₁ receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [³H]-choline (2.5 μCi/ml). Selective activation of muscarinic M₁ and adenosine A₁ receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 μM) and R-N⁶-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [³H]-ACh release induced by muscarinic M₂ receptor activation with oxotremorine (10 μM). Attenuation of the inhibitory effect of oxotremorine (10 μM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M₁ autoreceptors, suggesting that suppression of M₂-inhibiton by A₁ receptor activation is independent on muscarinic M₁ receptor activity. Conversely, the negative crosstalk between M₁ and M₂ autoreceptors seems to involve endogenous adenosine tonically activating A₁ receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 μM) by McN-A-343 (3 μM) was suppressed by the A₁ receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 μM). The results suggest that the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A₁ receptors at the rat motor endplate.     

C9orf72 G4C2 repeat expansions in Alzheimer's disease and mild cognitive impairment

C9orf72 G₄C₂ repeat expansion is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Its role in Alzheimer's disease (AD) is less clear. We assessed the prevalence of G₄C₂ pathogenic repeat expansions in Flanders-Belgian patients with clinical AD or mild cognitive impairment (MCI). In addition, we studied the effect of non-pathogenic G₄C₂ repeat length variability on susceptibility to AD, and on AD cerebrospinal fluid (CSF) biomarker levels. A pathogenic repeat expansion was identified in 5 of 1217 AD patients (frequency <1%). No pathogenic expansions were observed in patients with MCI (n = 200) or control individuals (n = 1119). Nonpathogenic repeat length variability was not associated with AD, risk of conversion to AD in MCI individuals, or CSF biomarker levels. We conclude that pathogenic C9orf72 G₄C₂ repeat expansions can be detected in clinical AD patients and could act as a contributor to AD pathogenesis. Non-pathogenic repeat length variability did not affect risk of AD or MCI, nor AD biomarker levels in CSF, indicating that C9orf72 is not a direct AD risk factor.     

PCR-Based Methods for Identifying Genetic Variations in Human α1B- and β2-Adrenergic Receptors

α- and β-adrenergic receptors belong to the superfamily of G-protein-coupled, seven transmembrane domain receptors and regulate a variety of cellular processes. Previous studies have demonstrated that changes in the amino acid sequence can result in substantial changes in the function of the receptors and it has been suggested that there may be an association between different disease states and the altered structure of α- and β-adrenergic receptors. Accordingly, we have developed a simple PCR method for the identification of polymorphisms in the coding sequences of the human β₂-adrenergic receptor and the α_1B-adrenergic receptor. This method may be useful for screening individual patients or at-risk populations for endocrine-metabolic disorders, as well as for asthma, cardiovascular disorders, and neuropsychiatric diseases.     

Changes in 5-HT2A-mediated behavior and 5-HT2A- and 5-HT1A receptor binding and expression in conditional brain-derived neurotrophic factor knock-out mice

Changes in brain-derived neurotrophic factor (BDNF) expression have been implicated in the etiology of psychiatric disorders. To investigate pathological mechanisms elicited by perturbed BDNF signaling, we examined mutant mice with central depletion of BDNF (BDNF^2L/2LCk-cre). A severe impairment specific for the serotonin 2A receptor (5-HT_2AR) in prefrontal cortex was described previously in these mice. This is of much interest, as 5-HT_2ARs have been linked to neuropsychiatric disorders and anxiety-related behavior. Here we further characterized the serotonin receptor alterations triggered by BDNF depletion. 5-HT_2A ([³H]-MDL100907) and 5-HT_1A ([³H]-WAY100635) receptor autoradiography revealed site-specific alterations in BDNF mutant mice. They exhibited lower 5-HT_2A receptor binding in frontal cortex but increased binding in hippocampus. Additionally, 5-HT_1A receptor binding was decreased in hippocampus of BDNF mutants, but unchanged in frontal cortex. Molecular analysis indicated corresponding changes in 5-HT_2A and 5-HT_1A mRNA expression but normal 5-HT_2C content in these brain regions in BDNF^2L/2LCk-cre mice. We investigated whether the reduction in frontal 5-HT_2AR binding was reflected in reduced functional output in two 5-HT_2A-receptor mediated behavioral tests, the head-twitch response (HTR) and the ear-scratch response (ESR). BDNF^2L/2LCk-cre mutants treated with the 5-HT_2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) showed a clearly diminished ESR but no differences in HTR compared to wildtypes. These findings illustrate the context-dependent effects of deficient BDNF signaling on the 5-HT receptor system and 5-HT_2A-receptor functional output.     

Differential distribution of γ-aminobutyric acidA, receptor subunit (α1, α2, α3, α5 and β2+3) immunoreactivity in the medial prefrontal cortex of the rat

A detailed mapping of the γ-aminobutyric acid (GABA)_A receptor subunits (α₁, α₂, α₃ and β₂₊₃) in the infralimbic/ventral prelimbic region (IL/vPL) of the rat frontal cortex was carried out using subunit-specific antibodies. The α₁ and β₂₊₃ subunit antibodies immunostained all layers of the IL/vPL region. Layers II and III displayed immunostaining of cell bodies whereas I, V and VI showed predominantly neuropil staining. The size of the α₁-positive cell bodies corresponded to that of small interneurons (range, 20–55 μm²; mean ± SEM, 37 ± 5.5 μm²) as well as pyramidal cells or large interneurons (range, 87–135 μm²; mean ± SEM, 103.4 ± 9.7 μm²). However, β₂₊₃ antibody immunostained only small cell bodies. Immunoreactivity for α₂ was restricted to layers I and II, whereas α₃ and α₅ subunit expression was seen only in layer VI. The antibody to the α₂ subunit immunostained small cell bodies (range, 29–63 μm²; mean ± SEM, 32 ± 4.5 μm²) in layer II, resembling interneurons. Conversely, both α₃ and α₅ antibodies immunostained large cell bodies (range, 94–151 gmm²; mean ± SEM, 115.7 ± 13.4 μm²), consistent with pyramidal cell labelling in layer VI.     

Comparison of the ability of wild type and stabilized human IgG4 to undergo Fab arm exchange with endogenous IgG4 in vitro and in vivo

Fab arm exchange by a stabilized anti-IL-31 IgG₄S228P monoclonal antibody (mAb) was studied using physiologically relevant antibody concentrations and thiol exchange conditions, and directly compared to that of matched wild type IgG₄ (IgG₄wt) and IgG₁ control antibodies. In vitro arm exchange between the test mAbs and a purified IgG₄wt exchange partner was monitored using capillary isoelectric focusing and a size-exclusion peak shift assay. Arm exchange between the test mAbs and IgG exchange partners with unknown specificity was monitored using only the shift assay. Studies were performed using single isotype human and mouse mAbs, unfractionated human, mouse, and cynomolgus monkey IgG, and human serum as the sources of the exchange partners. In vitro studies using human serum demonstrated that anti-IL-31 IgG₄S228P did not undergo significant Fab arm exchange with endogenous human IgG₄ whereas anti-IL-31 IgG₄wt underwent rapid and extensive Fab arm exchange. The in vitro results were corroborated by in vivo studies in which mice were injected with a mixture of either form of the test mAb and an excess of non-specific human IgG₄ exchange partner.     

Optical and biological sensing capabilities of Au2S/AuAgS coated gold nanorods

Gold nanorods coated with a multiplex component, namely Au₂S/AuAgS coated gold nanorods, are produced without precipitation and aggregation among the nanorods. Both the thickness of the shell and size of the core can be readily controlled by this technique allowing one to tune the plasmon resonance of the nanocomposites over a range of several hundred nanometers. These Au₂S/AuAgS coated gold nanorods exhibit interesting optical properties and are suitable for many biological sensing applications. Functionalization of the Au₂S/AuAgS coated gold nanorods is achieved by manipulating the affinity between the Au₂S/AuAgS and thiol compounds. Biomolecules can be covalently attached via the NH₂ bond of the antibodies to the NHS-terminated nanorods. The longitudinal peaks of the Au₂S/AuAgS coated gold nanorods are extremely sensitive to the refractive index changes induced by target binding, suggesting that they are excellent sensors for target-specific binding events and have the potential to achieve single-molecule sensitivity in microspectroscopy.     

Leukotrienes (LTC4, LTD4) confer glass non-adherence on leukocytes of asthmatic individuals. Dependency on cycloxygenase products and calcium ion

It has been suggested that cysteinyl-containing leukotrienes (LT) are important mediators in bronchial asthma. Since leukotrienes have been shown to mediate the leukocyte adherence inhibition (LAI) phenomenon observed in cancer-bearing host we have devised a modified LAI assay which determines the acquisition of non-adherence properties of leukocytes following a challenge with pure synthetic LT. Our results demonstrate that peripheral blood leukocytes of asthmatic individuals acquire non-adherence properties when challenged with pure synthetic leukotriene C₄ and D₄, a property not shared by peripheral blood leukocytes of control healthy individuals. Furthermore, we demonstrate that LT activity as manifested by the LAI assay is dependent on cycloxygenase products, since 2×10⁻⁶ M Indomethacin abrogated the LT-induced LAI and is restored by the addition of 2×10⁻⁶ M prostaglandin E₂ which is also synergistic to LT activity. Our results further suggest the possibility that leukotriene activity is dependent on calcium ions since it was negated by known calcium antagonists. It is thus suggested that the LT-induced LAI may serve as a tool for the study of the interrelationship between the metabolic pathways of arachidonic acid and calcium ion homeostasis.