Decreased T cell expression of H/ACA box small nucleolar RNA 12 promotes lupus pathogenesis in patients with systemic lupus erythematosus.

Objective To investigate whether the aberrant expression of non-coding RNAs (ncRNAs) in T cells from patients with systemic lupus erythematosus (SLE) could contribute to the pathogenesis of lupus. Methods Expression profiles of RNA transcripts in T cells from three patients with SLE and three controls were analyzed by microarray analysis. Potentially aberrant-expressed ncRNAs were validated using T cell samples from 23 patients with SLE and 17 controls. Transfection studies and microarray analyses were conducted to search for any gene expression that is regulated by specific ncRNAs. Results Initial analysis revealed differential expression of 18 ncRNAs in SLE T cells. After validation, decreased expression of H/ACA box small nucleolar RNA 12 (SNORA12) was confirmed in SLE T cells (0.69-fold, P = 0.007) compared with normal T cells, and its expression level was inversely associated with higher SLE disease activity scores. Jurkat cells transfected with a plasmid encoding SNORA12 showed increased expression of two genes and decreased expression of 15 genes in Jurkat cells. These changes of gene expression were significantly associated with the SLE pathway in the Kyoto Encyclopedia of Genes and Genomes map using microarray analysis. Overexpression of SNORA12 altered the expression of CD69, decreased the expression of histone cluster 1 H4 family member k (HIST1H4K), inhibited the secretion of interferon gamma and the expression of HIST1H4K was increased in SLE T cells. Conclusion Among the ncRNAs, we found that the expression level of SNORA12, which belongs to the family of small nucleolar RNAs, was lower in SLE T cells and affected T cell function. This novel finding suggests that aberrant-expressed snoRNAs lead to dysfunction of T cells and may be involved in the immunopathogenesis of SLE.

Increased prevalence of JC polyomavirus viruria was associated with arthritis/arthralgia in patients with systemic lupus erythematosus.

OBJECTIVE: The objective of this paper is to investigate the prevalence of reactivation of the human polyomavirus John Cunningham virus (JCV) in patients with systemic lupus erythematosus (SLE) and its associated clinical manifestations. METHODS: Sixty-one patients with SLE and 22 controls were enrolled. Urine JCV viral load was quantified by real-time polymerase chain reaction (PCR). Length variants of the VP1 gene were analyzed using capillary electrophoresis. RESULTS: The prevalence of JCV viruria (63.9% vs. 18.2%, p < 0.001) and urine JCV viral load (2.92 ± 2.76 vs. 0.81 ± 1.85 copies/ml by log10 scale, p < 0.001) were significantly higher in patients with SLE compared with controls. JCV viruria (+) SLE patients had a higher occurrence of arthritis/arthralgia compared with JCV viruria (-) SLE patients (64.1% vs. 22.7%, p = 0.003). In SLE patients, the urine JCV viral load was significantly associated with the occurrence of arthritis/arthralgia. SLE patients with urine JCV viral load >10,000 copies/ml exhibited a 12.75-fold (95% confidence interval 2.88-56.40) risk in clinical arthritis/arthralgia, 18.90-fold (95% confidence interval 2.10-170.39) risk in persistent arthritis, and significantly greater number of length variants in the VP1 gene of JCV compared with JCV viruria (-) SLE patients. CONCLUSION: Reactivation of JCV in the urinary tract of SLE patients was very common. Both JCV viruria and urine JCV viral load were associated with the occurrence of arthritis/arthralgia in patients with SLE. High urine JCV viral load also was associated with the genetic variant in the VP1 gene.

Decreased microRNA(miR)-145 and increased miR-224 expression in T cells from patients with systemic lupus erythematosus involved in lupus immunopathogenesis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with abnormal T cell immune responses. We hypothesized that aberrant expression of microRNAs (miRNAs) in T cells may contribute to the pathogenesis of SLE. First, we analysed the expression profiles of 270 human miRNAs in T cells from five SLE patients and five healthy controls and then validated those potentially aberrant-expressed miRNAs using real-time polymerase chain reaction (PCR). Then, the expression of mRNAs regulated by these aberrant-expressed miRNAs was detected using real-time PCR. Finally, miRNA transfection into Jurkat T cells was conducted for confirming further the biological functions of these miRNAs. The initial analysis indicated that seven miRNAs, including miR-145, miR-224, miR-513-5p, miR-150, miR-516a-5p, miR-483-5p and miR-629, were found to be potentially abnormally expressed in SLE T cells. After validation, under-expressed miR-145 and over-expressed miR-224 were noted. We further found that STAT1 mRNA targeted by miR-145 was over-expressed and apoptosis inhibitory protein 5 (API5) mRNA targeted by miR-224 was under-expressed in SLE T cells. Transfection of Jurkat cells with miR-145 suppressed STAT1 and miR-224 transfection suppressed API5 protein expression. Over-expression of miR-224 facilitates activation-induced cell death in Jurkat cells. In the clinical setting, the increased transcript levels of STAT1 were associated significantly with lupus nephritis. In conclusion, we first demonstrated that miR-145 and miR-224 were expressed aberrantly in SLE T cells that modulated the protein expression of their target genes, STAT1 and API5, respectively. These miRNA aberrations accelerated T cell activation-induced cell death by suppressing API5 expression and associated with lupus nephritis by enhancing signal transducer and activator of transcription-1 (STAT)-1 expression in patients with SLE.

Combination of nifedipine and subtherapeutic dose of cyclosporin additively suppresses mononuclear cells activation of patients with rheumatoid arthritis and normal individuals via Ca(2+) -calcineurin-nuclear factor of activated T cells pathway.

Abnormal Ca(2+) -mediated signalling contributes to the pathogenesis of rheumatoid arthritis (RA). However, the potential implication of calcium channel blocker in RA remained unknown. We hypothesized that nifedipine, an L-type calcium channel blocker, combined with a calcineurin inhibitor, could suppress T cell activation via targeting different level of the Ca(2+) signalling pathway. The percentage of activated T cells and the apoptotic rate of mononuclear cells (MNCs) was measured by flow cytometry. The MNC viability, cytokine production, cytosolic Ca(2+) level and activity of the nuclear factor of activated T cells (NFAT) were measured by enzyme-linked immunosorbent assay (ELISA). The NFAT-regulated gene expression, including interleukin (IL)-2, interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF), was measured by real-time polymerase chain reaction (PCR). We found that the percentage of activated T cells in anti-CD3 + anti-CD28-activated MNC was higher in RA patients. High doses of nifedipine (50 µM) increased MNCs apoptosis, inhibited T cell activation and decreased T helper type 2 (Th1) (IFN-γ)/Th2 (IL-10) cytokine production in both groups. The Ca(2+) influx was lower in anti-CD3 + anti-CD28-activated MNC from RA patients than healthy volunteers and suppressed by nifedipine. When combined with a subtherapeutic dose (50 ng/ml) of cyclosporin, 1 µM nifedipine suppressed the percentage of activated T cells in both groups. Moreover, this combination suppressed more IFN-γ secretion and NFAT-regulated gene (GM-CSF and IFN-γ) expression in RA-MNCs than normal MNCs via decreasing the activity of NFATc1. In conclusion, we found that L-type Ca(2+) channel blockers and subtherapeutic doses of cyclosporin act additively to suppress the Ca(2+) -calcineurin-NFAT signalling pathway, leading to inhibition of T cell activity. We propose that this combination may become a potential treatment of RA.

Imaging gene delivery in a mouse model of congenital neuronal ceroid lipofuscinosis.

Adeno-associated virus (AAV)-mediated gene replacement for lysosomal disorders have been spurred by the ability of some serotypes to efficiently transduce neurons in the brain and by the ability of lysosomal enzymes to cross-correct among cells. Here, we explored enzyme replacement therapy in a knock-out mouse model of congenital neuronal ceroid lipofuscinosis (NCL), the most severe of the NCLs in humans. The missing protease in this disorder, cathepsin D (CathD) has high levels in the central nervous system. This enzyme has the potential advantage for assessing experimental therapy in that it can be imaged using a near-infrared fluorescence (NIRF) probe activated by CathD. Injections of an AAV2/rh8 vector-encoding mouse CathD (mCathD) into both cerebral ventricles and peritoneum of newborn knock-out mice resulted in a significant increase in lifespan. Successful delivery of active CathD by the AAV2/rh8-mCathD vector was verified by NIRF imaging of mouse embryonic fibroblasts from knock-out mice in culture, as well as by ex vivo NIRF imaging of the brain and liver after gene transfer. These studies support the potential effectiveness and imaging evaluation of enzyme replacement therapy to the brain and other organs in CathD null mice via AAV-mediated gene delivery in neonatal animals.

In vivo molecular target assessment of matrix metalloproteinase inhibition.

A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.

Pulse electroplating of copper film: a study of process and microstructure.

Copper films with high density of twin boundaries are known for high mechanical strength with little tradeoff in electrical conductivity. To achieve such a high density, twin lamellae and spacing will be on the nanoscale. In the current study, 10 microm copper films were prepared by pulse electrodeposition with different applied pulse peak current densities and pulse on-times. It was found that the deposits microstructure was dependent on the parameters of pulse plating. Higher energy pulses caused stronger self-annealing effect on grain recrystallization and growth, thus leading to enhanced fiber textures, while lower energy pulses gave rise to more random microstructure in the deposits and rougher surface topography. However in the extremes of pulse currents we applied, the twin densities were not as high as those resulted from the medium or relatively high pulse currents. The highest amount of nanoscale twinning was found to form from a proper degree of self-annealing induced grain structure evolution. The driving force behind the self-annealing is discussed.

In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.

We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.

Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells.

The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells.

In vivo imaging of proteolytic enzyme activity using a novel molecular reporter.

The single biggest challenge facing in vivo imaging techniques is to develop biocompatible molecular beacons that are capable of specifically and accurately measuring in vivo targets at the protein, RNA, or DNA level. Our efforts have focused on developing activatable imaging probes to measure specific enzyme activities in vivo. Using cathepsin D as a model target protease, we synthesized a long-circulating, synthetic graft copolymer bearing near-infrared (NIR) fluorochromes positioned on cleavable substrate sequences. In its native state, the reporter probe was essentially nonfluorescent at 700 nm due to energy resonance transfer among the bound fluorochromes (quenching) but became brightly fluorescent when the latter were released by cathepsin D. NIR fluorescence signal activation was linear over at least 4 orders of magnitude and specific when compared with scrambled nonsense substrates. Using matched rodent tumor models implanted into nude mice expressing or lacking the targeted protease, it could be shown that the former generated sufficient NIR signal to be directly detectable and that the signal was significantly different compared with negative control tumors. The developed probes should find widespread applications for real-time in vivo imaging of a variety of clinically relevant proteases, for example, to detect endogenous protease activity in disease, to monitor the efficacy of protease inhibitors, or to image transgene expression.

Adenovirus-mediated expression of antisense urokinase plasminogen activator receptor and antisense cathepsin B inhibits tumor growth, invasion, and angiogenesis in gliomas.

We have shown previously that urokinase plasminogen activator receptor (uPAR) and cathepsin B are overexpressed during glioma progression, particularly at the leading edge of the tumor. In the present study, we simultaneously down-regulated uPAR and cathepsin B in SNB19 glioma cell monolayer or SNB19 spheroids using an adenoviral vector carrying antisense uPAR and antisense cathepsin B and a combination of these genes as determined by Western blot analysis. The Ad-uPAR-Cath B-infected cells revealed a marked reduction in tumor growth and invasiveness as compared with the parental and vector controls. In vitro and in vivo angiogenic assays demonstrated inhibition of capillary-like structure formation and microvessel formation after Ad-uPAR-Cath B infection of SNB19 cells when compared with Ad-cytomegalovirus (CMV)-infected or mock-infected controls. Furthermore, using a near infrared fluorescence probe, in vivo imaging for cathepsin B indicated low/undetectable levels of fluorescence after injection of the Ad-uPAR-Cath B construct into pre-established s.c. tumors as compared with Ad-CMV-treated and untreated tumors. The effect with bicistronic construct (Ad-uPAR-Cath B) was much higher than with single (Ad-uPAR/Ad-Cath B) constructs. These results indicate that the down-regulation of cathepsin B and uPAR plays a significant role in inhibiting tumor growth, invasion, and angiogenesis. Hence, the targeting of these two proteases may be a potential therapy for brain tumors and other cancers.

Application of convergent-beam collimation and simultaneous transmission emission tomography to cardiac single-photon emission computed tomography.

Single-photon emission computed tomography (SPECT) is the most commonly performed imaging technique for perfusion studies of the heart and brain. However, these organs are much smaller than the crystal surface of gamma cameras. SPECT sensitivity and resolution can be improved by using fan- and cone-beam collimators to magnify the image of these organs over a larger portion of the crystal face. Special orbits and reconstruction algorithms must be used with convergent-beam acquisitions to prevent image distortion. Differential attenuation of source activity in the chest is one of the most vexing problems in cardiac SPECT, especially with Thallium-201. Multi-headed cameras equipped with convergent-beam collimators allow a transmission image to be obtained at the same time as emission images. Applying a transmission map of the chest attenuation values to the emission images produces a truer picture of source distribution in the heart. This article reviews the technical problems associated with convergent-beam geometry and simultaneous transmission emission tomography SPECT imaging of the heart.

Luminescence and photocatalytic properties of a platinum(II)-quaterpyridine complex incorporated in Nafion membrane.

The non-emissive platinum(II)-quaterpyridine complex shows strong photoluminescence at room temperature upon incorporation into Nafion membrane; this complex is stabilized toward photochemical decomposition in Nafion even in the presence of oxygen, and can be used as a sensitizer to generate singlet oxygen to oxidize alkenes.

A simulation of emission and transmission noise propagation in cardiac SPECT imaging with nonuniform attenuation correction.

Transmission computed tomography provides information needed for nonuniform attenuation correction of cardiac single photon emission computed tomography (SPECT). Nonuniform attenuation correction is accomplished using an iterative ML-EM algorithm and a projection-backprojection operation that incorporates attenuation factors measured from the reconstructed transmission map. The precision and accuracy of the attenuation corrected emission reconstruction is a function of emission and transmission statistics. This paper presents an error propagation analysis that uses a mathematical cardiac chest phantom to simulate various combinations of total emission counts C and transmission flux I0 under ideal imaging conditions (without geometric response distortion and without scatter). The spatial average, spatial variance, and accuracy measures for a 4 x 4 pixel region in the heart are tabulated after 30 iterations of the ML-EM algorithm. The confidence intervals for these measures were determined from 1000 realizations of reconstructions from projections randomly generated with the same transmission and emission statistics. It can be shown empirically from the simulation results that the spatial %rms uncertainty for the simulated cardiac region has a simple expression: %rms2 = K1/C+K2/I0(2)+B2 where K1 and K2 are least-square estimates based on the simulation results, and B is the measured spatial %rms uncertainty for the simulation at infinite statistics. For a transmission incident flux of 1500 events per projection bin of 0.712 cm and typical clinical emission events totaling 1 x 10(5), the spatial %rms uncertainty is approximately 14%. At clinical transmission and emission statistics, the statistical noise in the simulated attenuation-corrected reconstructions are dominated by the emission statistics.

Review of convergent beam tomography in single photon emission computed tomography.

Investigation of convergent-beam single photon emission computed tomography (SPECT) is actively being pursued to evaluate its clinical potentials. Fan-beam, cone-beam, pin-hole and astigmatic collimators are being used with rotating gamma cameras having large crystal areas, to increase the sensitivity for emission and transmission computed tomography of small organs such as the thyroid, brain or heart. With new multi-detector SPECT systems, convergent-beam geometry offers the ability to simultaneously obtain emission and transmission data necessary to quantify uptake of radiopharmaceutical distributions in the heart. The development of convergent-beam geometry in SPECT requires the integration of hardware and software. In considering hardware, the optimum detector system for cone-beam tomography is a system that satisfies the data sufficiency condition for which the scanning trajectory intersects any plane passing through the reconstructed region of interest. However, the major development of algorithms has been for the data insufficient case of single planar orbit acquisitions. The development of these algorithms have made possible the preliminary evaluation of this technology and the imaging of brain and heart are showing significant potential for the clinical application of cone-beam tomography. Presently, significant research activity is pursuing the development of algorithms for data acquisitions that satisfy the data sufficiency condition and that can be implemented easily and inexpensively on clinical SPECT systems.

Synthesis of oligoarginine-oligonucleotide conjugates and oligoarginine-bridged oligonucleotide pairs.

Conjugates consisting of oligoarginine peptides linked to oligodeoxynucleotides have been synthesized, including a new type of conjugate, in which a pair of oligonucleotides is bridged by a cationic peptide. Two different 9-mer oligonucleotides were conjugated to the terminal cysteine residues of the peptide series H-Cys-(Arg)n-Cys-NH2 (n = 3, 5, 7). Different thiol protecting groups were utilized on the amino- and carboxy-terminal cysteine residues of the peptide to allow selective attachment to the 3'- or 5'-terminus of each specific oligonucleotide. The conjugates containing oligoarginine peptides were purified by anion-exchange chromatography, and their structures were confirmed by polyacrylamide gel electrophoresis and amino acid analysis.

Polyamine-linked oligonucleotides for DNA triple helix formation.

The concept of antigene therapy of disease is based on the ability of an oligonucleotide (the therapeutic agent) to bind to double-stranded genomic DNA (the target associated with the disease). Examples are herein given of the linkage of a series of polyamines to a 21-mer homopyrimidine oligonucleotide. These conjugated 21-mers can each form a triple helix with an appropriate double-stranded homopurine-homopyrimidine DNA according to Hoogsteen base-pairing rules. No triple helix was found when unmodified third strand was used at 10 mM sodium phosphate, pH 6.5, 100 mM sodium chloride solution. In contrast, the spermine-conjugated oligonucleotide had a melting temperature of 42 degrees C. According to the melting profile, the appended spermine moiety was found to affect the Tm only of the triple helix, but not of the subsequent melting of the underlying double helix. The Tm enhancing ability of the spermine-conjugate was found to be better than that of other polyamine-conjugates.

Preparation of oligonucleotide-peptide conjugates.

A procedure for preparing oligonucleotide-peptide conjugates is presented. It is based on appending a maleimide group to the oligonucleotide for selective coupling to the thiol side chain of a cysteine residue in the peptide. A convenient chromatographic purification procedure, based on Fmoc-on/Fmoc-off, is described.