Immune Checkpoints of the B7 Family. Part 1. General Characteristics and First Representatives: B7-1, B7-2, B7-H1, B7-H2, and B7-DC

Russian Journal of Bioorganic Chemistry - T-cell response along with humoral response compose the basis of acquired immunity. Effective activation of T lymphocytes requires at least two signals....     

Multiplexed echo planar imaging for sub-second whole brain FMRI and fast diffusion imaging

Echo planar imaging (EPI) is an MRI technique of particular value to neuroscience, with its use for virtually all functional MRI (fMRI) and diffusion imaging of fiber connections in the human brain. EPI generates a single 2D image in a fraction of a second; however, it requires 2-3 seconds to acquire multi-slice whole brain coverage for fMRI and even longer for diffusion imaging. Here we report on a large reduction in EPI whole brain scan time at 3 and 7 Tesla, without significantly sacrificing spatial resolution, and while gaining functional sensitivity. The multiplexed-EPI (M-EPI) pulse sequence combines two forms of multiplexing: temporal multiplexing (m) utilizing simultaneous echo refocused (SIR) EPI and spatial multiplexing (n) with multibanded RF pulses (MB) to achieve m×n images in an EPI echo train instead of the normal single image. This resulted in an unprecedented reduction in EPI scan time for whole brain fMRI performed at 3 Tesla, permitting TRs of 400 ms and 800 ms compared to a more conventional 2.5 sec TR, and 2-4 times reductions in scan time for HARDI imaging of neuronal fibertracks. The simultaneous SE refocusing of SIR imaging at 7 Tesla advantageously reduced SAR by using fewer RF refocusing pulses and by shifting fat signal out of the image plane so that fat suppression pulses were not required. In preliminary studies of resting state functional networks identified through independent component analysis, the 6-fold higher sampling rate increased the peak functional sensitivity by 60%. The novel M-EPI pulse sequence resulted in a significantly increased temporal resolution for whole brain fMRI, and as such, this new methodology can be used for studying non-stationarity in networks and generally for expanding and enriching the functional information.     

Simultaneous amplification and screening of whole plasmids using the T7 bacteriophage replisome

This study describes a novel helicase-mediated isothermal DNA amplification method that exponentially amplifies circular DNAs. The circular helicase-dependent amplification (cHDA) system is based on the T7 replication machinery, which includes the processive T7 helicase, an exonuclease-deficient T7 DNA polymerase (T7 Sequenase) and the T7 Gp2.5 single-stranded DNA-binding (SSB) protein. After the duplex DNA template is unwound by the T7 helicase, specific primers anneal to the separated DNA strands and T7 Sequenase extends the 3′ end of each primer by a rolling circle mechanism to amplify not only a region defined by the primers but also continuous concatemers of the template. The cHDA reaction can be carried out at one temperature (25°C) for the entire process and can achieve up to 10000-fold amplification. Amplification can be performed using purified plasmid DNA or a crude cell lysate and can amplify inserts as large as 10 kb. Following a cHDA reaction, the amplified products can be used directly for sequencing and restriction enzyme digestion without further purification. By utilizing the helicase enzyme, circular DNA samples can be simultaneously screened and amplified at one constant temperature in one easy step.     

Identification and mapping of five new genes in bacteriophage T7

Mutations that affect the single-stranded DNA-binding protein of bacteriophage T7 (gene 2.5) and four T7 proteins of unknown function (the gene 4.3, 4.5, 4.7 and 5.5 proteins) are described and mapped by three-factor crosses. An extensive search for mutants defective in the DNA-binding protein (M_r = 25,562) produced several strains in which this protein has an altered electrophoretic mobility but no strains that appear to lack it completely. The gene 2.5 mutation that was mapped produces a slightly short DNA-binding protein that appears functional by tests in vitro. It seems likely that a functional DNA-binding protein is needed for T7 growth but that conditional-lethal amber mutations in it are rare; the nucleotide sequence known to code for the gene 2.5 protein contains only 1 to 3 sites that would be expected to be readily mutable to conditional-lethal amber codons by N-methyl-N?nitro-N-nitrosoguanidine. The gene 4.3, 4.5 and 4.7 proteins (M_r ～ 8000 to 15,000) are eliminated by a deletion mutant that removes most of the DNA between genes 4 and 5. The gene 5.5 protein (M_r ～ 11,700) is made in relatively large amounts and is affected by two different mutations that were mapped between genes 5 and 6. One of these mutations appears to be an amber mutation that eliminates the protein entirely; the other decreases the electrophoretic mobility of the protein (an apparent increase in size). A larger protein (M_r ～ 18,000), found in small amounts and difficult to observe, is also affected by these two mutations; the relationship of this minor protein to the major gene 5.5 protein is not yet known. The genes 2 and 18 proteins have also been identified in patterns of protein synthesis during infection. The proteins specified by at least 34 different T7 genes have now been identified.     

Host B7-1 and B7-2 costimulatory molecules contribute to the eradication of B7-1-transfected P815 tumor cells via a CD8+ T cell-dependent mechanism

B7-1 (CD80)-transfected P815 tumor cells were previously shown to elicit tumor-eradicating immunity that leads to the regression of B7-1+ P815 tumors after transient growth in normal syngeneic (DBA/2) mice. Here, we show that not only the B7-1 molecule but also the B7-2 (CD86) molecule contributed to the eradication of B7-1+ P815 tumors. The B7-1 molecule that contributed to the eradication of B7-1+ P815 tumors was expressed not only on the tumor cells but also on host APCs, including MAC-1+ cells. The B7-2 molecule that contributed to the eradication of B7-1+ P815 tumors was expressed only on host APCs, such as B220+ cells, and not on the tumor cells. In spite of the fact that B7-expressing host APCs contributed to the eradication of B7-1+ P815 tumors, only CD8+ T cells without help from CD4+ T cells were important for tumor eradication. Taken together, these findings indicate that in addition to the ability of B7-1-transfected tumor cells to stimulate CD8+ T cell-mediated tumor-eradicating immunity directly, such tumor cells can also stimulate CD8+ T cell-mediated tumor-eradicating immunity indirectly as a result of cross-priming through B7-expressing host APCs.     

Restriction enzyme cleavage mapping of T7 virus early region

Summary DNA molecules of seven T7 mutants with overlapping deletions in the early region were cleaved by restriction enzymes HindII, HpaI and II, and HaeIII. The differences in the cleavage patterns after electrophoresis have been used to generate a cleavage map of the restriction sites of this enzyme. It covers the first 9% of the T7 DNA molecule. Cleavage points for HindII are at 0.60, 1.33, 1.59, 1.76, 5.26, 6.27, 7.4 and 8.38%; for HpaI and II at 1.36, 1.62, 4.46, 6.29, 6.62, 7.56, and 8.76%; for HaeIII at 3.85, 6.98, 7.88 and 8.26%. Some fragments have been located in the region containing the early promoters, others carry the complete sequences of gene 0.3.     

Accuracy of real-time MR temperature mapping in the brain: A comparison of fast sequences

PurposeTo compare magnetic resonance (MR) thermometry based on the proton resonance frequency (PRF) method using a single shot echoplanar imaging (ss EPI) sequence to both of the standard sequences, gradient echo (GRE) and segmented echoplanar imaging (seg EPI) in the in vivo human brain, at 1.5T and 3T.Material and methodsRepetitive MR thermometry was performed on the brain of six volunteers using GRE, seg EPI, and ss EPI sequences on whole-body 1.5T and 3T clinical systems using comparable acquisition parameters. Phase stability and temperature data precision in the human head were determined over 12 min for the three sequences at both field strengths. An ex-vivo swine skeletal muscle model was used to evaluate temperature accuracy of the ss EPI sequence during heating by high intensity focused ultrasound (HIFU).ResultsIn-vivo examinations of brain revealed an average temperature precision of 0.37 °C/0.39 °C/0.16 °C at 3T for the GRE/seg EPI/ss EPI sequences. At 1.5T, a precision of 0.58 °C/0.63 °C/0.21 °C was achieved. In the ex-vivo swine model, a strong correlation of temperature data derived using ss EPI and GRE sequences was found with a temperature deviation <1 °C.ConclusionThe ss EPI sequence was the fastest and the most precise sequence for MR thermometry, with significantly higher accuracy compared to GRE.     

Thermophysiological responses of human volunteers to whole body RF exposure at 220 MHz

Since 1994, our research has demonstrated how thermophysiological responses are mobilized in human volunteers exposed to three radio frequencies, 100, 450, and 2450 MHz. A significant gap in this frequency range is now filled by the present study, conducted at 220 MHz. Thermoregulatory responses of heat loss and heat production were measured in six adult volunteers (five males, one female, aged 24-63 years) during 45 min whole body dorsal exposures to 220 MHz radio frequency (RF) energy. Three power densities (PD = 9, 12, and 15 mW/cm(2) [1 mW/cm(2) = 10 W/m(2)], whole body average normalized specific absorption rate [SAR] = 0.045 [W/kg]/[mW/cm(2)] = 0.0045 [W/kg]/[W/m(2)]) were tested at each of three ambient temperatures (T(a) = 24, 28, and 31 degrees C) plus T(a) controls (no RF). Measured responses included esophageal (T(esoph)) and seven skin temperatures (T(sk)), metabolic rate (M), local sweat rate, and local skin blood flow (SkBF). Derived measures included heart rate (HR), respiration rate, and total evaporative water loss (EWL). Finite difference-time domain (FDTD) modeling of a seated 70 kg human exposed to 220 MHz predicted six localized "hot spots" at which local temperatures were also measured. No changes in M occurred under any test condition, while T(esoph) showed small changes (< or =0.35 degrees C) but never exceeded 37.3 degrees C. As with similar exposures at 100 MHz, local T(sk) changed little and modest increases in SkBF were recorded. At 220 MHz, vigorous sweating occurred at PD = 12 and 15 mW/cm(2), with sweating levels higher than those observed for equivalent PD at 100 MHz. Predicted "hot spots" were confirmed by local temperature measurements. The FDTD model showed the local SAR in deep neural tissues that harbor temperature-sensitive neurons (e.g., brainstem, spinal cord) to be greater at 220 than at 100 MHz. Human exposure at both 220 and 100 MHz results in far less skin heating than occurs during exposure at 450 MHz. However, the exposed subjects thermoregulate efficiently because of increased heat loss responses, particularly sweating. It is clear that these responses are controlled by neural signals from thermosensors deep in the brainstem and spinal cord, rather than those in the skin.     

MR T1 mapping for quantifying brain manganese deposition in type C hepatic encephalopathy rats

BioMetals - To evaluate magnetic resonance (MR) T1 mapping for quantifying brain manganese (Mn) deposition in type C hepatic encephalopathy (CHE) rats and to investigate the mechanism of magnesium...     

Acquisition of CD80 (B7-1) by T cells

Activation of T cells usually requires two signals. Signal 1 is mediated via a peptide-MHC on the APC; signal 2 is mediated via a costimulatory molecule on the APC surface. We demonstrate here that naive CD4(+) T cells actually acquire the costimulatory molecule CD80 (B7-1) from syngeneic APCs after activation. This phenomenon was demonstrated showing acquisition of CD80 by T cells from CD80/CD86 (B7-2) knockout mice, and by treating T cells with cyclohexamide to further rule out endogenous expression of CD80 by T cells. Moreover, no CD80 mRNA could be detected in T cells that had acquired CD80. The amount of acquisition of CD80 by T cells was shown to be directly related to both the strength of signal 1 and the amount of CD80 on the APC. Specificity of this acquisition was also shown by the lack of acquisition by T cells from CD28 knockout mice (implicating CD28 in this process), the lack of acquisition of CD40 (another molecule on the APC surface) by T cells, and confocal microscopy studies. We demonstrate for the first time that 1) naive T cells, following acquisition of CD80 from APCs, were themselves shown to be capable of acting as APCs; and 2) memory T cells that have acquired CD80 from APCs undergo apoptosis in the presence of increased levels of signal 1. Thus we demonstrate both immunostimulatory and immunoregulatory functions as a result of CD80 acquisition by different T cell populations.     

Dysregulated B7-1 and B7-2 expression on nonobese diabetic mouse B cells is associated with increased T cell costimulation and the development of insulitis

Little is known about the pathogenic role of B cell dysfunction in T cell-mediated autoimmune disease. We previously reported that B cell hyper-responsiveness, resistance to apoptosis, and accumulation in islets occur during the onset of insulitis, but not in type 1 diabetes (T1D), in NOD mice. In this study we extended these studies to further determine how islet-infiltrated B cells contribute to this inflammatory insulitis. We demonstrate the presence of an increased percentage of B7-1(+) and a decreased percentage of B7-2(+) B cells in the spleen of autoimmune disease-prone NOD and nonobese diabetes-resistant mice compared with the spleen of nonautoimmune disease-prone C57BL/6 and BALB/c mice. An age-dependent differential expression of B7-1 and B7-2 was associated with the development of insulitis and CD4(+)CD25(+) T cell deficiency in autoimmune disease-prone mice. Whereas BCR and LPS stimulation increased B7-2 expression on B cells from autoimmune disease-prone and nonautoimmune disease-prone mice, LPS-induced B7-1 expression was higher on NOD than C57BL/6 B cells. Interestingly, increased expression of B7-1 and B7-2 was found on islet-infiltrated B cells, and this increase was associated with enhanced T cell costimulation. Islet-infiltrated B cells were shown to be a source of TNF-alpha production in islets. B7 blockade of BCR-stimulated NOD B cells by anti-B7-1 and anti-B7-2 mAbs during coadoptive transfer with diabetogenic T cells into NOD.scid mice protected these recipients from T1D. These results suggest that increased B7-1 and B7-2 expression on islet-infiltrated NOD B cells is associated with increased T cell costimulation and the development of inflammatory insulitis in NOD mice.