Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2⁺ cells after intravenous injection and in CD11c⁺ dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8⁺ T-cell response in HLA-A2 transgenic mice and stimulated a CD4⁺ T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A^b. As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.     

Is IP-10 a Better Biomarker for Active and Latent Tuberculosis in Children than IFNγ?

Background

The blood based interferon-gamma release assays (IGRA) for the diagnosis of tuberculosis do not discriminate between active TB disease and latent TB infection (LTBI). The search for distinguishing biomarkers therefore continues, as the accurate diagnosis of tuberculosis is particularly challenging in children. IFN-γ-inducible protein 10 (IP-10/CXCL10) has recently been evaluated as a marker for active TB in adults with promising results.

Aim

To investigate this new biomarker for active TB and LTBI in paediatrics.

Method

We measured IP-10 levels using ELISA in supernatants of whole blood samples stimulated with TB-specific-antigens and negative control antigen.

Results

IP-10 is produced in high levels following mycobacterial antigen stimulation in active TB (n = 17) and LTBI (n = 16) compared to controls (n = 16) and to IFN-γ. The baseline levels of IP-10 are increased in active TB and in LTBI, but there is no significant difference of stimulated levels of IP-10 between active TB and LTBI.

Conclusions

IP-10 is a biomarker for tuberculosis in children. However like IFNγ, IP-10 also does not distinguish between active TB and LTBI.     

Oxidative stress induced by lead, cadmium and arsenic mixtures: 30-day, 90-day, and 180-day drinking water studies in rats: An overview

Humans are frequently exposed to combinations of lead (Pb), cadmium (Cd) and Arsenic (As) but there is a paucity of actual data on the molecular effects of these agents at low dose levels. The present factorial design studies were undertaken in rats to examine the effects of these agents at LOEL dose levels on a number of molecular parameters of oxidative stress in hematopoietic and renal organ systems following oral exposure in drinking water at 30, 90 and 180 day time points. Results of these studies demonstrated dynamic, time-dependent alterations in both molecular targets and inducible oxidative stress protective systems in target cell populations. In general, cellular protective systems, which protected against oxidative damage at the 90 day time point, appeared to be finite such that molecular manifestations of oxidative stress became statistically significant at the 180 day time point for several of the combination exposure groups. These data demonstrate the importance of duration of exposure in assessing the toxic potential of Pb, Cd and As mixtures at low dose levels.     

Conformationally gated metal uptake by apomanganese superoxide dismutase

Metal uptake by apomanganese superoxide dismutase in vitro is a complex process exhibiting multiphase "gated" reaction kinetics and a striking sigmoidal temperature profile that has led to a model of conformationally gated metal binding, requiring conversion between "closed" and "open" forms. This work systematically explores the structural determinants of metal binding in both wild-type (WT) apoprotein and mutational variants as a test of mechanistic models. The pH dependence of metalation under physiological conditions (37 degrees C) shows it is linked to ionization of a single proton with a p K a of 7.7. Size exclusion chromatography demonstrates that the apoprotein is dimeric even when it is fully converted to the open form. The role of molecular motions in metal binding has been probed by using disulfide engineering to introduce covalent constraints into the protein. While restricting motion at domain interfaces has no effect, constraining the subunit interface significantly perturbs metal uptake but does not prevent the process. Mutagenesis of residues in the active site environment results in a dramatic shift in the transition temperature by as much as 20 degrees C or a loss of pH sensitivity. On the basis of these results, a mechanism for metal uptake by manganese superoxide dismutase involving reorientation of active site residues to form a metal entry channel is proposed.     

The electronic structure of the Cys-Tyr(*) free radical in galactose oxidase determined by EPR spectroscopy

Galactose oxidase is a metalloenzyme containing a novel metalloradical complex in its active site, comprised of a mononuclear copper ion associated with a protein free radical. The free radical has been shown to be localized on an intrinsic redox cofactor, 3'-(S-cysteinyl)tyrosine (Cys-Tyr), formed by a posttranslational covalent coupling of tyrosine and cysteine side chains in a self-processing reaction. The role of the thioether linkage in the function of the Cys-Tyr cofactor is unresolved, and some computational studies have suggested that the thioether substituent has a negligible effect on the properties of the tyrosyl free radical. In order to address this question experimentally, we have incorporated site-selectively labeled tyrosine ((2)H, (13)C, (17)O) into galactose oxidase using an engineered tyrosine auxotroph strain of Pichia pastoris . (33)S was also incorporated into the protein. EPR spectra for the Cys-Tyr(*) free radical in each of these isotopic variants were analyzed to extract nuclear hyperfine parameters for comparison with theoretical predictions, and the unpaired spin distribution in the free radical was reconstructed from the hyperfine data. These labeling studies allow the first comprehensive experimental evaluation of the effect of the thioether linkage on the properties of Cys-Tyr(*) and indicate that previous calculations significantly underestimated the contribution of this feature to the electronic ground state of the free radical.     

Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus stapfianus and Xerophyta viscosa.

The phosphorylation of glucose and fructose is an important step in regulating the supply of hexose sugars for biosynthesis and metabolism. Changes in leaf hexokinase (EC 2.7.1.1) activity and in vivo metabolite levels were examined during drying in desiccation-tolerant Sporobolus stapfianus and Xerophyta viscosa. Leaf hexokinase activity was significantly induced from 85% to 29% relative water content (RWC) in S. stapfianus and from 89% to 55% RWC in X. viscosa. The increase in hexokinase corresponded to the region of sucrose accumulation in both species, with the highest activity levels coinciding with region of net glucose and fructose removal. The decline of hexose sugars and accumulation of sucrose in both plant species was not associated with a decline in acid and neutral invertase. The increase in hexokinase activity may be important to ensure that the phosphorylation and incorporation of glucose and fructose into metabolism exceeded production from potential hydrolytic activity. Total cellular glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P) levels were held constant throughout dehydration. In contrast to hexokinase, fructokinase activity was unchanged during dehydration. Hexokinase activity was not fully induced in leaves of S. stapfianus dried detached from the plant, suggesting that the increase in hexokinase may be associated with the acquisition of desiccation-tolerance.     

Potent, orally active inhibitors of lipoprotein-associated phospholipase A(2): 1-(biphenylmethylamidoalkyl)-pyrimidones.

A series of 1-(biphenylmethylamidoalkyl)-pyrimidones has been designed as nanomolar inhibitors of recombinant lipoprotein-associated phospholipase A(2) with high potency in whole human plasma. 5-(Pyrazolylmethyl) derivative 16 and 5-(methoxypyrimidinylmethyl) derivative 27 demonstrated excellent pharmacodynamic profiles which correlated well with their pharmacokinetic effects.     

COMPARATIVE STUDIES IN SYNAPTOSOME FORMATION: THE PREPARATION OF SYNAPTOSOMES FROM THE HEAD GANGLION OF THE SQUID, LOLIGO PEALII

Abstract— Relatively high concentrations of ACh have been found in the head ganglion of the squid ( Loligo pealii ) and the identity of the ACh has been verified by ion-exchange chromatography. Following homogenization in media iso-osmotic with sea water about 40 per cent of the ACh survives in particle-bound form. Experiments using media of varying osmolarity suggest that this bound ACh is osmotically sensitive. A study has been made of the subcellular fractionation of squid head ganglion using sucrose homogenates. A rapid and novel method is described for the preparation of a synaptosome fraction freed from mitochondria. This preparation contains synaptosomes of well-preserved morphology with occluded cytoplasm and a high specific content of ACh. The synaptosomes are osmotically sensitive and when suspended in water they burst, releasing cytoplasmic constituents and ACh-containing synaptic vesicles. The synaptic vesicles can be separated from other sub-synaptic constituents by density gradient centrifugation.     

Development of vestigial tail muscle acetylcholinesterase in embryos of an anural ascidian species.

1. The ascidian Molgula arenata produces an anural larva lacking a tail and other structural features of typical urodele larvae in the family Molglidae, yet its embryos developed a histochemically detectable acetylcholinesterase in the tail muscle rudiment. Development of the myoblasts seemed to fail during the neurula stage. 2. Larval enzyme activity occurred at a mean of 5--6% of the level found in the urodele species Molgula occidentalis and Molgula manhattensis, as measured by scanning integrating microdensitometry of the histochemical reaction product. Some anural larvae had as much as 20% of the enzyme activity in urodele larvae. 3. This example of vestigial expression in the absence of other urodele larval features further illustrates the autonomy of a histospecific enzyme development thought to be controlled by an egg cytoplasmic determinant. Partial suppression of the determinant might be the cause of this diminished expression. 4. Two other anural molgulid species, Molgula occulta and Bostrichobranchus pilularis, did not have vestigial larval enzyme and possibly have lost the determinant completely.