Adoptive Transfer of mRNA-Transfected T Cells Redirected against Diabetogenic CD8 T Cells Can Prevent Diabetes.

Chimeric major histocompatibility complex (MHC) molecules supplemented with T cell receptor (TCR) signaling motifs function as activation receptors and can redirect gene-modified T cells against pathogenic CD8 T cells. We have shown that ß2 microglobulin (ß2m) operates as a universal signaling component of MHC-I molecules when fused with the CD3-ζ chain. Linking the H-2Kd-binding insulin B chain peptide insulin B chain, amino acids 15-23 (InsB15-23) to the N terminus of ß2m/CD3-ζ, redirected polyclonal CD8 T cells against pathogenic CD8 T cells in a peptide-specific manner in the non-obese diabetic (NOD) mouse. Here, we describe mRNA electroporation for delivering peptide/ß2m/CD3-ζ genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/ß2m/CD3-ζ products paired with endogenous MHC-I chains and transmitted strong activation signals upon MHC-I cross-linking. The reporter T cell line transfected with InsB15-23/ß2m/CD3-ζ mRNA was activated by an InsB15-23-H-2Kd-specific CD8 T cell hybrid only when the transfected T cells expressed H-2Kd. Primary NOD CD8 T cells expressing either InsB15-23/ß2m/CD3-ζ or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206-214 (IGRP206-214)/ß2m/CD3-ζ killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15-23/ß2m/CD3-ζ mRNA significantly reduced insulitis and protected NOD mice from diabetes. Our results demonstrate that mRNA encoding chimeric MHC-I receptors can redirect effector CD8 against diabetogenic CD8 T cells, offering a new approach for the treatment of type 1 diabetes.

Early Pleistocene human occupation at the edge of the boreal zone in northwest Europe.

The dispersal of early humans from Africa by 1.75 Myr ago led to a marked expansion of their range, from the island of Flores in the east to the Iberian peninsula in the west. This range encompassed tropical forest, savannah and Mediterranean habitats, but has hitherto not been demonstrated beyond 45 degrees N. Until recently, early colonization in Europe was thought to be confined to the area south of the Pyrenees and Alps. However, evidence from Pakefield (Suffolk, UK) at approximately 0.7 Myr indicated that humans occupied northern European latitudes when a Mediterranean-type climate prevailed. This provided the basis for an 'ebb and flow' model, where human populations were thought to survive in southern refugia during cold stages, only expanding northwards during fully temperate climates. Here we present new evidence from Happisburgh (Norfolk, UK) demonstrating that Early Pleistocene hominins were present in northern Europe >0.78 Myr ago when they were able to survive at the southern edge of the boreal zone. This has significant implications for our understanding of early human behaviour, adaptation and survival, as well as the tempo and mode of colonization after their first dispersal out of Africa.

Taphonomic and technological analyses of Lower Palaeolithic bone tools from Clacton-on-Sea, UK.

The exceptional survival of Middle Pleistocene wooden spears at Schöningen (Germany) and Clacton-on-Sea (UK) provides tantalizing evidence for the widespread use of organic raw materials by early humans. At Clacton, less well-known organic artefacts include modified bones that were identified by the Abbé Henri Breuil in the 1920s. Some of these pieces were described and figured by Hazzledine Warren in his classic 1951 paper on the flint industry from the Clacton Channel, but they have been either overlooked in subsequent studies or dismissed as the product of natural damage. We provide the first detailed analysis of two Clactonian bone tools found by Warren and a previously unrecognized example recovered in 1934 during excavations directed by Mary Leakey. Microscopic examination of percussion damage suggests the bones were used as knapping hammers to shape or resharpen flake tools. Early Palaeolithic bone tools are exceedingly rare, and the Clacton examples are the earliest known organic knapping hammers associated with a core-and-flake (Mode 1) lithic technology. The use of soft hammers for knapping challenges the consensus that Clactonian flintknapping was undertaken solely with hard hammerstones, thus removing a major technological and behavioural difference used to distinguish the Clactonian from late Acheulean handaxe (Mode 2) industries.

Adenosine regulates thrombomodulin and endothelial protein C receptor expression in folliculostellate cells of the pituitary gland.

Adenosine stimulates the release of interleukin 6 (IL-6) and vascular endothelial growth factor from folliculostellate cells of the anterior pituitary gland indicating that such cells are also involved in the communication between the immune and endocrine systems during stress and inflammation. In order to understand the precise actions of adenosine on folliculostellate cells, DNA microarray analysis was used to determine global changes in gene expression. Hierarchical clusters revealed, of the genes that had altered expression, the majority were suppressed and many, such as B cell translocation gene 2 and cyclin-dependent kinase inhibitor 2b were related to cell cycle arrest or inhibition of proliferation. Several of the up-regulated genes were associated with cytokine signalling or membrane receptor activity. The most notable of these being IL-6, sulfiredoxin 1, endothelial protein C receptor (EPCR) and thrombomodulin (THBD) which can all play a role in controlling inflammation. The EPCR and THBD pathway is well known in anti-coagulation but also has anti-inflammatory and anti-apoptotic properties. Up-regulation of EPCR and THBD in folliculostellate cells was confirmed by qRT-PCR and western blotting analysis and their expression were also demonstrated in many of the hormone-secreting cells of the anterior pituitary gland. Our findings suggest that adenosine can stimulate expression of stress and inflammation related genes from folliculostellate cells of the anterior pituitary gland. These genes include EPCR and THBD, neither of which has been previously identified in the pituitary gland.

Growth hormone (GH1) gene variation and the growth hormone receptor (GHR) exon 3 deletion polymorphism in a West-African population.

Among Europeans, functionally significant GH1 gene variants occur not only in individuals with idiopathic growth hormone (GH) deficiency and/or short stature but also fairly frequently in the general population. To assess the generality of these findings, 163 individuals from Benin, West Africa were screened for mutations and polymorphisms in their GH1 genes. A total of 37 different sequence variants were identified in the GH1 gene region, 24 of which occurred with a frequency of >1%. Although four of these variants were novel missense substitutions (Ala13Val, Arg19His, Phe25Tyr and Ser95Arg), none of these had any measurable effect on either GH function or secretion in vitro. Some 37 different GH1 promoter haplotypes were identified, 23 of which are as yet unreported in Europeans. The mean in vitro expression level of the GH1 promoter haplotypes observed in the African population was significantly higher than that previously measured in Britons (p<0.001). A gene conversion in the GH1 promoter, previously reported in a single individual of British origin, was found to occur at polymorphic frequency (5%) in the West-African population and was associated with a 1.7-fold increase in promoter activity relative to the wild-type. The d3 allele of the GHR exon 3 deletion polymorphism, known to be associated with increased GH responsiveness, was also found to occur at an elevated frequency in these individuals from Benin. We speculate that both elevated GH1 gene expression and increased GHR-mediated GH responsiveness may constitute adaptive responses to the effects of scarce food supply in this West-African population since increased circulating GH appears to form part of a physiological response to nutritional deprivation.

A reproducible method for the expansion of mouse CD8+ T lymphocytes.

Murine adoptive CD8+ T-cell immunotherapy studies require the generation of large numbers of high viability CD8+ cells. Here we report a tissue culture protocol for the reliable expansion of CD8+ T-cells derived from murine spleen to give a 20-fold expansion after 4 days in culture. The cells were transfected with an mRNA GFP construct and transferred into NOD mice. GFP positive cells could be detected 7 days after transfer thus confirming that the cells survive and are functional for up to 1 week.

Novel mutations of the growth hormone 1 (GH1) gene disclosed by modulation of the clinical selection criteria for individuals with short stature.

Subtle mutations in the growth hormone 1 (GH1) gene have been regarded as a comparatively rare cause of short stature. Such lesions were sought in a group of 41 individuals selected for short stature, reduced height velocity, and bone age delay; a group of 11 individuals with short stature and idiopathic growth hormone deficiency (IGHD); and a group of 154 controls. Heterozygous mutations were identified in all three groups but disproportionately in the individuals with short stature, both with (odds ratio 25.2; 95% CI, 5.1-132.2) and without (odds ratio 3.6; 95% CI, 1.0-12.9) IGHD. Twenty-four novel GH1 gene lesions were found. Thirteen novel missense mutations were characterized by assaying the signal transduction activity of in vitro expressed variants; six (T27I, K41R, N47D, S71F, S108R, and T175A) exhibited a reduced ability to activate the JAK/STAT pathway. Molecular modeling suggested that both K41R and T175A might compromise GH receptor binding. Seven GH variants (R16C, K41R, S71F, E74K, Q91L, S108C, and a functional polymorphism, V110I) manifested reduced secretion in rat pituitary cells after allowance had been made for the level of expression attributable to the associated GH1 proximal promoter haplotype. A further leader peptide variant (L-11P) was not secreted. Eleven novel mutations in the GH1 gene promoter were assessed by reporter gene assay but only two, including a GH2 gene-templated gene conversion, were found to be associated with a significantly reduced level of expression. Finally, a novel intron 2 acceptor splice-site mutation, detected in a family with autosomal dominant type II IGHD, was shown to lead to the skipping of exon 3 from the GH1 transcript. A total of 15 novel GH1 gene mutations were thus considered to be of probable phenotypic significance. Such lesions are more prevalent than previously recognized and although most may be insufficient on their own to account for the observed clinical phenotype, they are nevertheless likely to play a contributory role in the etiology of short stature.

A novel dysfunctional growth hormone variant (Ile179Met) exhibits a decreased ability to activate the extracellular signal-regulated kinase pathway.

The pituitary-expressed GH1 gene was screened for mutation in a group of 74 children with familial short stature. Two novel mutations were identified: an Ile179Met substitution and a -360A-->G promoter variant. The Ile179Met variant was shown to exhibit a similar degree of resistance to proteolysis as wild-type GH, indicating that the introduction of Met does not cause significant misfolding. Secretion of Ile179Met GH from rat pituitary cells was also similar to that of wild type. Although receptor binding studies failed to show any difference in binding characteristics, molecular modeling studies suggested that the Ile179Met substitution might nevertheless perturb interactions between GH and the GH receptor loop containing the hotspot residue Trp169, thereby affecting signal transduction. The ability of the Ile179Met variant to activate a signal transducer and activator of transcription (STAT) 5-responsive luciferase reporter gene and induce phosphorylation of STAT 5 and ERK was therefore studied. In contrast to its ability to activate STAT 5 normally, activation of ERK by the Ile179Met variant was reduced to half that observed with wild type. Although differential effects on the activation of distinct signaling pathways by a mutant receptor agonist are unprecedented, these findings also suggest that the ERK pathway could play a role in mediating the action of GH.

Adenosine-induced IL-6 expression in pituitary folliculostellate cells is mediated via A2b adenosine receptors coupled to PKC and p38 MAPK.

Activation of adenosine receptors in folliculostellate (FS) cells of the pituitary gland leads to the secretion of IL-6 and vascular endothelial growth factor (VEGF). We investigated the action of adenosine A2 receptor agonists on IL-6 and VEGF secretion in two murine FS cell lines (TtT/GF and Tpit/F1), and demonstrated a rank order of potency, 5'-N-ethylcarboxamidoadenosine (NECA)>2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine>adenosine, suggesting mediation via the A2b receptor. NECA-mediated IL-6 release was inhibited by the PLC inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-tiene-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione, the PI3 kinase inhibitor wortmannin and the PKC inhibitors bisindolylmaleimide 1 and bisindolymaleimide X1 HCl (Ro-32-0432). NECA-mediated IL-6 release was attenuated (<50%) by the extracellular signal-regulated kinase MAPK inhibitor 2'-amino-3'-methoxyflavone, and completely (>95%) inhibited by the p38 MAPK inhibitor 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole. NECA stimulates p38 MAPK phosphorylation that is inhibited by Ro-32-0432 but not by wortmannin. Dexamethasone inhibits NECA-stimulated IL-6 and VEGF secretion. These findings indicate that adenosine can stimulate IL-6 secretion in FS cells via the A2b receptor coupled principally to PLC/PKC and p38 MAPK; such an action may be important in the modulation of inflammatory response processes in the pituitary gland.

Production and application of polyclonal antibody to human thyroid transcription factor 2 reveals thyroid transcription factor 2 protein expression in adult thyroid and hair follicles and prepubertal testis.

Germline mutations in thyroid transcription factor 2 (TTF2) cause thyroid agenesis, spiky hair, and cleft palate, indicating thyroidal and extrathyroidal expression. We sought to investigate this by producing and applying an antibody to human TTF2. The coding region of human TTF2 was cloned into a bacterial expression vector, production of the soluble TTF2 protein optimized, and pure TTF2 obtained by nickel chromatography. Rabbits were immunized and the resulting TTF2 polyclonal titrated on formalin-fixed, paraffin-embedded sections of thyroid. The optimized protocol was applied to a range of tissues. Nine milligrams of TTF2 protein was obtained per liter of culture and a high-titer antibody produced. This displayed specific staining of thyroid follicular cell nuclei/cytoplasm and not of the interstitium, connective tissue, smooth muscle, or endothelium. No staining was obtained with the preimmune serum in the same conditions, or with the majority of other tissues tested with the TTF2 polyclonal. The exceptions were testis and skin, in which nuclear TTF2 immunoreactivity was present in the seminiferous tubules and cells in the follicular outer root sheath, respectively. In conclusion, we have produced a polyclonal antibody for human TTF2 and demonstrated immunoreactivity for this transcription factor in adult human thyroid and hair follicles and prepubertal testis.

Gsalpha signalling suppresses PPARgamma2 generation and inhibits 3T3L1 adipogenesis.

Since TSH receptor (TSHR) expression increases during adipogenesis and signals via cAMP/phospho-cAMP-response element binding protein (CREB), reported to be necessary and sufficient for adipogenesis, we hypothesised that TSHR activation would induce preadipocyte differentiation. Retroviral vectors introduced constitutively active TSHR (TSHR*) into 3T3L1 preadipocytes; despite increased cAMP (RIA) and phospho-CREB (western blot) there was no spontaneous adipogenesis (assessed morphologically, using oil red O and QPCR measurement of adipogenesis markers). We speculated that Gbetagamma signalling may be inhibitory but failed to induce adipogenesis using activated Gsalpha (gsp*). Inhibition of phosphodiesterases did not promote adipogenesis in TSHR* or gsp* populations. Furthermore, differentiation induced by adipogenic medium with pioglitazone was reduced in TSHR* and abolished in gsp* expressing 3T3L1 cells. TSHR* and gsp* did not inactivate PPARgamma (PPARG as listed in the HUGO database) by phosphorylation but expression of PPARgamma1 was reduced and PPARgamma2 undetectable in gsp*. FOXO1 phosphorylation (required to inactivate this repressor of adipogenesis) was lowest in gsp* despite the activation of AKT by phosphorylation. PROF is a mediator that facilitates FOXO1 phosphorylation by phospho-Akt. Its transcript levels remained constantly low in the gsp* population. In most measurements, the TSHR* cells were between the gsp* and control 3T3L1 preadipocytes. The enhanced down-regulation of PREF1 (adipogenesis inhibitor) permits retention of some adipogenic potential in the TSHR* population. We conclude that Gsalpha signalling impedes FOXO1 phosphorylation and thus inhibits PPARgamma transcription and the alternative promoter usage required to generate PPARgamma2, the fat-specific transcription factor necessary for adipogenesis.

Genetic variation at the growth hormone (GH1) and growth hormone receptor (GHR) loci as a risk factor for hypertension and stroke.

An increased prevalence of both hypertension and cerebrovascular stroke is apparent in growth hormone (GH) deficiency whilst hypertension is a frequent complication in acromegaly. This has suggested a possible link between GH, stature and arterial function. Since the risk of both hypertension and stroke also appears to be inversely correlated with adult height, we have instigated an exploratory study to assess whether inter-individual variation in the genes encoding human growth hormone (GH1) and the GH receptor (GHR) might be associated with an increased risk of hypertension and stroke. GH1 promoter haplotypes were found to differ significantly not only between hypertensive patients (n = 111) and controls (n = 121) but also between stroke patients (n = 155) and controls (n = 158). Intriguingly, the association between GH1 promoter haplotype and risk of hypertension was much greater in females than in males. An inverse correlation between height and central systolic blood pressure was apparent in both hypertensive patients and normal controls but was much stronger in individuals carrying at least one GH1 promoter risk haplotype. The GH1 genotype therefore constitutes a risk factor for hypertension that interacts with stature. A strong association was found between the presence of at least one GH1 risk haplotype and a family history of stroke at an early age (odds ratio: 9.07, 95% confidence interval: 1.14-72.22). Three novel GH variants (Arg16His, Phe176Cys, Cys189Arg) were identified during the course of this study. Although two exhibited markedly reduced biological activity in vitro, their clinical significance remains unclear. No association was found between GHR genotype and either hypertension or stroke, nor was any interaction noted between GHR and GH1 genotypes in terms of a disease association. However, an association between GHRd3 genotype and hypertension was observed among stroke patients, particularly females. Elevated HDL was found to be a risk factor for hypertension in individuals lacking a copy of the GHRd3 allele. Weak associations with GHR genotype were also noted for peripheral systolic and diastolic blood pressure in hypertensive patients. Although the underlying mechanisms are still unclear, our findings are consistent with a complex relationship between height, hypertension, GH1 promoter haplotype, GHR polymorphism and the risk of stroke.