Expression of regulatory receptors on γδ T Cells and their cytokine production in Behcet's disease

Introduction

Behcet''s disease (BD) is a multi-systemic disorder with muco-cutaneous, ocular, arthritic, vascular or central nervous system involvement. The role of γδ T cells is implicated in BD. The activation status of γδ T cells and their cytokine secretion against phosphoantigens are evaluated in BD.

Methods

NKG2A, NKG2C, NKG2D, CD16 and CCR7 molecules on γδ T cells were analyzed in 70 BD, 27 tuberculosis (TB) patients and 26 healthy controls (HC). Peripheral γδ T cells were expanded with a phosphoantigen (BrHPP) and IL-2, restimulated with BrHPP and a TLR3 ligand, and cytokine production was measured.

Results

γδ T cells were not increased in both BD and TB patients, but the proportions of TCRVδ2⁺ T cells were lower (58.9 and 50.7 vs. 71.7%, P = 0.04 and P = 0.005) compared to HC. Higher proportion of TCRVδ2⁺ T cells were CD16⁺ (26.2 and 33.9 vs. 16.6%, P = 0.02 and P = 0.001) and CCR7^- (32.2 and 27.9 vs. 17.7%, P < 0.0001 and P = 0.014) in BD and TB patients compared to HC. NKG2C⁺ γδ⁺ T cells were relatively increased (0.5 and 0.6 vs. 0.3%, P = 0.008 and 0.018), whereas NKG2D positivity was decreased in patients with BD and TB (77.7 and 75.8 vs. 87.5%, P = 0.001 and 0.004). Expansion capacity of γδ T cells in BD and TB as well as production of IL-13, IFN-γ, granulocyte monocyte colony stimulating factor (GM-CSF), TNF-α, CCL4 and CCL5 in BD was lower compared to HC, when restimulated by TLR3 ligand and BrHPP.

Conclusion

The changes on γδ T cells of BD as well as TB patients implicate that γδ T cells have already been exposed to regulatory effects, which changed their activity. Lower cytokine response of γδ T cells implicates down modulation of these cells in BD.     

Cardiac telocytes — their junctions and functional implications

Telocytes (TCs) form a cardiac network of interstitial cells. Our previous studies have shown that TCs are involved in heterocellular contacts with cardiomyocytes and cardiac stem/progenitor cells. In addition, TCs frequently establish 'stromal synapses' with several types of immunoreactive cells in various organs ( www.telocytes.com ). Using electron microscopy (EM) and electron microscope tomography (ET), we further investigated the interstitial cell network of TCs and found that TCs form 'atypical' junctions with virtually all types of cells in the human heart. EM and ET showed different junction types connecting TCs in a network (puncta adhaerentia minima, processus adhaerentes and manubria adhaerentia). The connections between TCs and cardiomyocytes are 'dot' junctions with nanocontacts or asymmetric junctions. Junctions between stem cells and TCs are either 'stromal synapses' or adhaerens junctions. An unexpected finding was that TCs have direct cell-cell (nano)contacts with Schwann cells, endothelial cells and pericytes. Therefore, ultrastructural analysis proved that the cardiac TC network could integrate the overall 'information' from vascular system (endothelial cells and pericytes), nervous system (Schwann cells), immune system (macrophages, mast cells), interstitium (fibroblasts, extracellular matrix), stem cells/progenitors and working cardiomyocytes. Generally, heterocellular contacts occur by means of minute junctions (point contacts, nanocontacts and planar contacts) and the mean intermembrane distance is within the macromolecular interaction range (10-30 nm). In conclusion, TCs make a network in the myocardial interstitium, which is involved in the long-distance intercellular signaling coordination. This integrated interstitial system appears to be composed of large homotropic zones (TC-TC junctions) and limited (distinct) heterotropic zones (heterocellular junctions of TCs).     

Cloning and functional characterization of the maize carotenoid isomerase and β-carotene hydroxylase genes and their regulation during endosperm maturation

In order to gain further insight into the partly-characterized carotenoid biosynthetic pathway in corn (Zea mays L.), we cloned cDNAs encoding the enzymes carotenoid isomerase (CRTISO) and β-carotene hydroxylase (BCH) using endosperm mRNA isolated from inbred line B73. For both enzymes, two distinct cDNAs were identified mapping to different chromosomes. The two crtiso cDNAs (Zmcrtiso1 and Zmcrtiso2) mapped to unlinked genes each containing 12 introns, a feature conserved among all crtiso genes studied thus far. ZmCRTISO1 was able to convert tetra-cis prolycopene to all-trans lycopene but could not isomerize the 15-cis double bond of 9,15,9′-tri-cis-ζ-carotene. ZmCRTISO2 is inactivated by a premature termination codon in B73 corn, but importantly the mutation is absent in other corn cultivars and the active enzyme showed the same activity as ZmCRTISO1. The two bch cDNAs (Zmbch1 and Zmbch2) mapped to unlinked genes each coding sequences containing five introns. ZmBCH1 was able to convert β-carotene into β-cryptoxanthin and zeaxanthin, but ZmBCH2 was able to form β-cryptoxanthin alone and had a lower overall activity than ZmBCH1. All four genes were expressed during endosperm development, with mRNA levels rising in line with carotenoid accumulation (especially zeaxanthin and lutein) until 25 DAP. Thereafter, expression declined for three of the genes, with only Zmcrtiso2 mRNA levels maintained by 30 DAP. We discuss the impact of paralogs with different expression profiles and functions on the regulation of carotenoid synthesis in corn.     

Systematic analysis of differentially methylated expressed genes and site-specific methylation as potential prognostic markers in head and neck cancer

Head and neck cancer (HNC) remains one of the most malignant tumors with a significantly high mortality. DNA methylation exerts a vital role in the prognosis of HNC. In this study, we try to screen abnormal differential methylation genes (DMGs) and pathways in Head–Neck Squamous Cell Carcinoma via integral bioinformatics analysis. Data of gene expression microarrays and gene methylation microarrays were obtained from the Cancer Genome Atlas database. Aberrant DMGs were identified by the R Limma package. We conducted the Cox regression analysis to select the prognostic aberrant DMGs and site-specific methylation. Five aberrant DMGs were recognized that significantly correlated with overall survival. The prognostic model was constructed based on five DMGs (PAX9, STK33, GPR150, INSM1, and EPHX3). The five DMG models acted as prognostic biomarkers for HNC. The area under the curve based on the five DMGs predicting 5-year survival is 0.665. Moreover, the correlation between the DMGs/site-specific methylation and gene expression was also explored. The findings demonstrated that the five DMGs can be used as independent prognostic biomarkers for predicting the prognosis of patients with HNC. Our study might lay the groundwork for further mechanism exploration in HNC and may help identify diagnostic biomarkers for early stage HNC.     

Expression and characterization of 4-α-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses

4-α-Glucanotransferase or disproportionating enzyme (D-enzyme, DPE) catalyzes the α-1.4 glycosyl transfer between oligosaccharides. Type I D-enzyme (DPE1) can transfer maltosyl unit from one 1.4-α-d-glucan to an acceptor mono- or oligo-saccharide, which reflects the physiological role of DPE1 in plant starch metabolism. In this study, the genes encoding DPE1 from Arabidopsis thaliana (AtDPE1) and Manihot esculenta Crantz cultivar KU50 (MeDPE1) were cloned and expressed in Escherichia coli and purified to homogeneity. MeDPE1 encoded 585 amino acid residues, including a 56 residue signal peptide, while AtDPE1 encoded 576 amino acid residues with a 45 residue signal peptide. The molecular mass of both mature enzymes, estimated from deduced amino acid sequence, were the same at 59.4 kDa, with a pI of 5.13. The predicted structures of both enzymes showed the conserved 250's loop and three catalytic amino acid residues, characteristics of disproportionating enzymes in the GH77 glycoside hydrolase family. Biochemical characterization showed that both purified recombinant enzymes were homodimers in solution, with similar optimum pH and temperature for disproportionating activity at pH 6–8 and 37 °C. Using potato amylose as a substrate, AtDPE1 can produce cycloamyloses in the range 16–50 glucose residues, while products from the action of MeDPE1 on the same substrate were in a wider range of 16 to DP > 60. These recombinant enzymes are useful tools for elucidation of their functional roles in starch metabolism and for applications in the starch industry.     

Development of livestock production in the tropics: farm and farmers’ perspectives

Because of an increasing demand for animal-source foods, an increasing desire to reduce poverty and an increasing need to reduce the environmental impact of livestock production, tropical farming systems with livestock must increase their productivity. An important share of the global human and livestock populations are found within smallholder mixed-crop–livestock systems, which should, therefore, contribute significantly towards this increase in livestock production. The present paper argues that increased livestock production in smallholder mixed-crop–livestock systems faces many constraints at the level of the farm and the value chain. The present paper aims to describe and explain the impact of increased production from the farm and farmers’ perspective, in order to understand the constraints for increased livestock production. A framework is presented that links farming systems to livestock value chains. It is concluded that farming systems that pass from subsistence to commercial livestock production will: (1) shift from rural to urban markets; (2) become part of a different value chain (with lower prices, higher demands for product quality and increased competition from peri-urban producers and imports); and (3) have to face changes in within-farm mechanisms and crop–livestock relationships. A model study showed that feed limitation, which is common in tropical farming systems with livestock, implies that maximum herd output is achieved with small herd sizes, leaving low-quality feeds unutilised. Maximal herd output is not achieved at maximal individual animal output. Having more animals than required for optimal production – which is often the case as a larger herd size supports non-production functions of livestock, such as manure production, draught, traction and capital storage – goes at the expense of animal-source food output. Improving low-quality feeds by treatment allows keeping more animals while maintaining the same level of production. Ruminant methane emission per kg of milk produced is mainly determined by the level of milk production per cow. Part of the methane emissions, however, should be attributed to the non-production functions of ruminants. It was concluded that understanding the farm and farmers’ perceptions of increased production helps with the understanding of productivity increase constraints and adds information to that reported in the literature at the level of technology, markets and institutions.     

Simultaneous production of endoglucanase and β-glucosidase using synthetic two cistron genes

Summary Simultaneous production of endoglucanase and -glucosidase by using a synthetic two cistron system inEscherichia coli was attempted as a possible way of reducing production cost. The first cistron in this system we constructed is an endoglucanase gene fused to a tac promoter that provides for efficient expression. The second cistron is a -glucosidase structural gene. A ribosome binding site sequence of 33-base was inserted between the two cistron genes.E. coli cells transformed with the system produced 12.4 units/mg protein of endoglucanase and 327 units/mg protein of -glucosidase, which represent 15% and 22% of total cellular protein, respectively, in L medium within three hours after induction with IPTG.     

Essential oils—a brief survey of their chemistry and production in the united states

Essential oils can be produced in this country, but extensive investigations and careful planning are of prime importance.     

TGF—β receptors in mouse ES—5 cells and their differentiated derivatives

By radioreceptor binding studies with iodinated TGF－β1,it has been shown that an undifferentiated ES-5 cell expresses approximately 3270 receptors with a dissociation constant Kd-130pM,but after the induction of differentiation by retinoic acid and dBcAMP,the receptor number of a differentiated RA-ES-5 cell was increased about 80% and the Kd was also increased to 370 pM.Furthermore,more direct evidence supporting the expression of TGF-β type I and type Ⅱ receptors in both ES-5 and RA-ES-5 cells has come from dot blot hybridization of cellular mRNA with cDNA probes for type I and type Ⅱ receptors.Meanwhile,mRNA expression level of types I and Ⅱ receptors in R-ES-5 cells were higher than that in ES-5 cells.Down-regulation of TGF-β receptors with a significant decrease in the rate of cell proliferation in both cells,was found by employing a pretreatment with neutralizing antibody to TGF-β1.The possible role of receptors for TGF-β in cell differentiation is discussed here.     

Loci controlling lymphocyte production of interferon γ after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1–Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2 ^pz ) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2 ^b ) or BALB/cHeA (H2 ^d ) mice, or by ConA. IFNγ production in MLCs of individual (O20 × OcB-9)F₂ mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.     

Molecular and functional characterization of genes encoding horse MHC class I antigens

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.     

Granulocytic anaplasmosis — emerging tick-borne disease of humans and animals

Granulocytic anaplasmoses represent a group of emerging tick-borne infectious diseases caused by the obligate intracellular gram-negative bacterium, Anaplasma phagocytophilum (Rickettsiales) that infects granulocytes. It has been known as a ruminant pathogen in Europe since 1932, however, recently it has emerged as a pathogen of humans and domestic animals such as dogs and horses in the Northern Hemisphere, including United States and Europe. Rodents and game animals (especially deer) are presumed to play a crucial role in the maintenance of A. phagocytophilum in natural foci and serve as competent reservoirs. Up to now, the presence of bacterial DNA has been confirmed by molecular methods in a number of domestic and wildlife animals. Circulation of several genotypes has been confirmed in natural foci but the vector competence and the host spectrum involved in its circulation is still under investigation. Human granulocytic anaplasmosis (HGA) typically occurs in spring or summer and clinical manifestations range from mild or self-limiting to severe disease, especially in elderly patients with up to 50% requiring hospitalization and 7% intensive care. So far, no confirmed A. phagocytophilum infections of humans have been reported in Slovakia despite the fact that the presence of anti-anaplasma antibodies has been detected in investigated patients sera. This fact could be explained by non-specific clinical signs of the infection or lack of information in physicians and underdiagnosed or misdiagnosed cases. The purpose of this review is to present biology, ecology and life cycle of A. phagocytophilum and introduce clinical symptoms, diagnosis and treatment of the infection caused by this pathogenic bacterium.     

Can functional regions of proteins be predicted from their coding sequences? The case study of G-protein coupled receptors

A filter based on a set of unsupervised neural networks trained with a winner-take-all strategy discloses signals along the coding sequences of G-protein coupled receptors. By comparing with the existing experimental data it appears that these signals correlate with putative functional domains of the proteins. After protein alignment within subfamilies, signals cluster in protein regions which, according to the presently available experimental results, are described as possible functional domains of the folded proteins. The mapping procedure reveals characteristic regions in the coding sequences common and/or characteristic of the receptor subtype. This is particularly noticeable for the third cytoplasmic loop, which is likely to be involved in the molecular coupling of all the subfamilies with G-proteins. The results indicate that our mapping can highlight intrinsic representative features of the coding sequences which, in the case of G-protein coupled receptors, are characteristic of protein functional regions and suggest a possible application of the filter for predicting functional determinants in proteins starting from the coding sequence.     

Development of chromosome 6D-specific markers for α-gliadin genes and their use in assessing dynamic changes at the Gli-2 loci

To develop chromosome 6D-specific point mutation (PM) markers for α-gliadin genes, 79 α-gliadin sequences cloned from Aegilops tauschii and another 40 α-gliadin genes with known chromosome locations were used in multi-sequence alignment and phylogenic analysis. Additional multiple alignment adjustments were performed manually to facilitate discovery of putative chromosome 6D-specific point mutations. A total of 85 PM primers were designed to detect 68 candidate chromosome 6D-specific point mutations. Experimental tests revealed 11 chromosome 6D-specific PM markers by using genomic DNA from homoeologous group 6 nullisomic–tetrasomic lines of Chinese Spring and putative diploid and tetraploid ancestors of hexaploid wheat as PCR templates. Detection of PM markers in one synthetic hexaploid wheat and its parental lines indicated that some α-gliadin genes were lost from Gli-2 loci during the formation of hexaploid wheat by amphidiploidization of the genomes of Triticum turgidum and Ae. tauschii. Detection of these PM markers in Ae. tauschii, T. aestivum and its four subspecies indicated that at least two genetically distinct sources of Ae. tauschii contributed germplasm to the D genome of T. aestivum.     

MAOA, MTHFR, and TNF-β genes polymorphisms and personality traits in the pathogenesis of migraine

Migraine is a multifactorial disease with various factors, such as genetic polymorphisms and personality traits, but the contribution of those factors is not clear. To clarify the pathogenesis of migraine, the contributions of genetic polymorphisms and personality traits were simultaneously investigated using multivariate analysis. Ninety-one migraine patients and 119 non-headache healthy volunteers were enrolled. The 12 gene polymorphisms analysis and NEO-FFI personality test were performed. At first, the univariate analysis was performed to extract the contributing factors to pathogenesis of migraine. We then extracted the factors that independently contributed to the pathogenesis of migraine using multivariate stepwise logistic regression analysis. Using the multivariate analysis, three gene polymorphisms including monoamine oxidase A (MAOA) T941G, methylenetetrahydrofolate reductase (MTHFR) C677T, and tumor necrosis factor beta (TNF-β) G252Α, and the neuroticism and conscientiousness scores in NEO-FFI were selected as significant factors that independently contributed to the pathogenesis of migraine. Their odds ratios were 1.099 (per point of neuroticism score), 1.080 (per point of conscientiousness score), 2.272 (T and T/T or T/G vs G and G/G genotype of MAOA), 1.939 (C/T or T/T vs C/C genotype of MTHFR), and 2.748 (G/A or A/A vs G/G genotype of TNF-β), respectively. We suggested that multiple factors, such as gene polymorphisms and personality traits, contribute to the pathogenesis of migraine. The contribution of polymorphisms, such as MAOA T941G, MTHFR C677T, and TNF-β G252A, were more important than personality traits in the pathogenesis of migraine, a multifactorial disorder.