Sequencing-based typing of HLA-B*51 alleles and the significant association of HLA-B*5101 and -B*5108 with Behçet's disease in Greek patients

Beh?et's disease (BD) is widely known to be strongly associated with human leukocyte antigen (HLA) B51 in many different ethnic groups.Recently, HLA-B51 allele typing of Greek BD patients was performed to study the distribution of B*5101-B*5107 alleles in this Greek population, the B51 antigen strongly associated with BD was found to be predominantly encoded by allele B*5101. As it is now known that the B51 antigen can be encoded by 21 alleles, B*5101-B*5121, we performed HLA-B*51 allele genotyping among 58 Greek patients with BD. After serological HLA typing, typing of HLA-B*51 alleles was performed using the polymerase chain reaction-sequencing-based typing (PCR-SBT) method. The frequency of the B51 antigen was found to be significantly higher in the patient group as compared with the control group (75.9% of patients vs 22.0% of controls. In the genotyping of B51 alleles, 34 out of 44 B51-positive patients possessed B*5101, 13 out of the 44 carried B*5108. In contrast, all of the 9 B51-positive normal controls carried B*5101. This study revealed a strong association between Greeks with BD, both B*5101, B*5108, provided important insights into the molecular mechanism underlying the association between HLA status, this disease.     

Development and characterization of Thy*IκBα-SI transgenic mice

We developed and characterized a new transgenic model where NF-κB activity is inhibited only in mature neurons. Transgenic mouse strain Thy*IκBα-SI was created using transdominant super inhibitor NF-κB (IκBα-SI), which is a multimutant form of IκBα inhibitory protein cloned into specifi c neutral Thy-1.2 cassette. Detailed molecular analysis showed that the transgene and its product (IκBα-SI protein) are expressed in the nervous system of transgenic mice. In situ hybridization showed that Thy*IκBα-SI in the nervous system is expressed exclusively in neurons. The developed model provides wide opportunities for studying functional role of NF-κB in mature neurons in the central and peripheral nervous system in vivo.     

CHEK2*1100delC homozygosity in the Netherlands—prevalence and risk of breast and lung cancer

The 1100delC mutation in the CHEK2 gene has a carrier frequency of up to 1.5% in individuals from North-West Europe. Women heterozygous for 1100delC have an increased breast cancer risk (odds ratio 2.7). To explore the prevalence and clinical consequences of 1100delC homozygosity in the Netherlands, we genotyped a sporadic breast cancer hospital-based cohort, a group of non-BRCA1/2 breast cancer families, and breast tumors from a tumor tissue bank. Three 1100delC homozygous patients were found in the cohort of 1434 sporadic breast cancer patients, suggesting an increased breast cancer risk for 1100delC homozygotes (odds ratio 3.4, 95% confidence interval 0.4–32.6, P=0.3). Another 1100delC homozygote was found in 592 individuals from 108 non-BRCA1/2 breast cancer families, and two more were found after testing 1706 breast tumors and confirming homozygosity on their wild-type DNA. Follow-up data was available for five homozygous patients, and remarkably, three of them had developed contralateral breast cancer. A possible relationship between 1100delC and lung cancer risk was investigated in 457 unrelated lung cancer patients but could not be confirmed. Due to the small number of 1100delC homozygotes identified, the breast cancer risk estimate associated with this genotype had limited accuracy but is probably higher than the risk in heterozygous females. Screening for CHEK2 1100delC could be beneficial in countries with a relatively high allele frequency.     

Identification of two new HLA alleles: B*3546* and B*5611*. How reliable are the published HLA-B intron 2 sequences?

Polymerase chain reaction-sequence-specific primer (PCR-SSP) typing for human leukocyte antigen (HLA)-B in a male 25-year-old Caucasian individual of Iranian origin and in a 42-year-old German Caucasian bone marrow donor revealed reaction patterns that did not agree with any known HLA specificity, thus suggesting in both cases the existence of a novel allele. Sequence-based typing (SBT) after allelic separation revealed the sequences of the new alleles HLA-B*5611 and B*3546. The sequence patterns of both new alleles might have been generated as the results of double crossing over, possibly over several generations. During the analysis of the HLA-B*3546 intron 2 sequence for possible crossing over points, a base insert, an additional G after position 700, was found. This insert was analyzed using SBT and PCR-SSP and was found to be present not only in all samples carrying B*35, but also in all HLA-B specificities tested. It appears that all known HLA-B alleles may contain a G insert at position 700 of intron 2, and that the published intron 2 sequence alignments of the HLA-B locus may contain errors at this position.     

HLA-B allele frequencies in Côte d'Ivoire defined by direct DNA sequencing: identification of HLA-B*1405, B*4410, and B*5302

Direct automated DNA sequencing was used to analyze exons 2 and 3 of HLA-B alleles present in forty-four unrelated individuals residing in the village of Adiopodoume, C?te d'Ivoire (Ivory Coast). Of the 23 HLA-B alleles observed, the most frequently detected allele was HLA-B*5301 (22.7%), which is believed to confer resistance to severe Plasmodium falciparum malaria. B*4501 (9.1%), B*1503 (8.0%), B*0705 (5.7%), B*1510 (5.7%) and B*3501 (5.7%) occurred frequently in the population. A second allele of B53 was identified; B*5302 contains a single amino acid variation at residue 171 (Y-->H). Two additional novel alleles, B* 1405 (a single amino acid variant of B*1402) and B*4410 (a five amino acid variant of B*4403) were characterized.     

DRβ1*1501, DQβ1*0602 donor allelic haplotype and humoral antibody episodes of acute renal allograft rejection

The present investigation was designed to show the effect of molecular HLA class II DR and DQ allelic differences between donor and recipient on humoral antibody rejection identified by C4d peritubular capillary staining. The hypothesis is that expression of the DRβ1*1501, DQβ1*0602 allele in the donor kidney increases the likelihood of humoral antibody rejection. We found that 67% (n = 18) of DR15 and/or DQ6 haplotype donor kidneys induced humoral antibody renal allograft rejection; 35% (n = 40) of DR15 and/or DQ6 haplotype donor kidneys failed to induce humoral antibody renal allograft rejection (p = 0.02). 42% (n = 31) of C4d+ recipients had donors with DR15; 17% (n = 42) of C4d recipients had donors with HLA-DR15 (p = 0.01).We compared donor haplotype alleles of 4 C4d+ with 6 C4d− recipients by high resolution molecular typing; 3 of 4 C4d+ recipients had a donor with the DRβ1*1501/DQβ1*0602 allele. This allele was absent in all C4d− donors. 35% of C4d+ recipients had 2 DR mismatches when compared to 36% of C4d− recipients. Our results, suggest that the DRβ1*1501, DQβ1*0602 allele in the donor kidney increases the risk of humoral antibody episodes of acute rejection, and signals the need for C4d staining of renal biopsies. Future analysis of additional donor and recipient haplotypes will establish whether or not this is a useful predictor of humoral rejection episodes.     

Determination of CYP2D6 *3, *4, and *10 frequency in women with breast cancer in S?o Luís,Brazil, and its association with prognostic factors and disease-free survival

The CYP2D6 enzyme is crucial for the metabolism of tamoxifen. The CYP2D6 gene is highly polymorphic, and individuals can be extensive, intermediate, or poor tamoxifen metabolizers. The aim of this study was to determine the frequencies of the CYP2D6 *3, *4, and *10 alleles in women with breast cancer who were treated with tamoxifen and analyze the association of enzyme activity with prognostic factors and disease-free survival. We observed a high frequency of CYP2D6 *10, with an allelic frequency of 0.14 (14.4%). The *3 allele was not present in the studied population, and *4 had an allelic frequency of 0.13 (13.8%). We conclude that patients with reduced CYP2D6 activity did not present worse tumor characteristics or decreased disease-free survival than women with normal enzyme activity, as the difference was not statistically significant. We also observed a high frequency of CYP2D6 *10, which had not been previously described in this specific population. This study is the first in north-northeastern Brazil that aimed to contribute to the knowledge of the Brazilian regional profile for CYP2D6 polymorphisms and their phenotypes. These findings add to the knowledge of the distribution of different polymorphic CYP2D6 alleles and the potential role of CYP2D6 genotyping in clinical practice prior to choosing therapeutic protocols.     

Genomic full-length analysis of the B*08:79 allele suggests exon shuffling involving the B*08:01:01 and B*07:06 alleles

B*08:79 is composed by partial B*08:01:01 and B*07:06 sequences because of a possible recombination event within intron 2.     

Relationship between transdermal energy of electroporation and iontophoresis and permeably fluxes of tetracaini hydrochlordum and naproxenum

INTRODUCTIONWiththedevelopmentofmedicinetechnique,peoplehavebeenrealizedthatoralorinjectionlimitsmanypharmaceuticalsbybiotechnologyfromwidelyusing.Be-causealmostthesedrugsconsistofproteinsorpolypeptideseasydegraded.Trans-dermaldrugdelivery(TDD)providesano…     

Association of IL-1β +3953 C and HLA-DRB1*15 with Coronary Artery and Rheumatic Heart Diseases in South India

Among the various candidate genes predisposing for cardiovascular diseases, HLA-DRB1* and IL-1β +3953C/T alleles have been implicated repeatedly. To test these in South India, we carried out a case control study of 323 Coronary Artery Disease (CAD) patients, 56 Rheumatic Heart Disease (RHD) patients and 254 endemic controls. The polymorphisms were studied by PCR – SSP and ARMS-PCR methods and results analyzed for various clinical and demographic parameters. In CAD, HLA-DRB1*14 allele showed significant predisposition (OR: 2.19; 95% CI: 1.04–4.58; p value = 0.023), particularly in male patients (OR: 4.07; 95% CI: 1.20–13.81; p value = 0.01) and further in males with Triple Vessel Disease (OR: 5.49; 95% CI: 1.45–20.60; p value = 0.007). On the other hand, HLA-DRB1*15 predisposed for RHD (OR: 3.56; 95% CI: 1.87–6.78; p value = 0.001) in both the genders. Population stratification showed this higher risk association in Vanniyar caste (OR: 5.00; 95% CI: 1.27–19.59; p value = 0.022). Among the IL1-β +3953C/T polymorphism, the ancestral allele ‘C’ showed a significant high risk association with CAD (OR: 1.83; 95% CI: 1.24–2.70; p value = 0.001), particularly in Mudaliar (OR: 6.07; 95% CI: 1.77–20.74; p value = 0.003; AF = 0.7) and Vanniyar castes (OR: 3.67; 95% CI: 0.92–14.57; p value = 0.05; AF = 0.660). Two different cardiac ailments studied, RHD & CAD thus showed varied associations in this South Indian cohorts. RHD having an infectious aetiology shared a HLA-DRB1*15 high risk association, while HLA-DRB1*14 and IL-1β +3953C predisposed for CAD, an inflammatory disorder, reiterating the diverse genetic predisposition of the two cardiac ailments studied.     

Construction and Expression of Eukaryotic Expression Vector and Plasmid Expressing siRNA of Human Protection of Telomeres 1

1 IntroductionThe POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryocytes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells. Human POT1 had been identified in 2001 year. Its amino terminal is highly conservative in eukaryocytes. Since Pot1 can bind internal loops and directly adjacent DNA-binding sites, it is…     

Expression and Localization of α-amylase in the Submandibular and Sublingual Glands of Mice

In the major salivary glands of mice, acinar cells in the parotid gland (PG) are known to be the main site for the production of the digestive enzyme α-amylase, whereas α-amylase production in the submandibular gland (SMG) and sublingual gland (SLG), as well as the cell types responsible for α-amylase production, has been less firmly established. To clarify this issue, we examined the expression and localization of both the mRNA and protein of α-amylase in the major salivary glands of male and female mice by quantitative and histochemical methods. α-amylase mRNA levels were higher in the order of PG, SMG, and SLG. No sexual difference was observed in α-amylase mRNA levels in the PG and SLG, whereas α-amylase mRNA levels in the female SMG were approximately 30% those in the male SMG. Using in situ hybridization and immunohistochemistry, signals for α-amylase mRNA and protein were found to be strongly positive in acinar cells of the PG, serous demilune cells of the SLG, and granular convoluted tubule (GCT) cells of the male SMG, weakly positive in seromucous acinar cells of the male and female SMG, and negative in mucous acinar cells of the SLG. These results clarified that α-amylase is produced mainly by GCT cells and partly by acinar cells in the SMG, whereas it is produced exclusively by serous demilune cells in the SLG of mice.     

In and out of synchrony—Behavioral and physiological dynamics of dyadic interpersonal coordination

Interpersonal synchrony, the temporal coordination of actions, emotions, thoughts and physiological processes, is a widely studied ubiquitous phenomenon. Research has already established that more synchrony is not always more beneficial, especially in the fields of emotional and physiological synchrony. Despite this fact, the dominant tone in the literature is that behavioral interpersonal synchrony is a pro-social phenomenon, and hence, in social contexts, more behavioral synchrony is generally considered better. In accordance with that tone, the naturally occurring dynamics of moving in and out of synchrony have rarely been studied or considered as an adaptive state. In the present article, we aim to present a new model of interpersonal synchrony, based on the existing literature assessing synchrony as well as the ideas of complex dynamical systems. At the core of our model is the idea that two tendencies exist simultaneously, one to synchronize with others and another to move out of synchrony and act independently. We suggest that an adaptive interpersonal system is a flexible one, able to continuously adjust itself to the social context. We suggest that the concept of meta-stability might be a marker of such a flexible interpersonal system. Moreover, the model considers both behavioral and physiological aspects in order to provide a more extensive account. We present research implications of the model, as well as a demonstration of the model's applicability to data, and provide code researchers can use to analyze their own data in these methods. Finally, we discuss future directions in detail.     

Expression and purification of the recombinant outermembrane protein Tp0453 of Treponema pallidum and its characterization of immuno-competence

ELISA test has been shown to have some advan tages in relation to the tests used for the diagnosisof syphilis because of its easy and quick perfor mance and result readings. With recent develop ment of the gene engineering technology and eluci dation of the whole genome of Nichols strain ofTreponema pallidum, new protein coding openreading frames (ORFs) are available for testing,and the study on the serological tests based on therecombinant protein have been become the focus ofinter…     

Immunological and Regulatory Functions of Uninfected and Virus Infected Immature and Mature Subtypes of Dendritic Cells – a Review

In 1868, dendritic cells (DCs) were discovered in human skin by Paul Langerhans using gold staining. These cells were named Langerhans cells (LCs) after their discoverer who, due to their dendrites, regarded them as neurons. One hundred and eleven years were to pass until it was discovered that in vertebrates these cells originate in the bone marrow as monocytes. In the 1980s, DC research was mostly carried out on DCs that are present in different tissues of mice and humans. These studies revealed that after interaction with foreign antigens, skin LCs/DCs migrate through the lymph vessels to the draining lymph nodes and induce the two arms of the immune response. The isolation of DCs from tissue cell suspensions opened the way to studies on the cells' surface proteins and their ability to stimulate immune responses. During the 1990s, studies revealed the role of DCs in the activation of naïve T cells in the lymph nodes and the regulatory properties of DCs in lymph nodes, thymus, gut, and spleen.Part A of the review deals with the DC system of human and mice and immunological and regulatory functions of subsets of DCs in the skin with reference to migrating and stationary DCs, as well as the connection between DCs and the nervous system. Furthermore, the origin of both follicular DCs that are present in lymphoid tissues and thymic DCs are discussed. Part B is devoted to virus infections of DCs with an emphasis on infections caused by human herpes viruses. Part C presents the modulation of DC gene expression in response to the influenza virus. Contemporary research focuses on the role of DCs in the immune systems of vertebrates. Moreover, studies are being conducted on the regulatory functions of DCs by tissue cells in different organs of vertebrates.     

Assessment of the Reactivity and Localization of EEG α-Rhythm Frequency Components on Execution and Observation of Movements

Neuroscience and Behavioral Physiology - A total of 67 adult subjects took part in studies of the amplitude of the lower (α1) and upper (α2) frequency components of EEG α activity,...     

Iron distribution and histopathological characterization of the liver and heart of β-thalassemic mice with parenteral iron overload: Effects of deferoxamine and deferiprone

The liver and heart are the major target organs for iron accumulation and iron toxicity in β-thalassemia. To mimic the phenomenon of heavy iron overload resulting from repeated blood transfusions, a total of 180 mg of iron dextran was intraperitoneally injected into C57BL/6J mice (WT) and heterozygous β-globin knockout mice (^muβ^th-3/+, BKO). The effects of deferiprone and deferoxamine in this model were investigated. The iron was distributed homogenously throughout the 4 liver lobes (left, caudate, right and median) and was present in hepatocytes, Kupffer cells and the sinusoidal space. Iron accumulation in phagocytic macrophages, recruitment of hepatic lymphocytes and nucleus membrane degeneration were observed as a result of iron overload in the WT and BKO mice. However, the expansion of hepatic extramedullary hematopoiesis was observed only in the BKO mice with iron overload. In the heart, the iron accumulated in the cardiac interstitium and myocytes, and moderate hypertrophy of the myocardial fibers and cardiac myocyte degeneration were observed. Although the total liver iron was not significantly altered by iron chelation therapy, image analysis demonstrated a difference in the efficacies of two iron chelators. The major site of chelation was the extracellular compartment, but treatment with deferiprone also resulted in intracellular iron chelation. Interestingly, iron chelators reversed the pathological changes resulting from iron overload in WT and BKO mice despite being used for only a short treatment period. We suggest that some of these effects may be secondary to the anti-inflammatory activity of the chelators.     

Expression and purification of Ctomp2aa167-aa434 and preparation of its mAb

Chlamydia trachomatis outer membrane protein 2 (Ctomp2) is a major immunogen in chlamydial infections and a highly genus-conserved structural protein of all Chlamydia species . To purify the protein and to prepare monoclonal antibodies (mAbs) against it, the recombinant protein was induced by IPTG, which was confirmed by SDS-PAGE and purified by means of a Ni2 -charged resin column. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Western blotting. Then the BALB/c mice were immunized with the recombinant protein to prepare the mAb against Ctomp2. The obtained mAbs were characterized. Genital specimens were tested with indirect ELISA mostly made of the mAb and cell culture in 84 patients with genital symptoms. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 38 % of total bacterium protein. A mAb against Ctomp2 was obtained. It belongs to IgG 2b. The titers were as high as 1:40 000. The Western blotting showed that the mAb only reacted with the recombinant protein. It had no crossing reactions against E. coli, N. gonorhoea, M. hominis, U. urealyticum and M. penetrans . It had high specifity. In comparison with gold standard test-cell culture, the sensitivities, specificities, positive predictive values and negative predictive values of indirect ELISA were 95.24%, 100%, 100% and 98.44%, respectively. The above-mentioned research work contributed not only to the further study of the structure and function of this protein , but also to the establishment of the method for its clinical application, for it had not been reported before.