Lack of association of the KIR and HLA class I ligands with ZIKV infection in south and southeast of Brazil

BACKGROUND Zika virus (ZIKV) is an emerging arbovirus associated with foetal malformations and neurological complications. The infection is usually associated with mild symptoms. The comparison between the allelic frequency of polymorphic genes in symptomatic infected individuals in the population can clarify the pathogenic mechanisms of ZIKV. During ZIKV infection, cytokines are produced and natural killer (NK) cells are recruited, whose activation depends on signaling pathways activated by specific receptors, such as killer cell immunoglobulin-like receptors (KIR). These molecules interact with human leukocyte antigen (HLA) class I ligands and are encoded by polymorphic genes. OBJECTIVES This study aimed to evaluate the frequency of allelic variants of the genes encoding the KIR receptors and their HLA class I ligands in 139 symptomatic ZIKV-patients and 170 controls negative for the virus, and to evaluate the role of these variants for ZIKV susceptibility. METHODS KIR and HLA class I genes were genotyped using the polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) technique. FINDINGS No significant differences in the frequency distribution of KIRs and KIR-HLA in patients compared to controls were observed. MAIN CONCLUSIONS KIR and its HLA ligands might play a minor role in ZIKV infection in the south and southeast Brazilian individuals.

Dinámica estructural entre la conformación abierta y cerrada del canal KATP en células pancreáticas / Structural dynamics between the open and closed conformations of the KATP channel in pancreatic cells

INTRODUCCIÓN: el canal de Potasio sensible a ATP (canal KATP) regula la producción de Insulina por células ß pancreáticas. La Glibenclamida (GBM) (fármaco antidiabético) y el ATP actúan como inhibidores de este canal, mientras que el ADP lo activa. El canal KATP es un octámero constituido por 4 subunidades centrales Kir6.2 que forman el poro y 4 subunidades externas de regulación SUR1. OBJETIVO: determinar la dinámica estructural entre las conformaciones abierta y cerrada del canal KATP en células pancreáticas. MÉTODO: análisis estructural comparativo de diferentes estructuras cristalográficas del canal KATP de células pancreáticas humanas empleando el software Chimera v1.11.2 RESULTADOS: La subunidad Kir6.2 presenta un dominio de unión a PIP2 (activador), una Hélice Interfacial (IFH) y un dominio N-terminal (KNtp). Por otro lado, la subunidad SUR1 que contiene el sitio de unión a la GBM, tiene 2 Dominios de Unión a Nucleótidos (NBD1/2), un bucle M5-Lh1 y un Motivo de Lazo formado por la interface entre el Dominio Trans-membrana 0 y el Bucle 0 (TMD0-L0). Los resultados del análisis dinámico estructural mediante herramientas bioinformáticas, indican que estas regiones participan activamente en los cambios conformacionales que dan lugar al cierre (inhibición) o apertura (activación) de este canal. CONCLUSIÓN: El estudio de la dinámica de activación e inhibición de los canales KATP es imprescindible para la evaluación, descubrimiento y/o diseño de nuevos compuestos naturales, que como la GBM, puedan promover la secreción de Insulina para coadyuvar o mejorar el tratamiento de pacientes diabéticos.

INTRODUCTION: the ATP-sensitive Potassium channel (KATP channel) regulates insulin production by pancreatic ß cells. Glibenclamide (GBM) (antidiabetic drug) and ATP act as inhibitors of this channel, while ADP activates it. The KATP channel is an octamer consisting of 4 central Kir6.2 subunits that form the pore and 4 external regulation subunits SUR1. OBJECTIVE: to determine the structural dynamics between the open and closed conformations of the KATP channel in pancreatic cells. METHOD: comparative structural analysis of different crystallographic structures of the KATP channel of human pancreatic cells using Chimera v1.11.2. RESULTS: the Kir6.2 subunit has a PIP2 binding domain (activator), an Interfacial Helix (IFH) and an N-terminal domain (KNtp). On the other hand, the SUR1 subunit that contains the GBM binding site, has 2 Nucleotide Binding Domains (NBD1/2), an M5-Lh1 loop and a Lasso Motif formed by the interface between the Trans-membrane Domain 0 and Loop 0 (TMD0-L0). The results of the dynamic structural analysis using bioinformatics tools indicate that these regions participate actively in the conformational changes that lead to the closure (inhibition) or opening (activation) of this channel. CONCLUSION: the study of the dynamics of activation and inhibition of the KATP channels is essential for the evaluation, discovery and/or design of new natural compounds, which like GBM, can promote insulin secretion to aid or improve the treatment of diabetic patients.

Effects of hypothermia on the repolarization duration of ventricular myocytes in rats and its mechanism / 中国应用生理学杂志

To observe the effects of hypothermia on the repolarization duration and the expression of Kir2.1 protein of ventricular myocytes in isolated rat heart and explore the role of Kir2.1 protein. Eighteen healthy adult male Sprague-Dawley rats were randomly divided into three groups (n=6 per group): Control group (C group), 35℃ group (H group), 32℃ group (H group). Langendorff isolated heart models were established. After 15 min 37℃ K-H fluid banlanced perfusion, C group continued to perfuse the K-H solution at 37℃ for 30 minutes, H group continued to perfuse the K-H solution at 35℃ for 30 minutes, H group continued to perfuse the K-H solution at 32℃ for 30 minutes. At 15 min of balanced perfusion (T), and 30 min of continuous perfusion (T), the heart rate,and the MAP in the three layers of the left ventricular anterior wall were recorded, the action potential duration at 50% repolarization (MAPD), the action potential duration at 90% repolarization (MAPD) and transmural dispersion of repolarization(TDR) were calculated. At the same time, the occurrence of arrhythmia was recorded. The expression of Kir2.1 protein was measured by Western blot. The average optical density (AOD) and the distribution of Kir2.1 protein were measured by immunohistochemistry in the ventricular tissue measured by electrophysiology. Compared with T, the heart rate was decreased, MAPD and MAPD were prolonged significantly (P＜0.05), and TDR was increased significantly (P＜0.05) in H group, H group at T. Compared with C group, the HR was decreased, the MAPD was prolonged significantly (P＜0.05), TDR was increased significantly (P＜0.05),the expression and the AOD of Kir2.1 protein were decreased significantly (P＜0.05) in Hgroup, Hgroup at T. Compared with H group, the heart rate of H group was decreased significantly (P＜0.05), MAPD and MAPD were prolonged significantly (P＜0.05), and TDR was increased significantly (P＜0.05) at T. The distribution of Kir2.1 protein in group C was normal, while the distribution of Kir2.1 in H group and H group was disordered. Hypothermia prolonged the ventricular duration of repolarization and increased the dispersion of repolarization. The mechanism is related to the down-regulation the expression of Kir2.1 protein and the disorder of the distribution of Kir2.1 protein.

Association of kir2dl2 gene with anti-cyclic citrullinated protein antibodies for serodiagnosis in rheumatoid arthritis

Rheumatoid arthritis is a clinical autoimmune syndrome that causes joint damage. The positive or negative anti-cyclic citrullinated protein (CCP) antibodies serodiagnosis differentiates two subsets of the disease, each with different genetic background. Previous studies have identified associations between KIR genes and rheumatoid arthritis but not with anti-CCP serodiagnosis. Therefore, we investigated the proportion of patients seropositive and seronegative to anti-CCP and its possible association with KIR (killer cell immunoglobulin-like receptor) genes. We included 100 patients with rheumatoid arthritis from western Mexico, who were determined for anti-CCP serodiagnosis by ELISA, and 16 KIR genes were genotyped by PCR-SSP. The proportion of seropositive anti-CCP patients was 83%, and they presented a higher frequency of KIR2DL2 genes than the seronegative group (73.6% vs. 46.2%, p = 0.044) which, in turn, presented a higher KIR2DL2-/ KIR2DL3+ genotype frequency than the first ones (46.2% vs. 17.2%, p = 0.043). These results suggest different KIR genetic backgrounds for each subset of the disease according to anti-CCP serodiagnosis.

La artritis reumatoide es un síndrome clínico autoinmune que causa daño en las articulaciones. El serodiagnóstico positivo o negativo para anticuerpos proteicos anti-cíclicos citrulinados (CCP) diferencia dos subconjuntos de la enfermedad, cada uno con diferente fondo genético. Estudios previos han identificado asociaciones entre los genes killer cell immunoglobulin- like receptor (KIR) y la artritis reumatoide, pero no con el serodiagnóstico de anti-CCP. Por lo tanto, investigamos la proporción de seropositividad y seronegatividad anti-CCP y su posible asociación con genes KIR. Se incluyeron 100 pacientes con artritis reumatoide del occidente de México, a quienes se les determinó su serodiagnóstico anti-CCP por ELISA y también se les realizó genotipificación de 16 genes KIR por PCR-SSP. La proporción de pacientes seropositivos anti-CCP fue del 83% y presentaron una mayor frecuencia génica KIR2DL2 que el grupo seronegativo (73.6% vs. 46.2%, p = 0.044), estos últimos presentaron mayor frecuencia genotípica KIR2DL2-/KIR2DL3+ que los primeros (46.2% vs. 17.2%, p = 0.043). Los resultados sugieren diferente fondo genético KIR para cada subconjunto de la enfermedad, de acuerdo con el serodiagnóstico anti-CCP.

Kir2.1 Channel Regulation of Glycinergic Transmission Selectively Contributes to Dynamic Mechanical Allodynia in a Mouse Model of Spared Nerve Injury / 神经科学通报·英文版

Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5 days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.

Research progress of Müller cells in diabetic retinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

@#Müller cells are the most important glial cells in the vertebrate retina. They extend from the inner limiting membrane to the outer membrane through the entire retina, participate in the blood-retinal barrier, and actively participate in retinal development and promote the maintenance of retinal homeostasis through many intracellular mechanisms. Müller cells play an important role in the development of diabetic retinopathy. The pathophysiological changes in diabetic retinopathy remain to be further studied. This article reviews the pathophysiological changes of Müller cells in diabetic retinopathy and the recent research progress.

Construction of stable cell lines expressing large conductance Ca2+-activated K+ channel α-subunit and molecular mechanism of potassium excretion in this channel / 上海交通大学学报（医学版）

Objective: To construct stable cell lines expressing the large conductance Ca2+-activated K+ channel (MaxiK or BK) α-subunit and to explore the mechanism of potassium excretion via BKα channel. Methods:The BKα plasmid with Myc tag was constructed and transfected into HEK293 cell lines by lipofectamine 2000. The positive monoclonal cell lines were screened by G418, and the expression of BKα was detected by Western blotting and the location of BKα by immunofluorescence. The stable cell lines expressing BKα protein was cultured on slides to form a single cell layer, which was perfused with different potassium ion concentrations of 5 mmol/L and 100 mmol/L, and the single channel patch clamp recorded the ion flux of BKα. Wild type and mutants (G77R, G130R, C140R and R297C) of the inwardly rectifying potassium channel (Kir4.1) were transfected into HEK293 cells stably transfected with BKα, and then the membrane protein was extracted. The expression of BKα was detected by Western blotting. Results:Stable cell lines expressing BKα channel were selected from HEK293 cells after transfection and cellular immunofluorescence verified the expression of BKα channel and its expression on the cell membrane. The channel open frequency (Npo) of BKα increased rapidly when perfused with 100 mmol/L potassium. After being transfected with wild type or mutants of Kir4.1, the membrane expression of BKα in the stable cell lines showed significant difference among these groups (P<0.05). Conclusion:The HEK293 cell lines stably expressing BKα have been successfully constructed. BKα channel can be activated by high potassium solutions. The function of the BKα subunit can be related to Kir4.1 channel, which may be attributed to the depolarization of the cells transfected by Kir4.1 mutants.

A protective effect conferred by KIR3DL1 and its cognate ligand against cervical cancer among ethnic Han Chinese population and its potential mechanism / 中华医学遗传学杂志

Objective@#To explore the role of inhibitory KIR (iKIR) and its cognate HLA ligand in the occurrence and development of cervical cancer among ethnic Han Chinese and its potential mechanism.@*Methods@#Peripheral blood samples from 265 Han Chinese patients with cervical intraepithelial neoplasia (CIN)/cervical cancer and 200 ethnically matched healthy controls were collected. The results of KIR PCR-SSP, HLA PCR-rSSO and KIR3DL1 PCR-SBT, together with cervical cancer data from the TCGA database, were used to assess the association of iKIR genes, receptor-ligand gene combinations, iKIR transcription level in the tumor tissue and the KIR3DL1 alleles with the occurrence and development of cervical cancer.@*Results@#Among the four iKIR genes (KIR2DL1, 2DL2/3, 3DL1 and 3DL2), the frequencies of KIR3DL1 and KIR3DL1-HLA-Bw4 genes among controls were significantly higher than those of the cervical cancer group (96.5% vs. 87.0%, P = 0.018; 81.5% vs. 64.8%, P=0.009). The survival rate of cervical cancer patients with a high transcription level of KIR3DL1 in tumor tissues was significantly higher than those with a low/medium transcription level (P = 0.028). The frequency of strong-inhibitory and high-expression KIR3DL1*01502 allele among the healthy population was significantly higher than that of the cervical cancer group (76.0% vs. 59.3%, P = 0.015).@*Conclusion@#Combined KIR3DL1 and KIR3DL1-HLA-Bw4 can confer a protective effect against the development of cervical cancer, which may be attributed to the strong-inhibitory and high-expression allele of KIR3DL1*01502.

Effects of hyperoside on the myocardical activities of ATPases and expressions of Cx43 and Kir2.1 in arrhythmia rats induced by ischemia-reperfusion / 中成药

AIM To investigate the effects of hyperoside,an anti-arrhythmic agent capable of reducing myocardial infarct size,on arrhythmic rats induced by ischemia-reperfusion (I/R) and the corresponding mechanism.METHODS Male SD rats were randomly assigned to sham operation group,model group and hyperoside group (50 mg/kg,n =15).The I/R model was reconstructed by the ligation of left anterior descending coronary artery for 30 min ischemia.Rats in the hyperoside group were injected with 50 mg/kg hyperoside intraperitoneally 10 min before ischemia.Heart rate,mean arterial pressure (MAP) and heart rate systolic blood pressure product (RPP) at time points of 10 min before ischemia (T0),30 min after ischemia (T1),30 min (T2),60 min (T3),120 min (T4) after reperfusion were recorded.ELISA method was used to determine serum CK-MB and cTnI,spectrophotometry to measure Na+-K+-ATPase and Ca2+-Mg2+-ATPase levels,HE staining to observe myocardial tissue changes,immunohistochemistry to investigate Cx43 protein,and Western blot to detect Kir2.1 protein expression.RESULTS At T1,T2,T3 and T4,the model group demonstrated significantly lower HR,MAP and RPP than those in the sham operation group (P ＜ 0.05),whereas the hyperoside group had higher HR,MAP and RPP than the model group.Both hyperoside group and the model group shared significantly higher arrhythmia score,levels of CK-MB and cTnI than the sham operation group (P ＜ 0.05) while their lower activities of Na +-K +-ATPase and Ca2+-Mg2+-ATPase,protein expressions of Cx43 and Kir2.1 than the sham operation group were noticed as well.But the hyperoside group displayed its lower arrhythmia score,levels of CK-MB and cTnI,and yet higher activities of Na +-K +-ATPase and Ca2 +-Mg2 +-ATPase,protein expressions of Cx43 and Kir2.1 than the model group (P ＜0.05).CONCLUSION Hyperoside in improving ventricular arrhythmia of I/R rats may contribute to the activity restoration of Na +-K +-ATPase and Ca2 +-Mg2 +-ATPase,and the up-regulation of Cx43 and Kir2.1 protein expressions.

Changes of Kir4.1 and TASK-1 expression in rat Müller cell induced by SCH442416 at an elevated hydrostatic pressure in vitro / 中华实验眼科杂志

Objective To evaluate the effect of adenosine receptor antagonist SCH442416 on the expression of Kir2.1,Kir4.1 and TASK-1 in rat Müller cell at an elevated hydrostatic pressure in vitro.Methods Thirty SPF Sprague Dawley rats were purchased from Shanghai Slack Laboratory Animals Ltd.Cultured Müller cells were divided into normal control group (n =6),40 mmHg/24 hours (1 mmHg =0.133 kPa) group (n =6) and adenosine + SCH442416 intervention group (n =6).Müller cells were treated with 40 mmHg pressure for 24 hours in 40 mmHg/24 hours group,and Müller cells were treated with 40 mmHg pressure for 24 hours + 10 μ mol/L adenosine + 100 nmol/L SCH442416 in adenosine + SCH442416 intervention group.The real-time PCR,Western blot,whole-cell patch-clamp recordings and immunohistochemistry were used to detect Kir2.1,Kir4.1 and TASK-1 expression and Müller cells Kir currents.The experimental procedures were in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory,and follow the 3R principle.Results Western blot assay showed that,following 40 mmHg pressure cultured for 24 hours,the expression of Kir4.1 and TASK-1 protein in Müller cell were significantly decreased by 38.6％ and 52.6％ compared with the normal control group,with significant differences between the two groups (both at P =0.000);Kir2.1 protein expression decreased by 14.7％,with insignificant difference between the two groups (P =0.082).Kir4.1 and TASK protein expression in adenosine + SCH442416 intervention group was increased by 60.7％ and 61.4％ compared with the 40 mmHg/24 hours group,with significant differences between the two groups (both at P =0.000);Kir2.1 protein expression in adenosine + SCH442416 intervention group was increased by 8.8％ compared with the 40 mmHg/24 hours group,with insignificant difference between them (P =0.354).Real-time PCR assay showed that,following 40 mmHg pressure cultured for 24 hours,Kir2.1,Kir4.1 and TASK-1 mRNA expression in Müller cells were significantly decreased compared with the normal control group,with significant differences between the two groups (P =0.047,0.001,0.000);Kir4.1 and TASK-1 mRNA expression in Müller cells in the adenosine + SCH442416 intervention group was significantly increased compared with the 40 mmHg/24 hours group,with significant differences between the two groups (P =0.038,0.030);however,there is no significant change in Kir2.1 mRNA expression (P =0.612).Conclusions SCH442416 upregulates the expression of Kir4.1 and TASK-1 mRNA and protein,but weakly affects the expression of Kir2.1.

Human Leukocyte Antigen-C Genotype and Killer Immunoglobulin-like Receptor-Ligand Matching in Korean Living Donor Liver Transplantation

BACKGROUND: The interaction between killer immunoglobulin-like receptors (KIRs) and HLA class I regulates natural killer (NK) cell cytotoxicity and function. The impact of NK cell alloreactivity through KIR in liver transplantation remains unelucidated. Since the frequency of HLA-C and KIR genotypes show ethnic differences, we assessed the impact of HLA-C, KIR genotype, or KIR-ligand mismatch on the allograft outcome of Korean liver allografts. METHODS: One hundred eighty-two living donor liver transplant patients were studied. Thirty-five patients (19.2%) had biopsy-confirmed acute rejection (AR), and eighteen (9.9%) had graft failure. The HLA-C compatibility, KIR genotypes, ligand-ligand, and KIR-ligand matching was retrospectively investigated for association with allograft outcomes. RESULTS: Homozygous C1 ligands were predominant in both patients and donors, and frequency of the HLA-C2 allele in Koreans was lower than that in other ethnic groups. Despite the significantly lower frequency of the HLA-C2 genotype in Koreans, donors with at least one HLA-C2 allele showed higher rates of AR than donors with no HLA-C2 alleles (29.2% vs 15.7%, P=0.0423). Although KIR genotypes also showed ethnic differences, KIR genotypes and the number of activating KIR/inhibitory KIR were not associated with the allograft outcome. KIR-ligand mismatch was expected in 31.6% of Korean liver transplants and had no impact on AR or graft survival. CONCLUSIONS: This study could not confirm the clinical impact of KIR genotypes and KIR-ligand mismatch. However, we demonstrated that the presence of HLA-C2 allele in the donor influenced AR of Korean liver allografts.

Immune reconstruct regularity profile of KIR2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

Objective@#To investigate the immune reconstruct regularity profile of KIR2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation (allo-HSCT) with KIR-AA genotype.@*Method@#75 donor-recipient pairs were performed by KIR genotying using PCR-SSP, and all donors were identified with KIR-AA genotype. Dynamic detections (including unrelated-donor on the day of transplantation and the recipient each month post allo-HSCT) of the expression of KIR2DL1/3DL1 on NK cell and mRNA level were performed in 291 cases using flow cytometry (FCM) and real-time fluorescent quantitation PCR (RT-qPCR) .@*Result@#①The median expression of KIR2DL1 in unrelated-donor on transplant’s day was 21.60%, the median expression of KIR2DL1 in recipient 1M, 2M, 3M and 3-6M after transplantation were 7.40%, 12.00%, 16.92%, 17.64% respectively. The median expression of KIR2DL1 in unrelated-donor on transplant’s day was 265.14 copies/10 000abl copies, the median expression of KIR2DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 332.17, 438.31, 723.25, 414.17, 180.76 and 234.67 copies/10 000abl copies respectively. The median expression of KIR2DL1 on NK cells and mRNA level gradually increased at all time points after transplantation, and reached the highest expression at 3 months after transplantation. But mRNA expression levels increased earlier than NK cell membrane proteins. ②The median expression of KIR3DL1 in unrelated-donors on transplant’s day was 18.56%, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M after transplantation were 23.83%, 22.57%, 23.02%, 21.60% respectively. The median expression of KIR3DL1 in unrelated-donor on transplant’s day was 572.29 copies/10 000abl copies, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 1 233.74, 1 140.42, 876.73, 1 057.07, 739.02 and 514.43 copies/10 000abl copies respectively. The median expression of KIR3DL1 on NK cells and mRNA level were higher than donors at 1 month after transplantation, and stable expression at all time points after transplantation, so mRNA and NK cell membrane proteins expression increased at the same time.@*Conclusion@#The immune reconstruct regularity of KIR2DL1 and KIR3DL1 gene were different, which provided an experimental basis for selecting the best time to detect the expressions of KIR2DL1 and 3DL1 after transplantation.

Distribution of donor-specific aKIR after unrelated allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

Objective@#To analyze the distribution and proportion of donor-specific activated killer cell immunoglobulin like receptor (aKIR) genes and their clinical application values in unrelated allogeneic hematopoietic stem cell transplantation (allo-HSCT) .@*Methods@#Retrospective analyses of KIR genotyping using polymerase chain reaction with sequence specific primers (PCR-SSP) were performed in 216 pairs of donors and recipients.@*Results@#The frequency of donor-specific KIR genes was 53.7% (116/216) in 216 patients receiving unrelated allo-HSCT, with the frequency of 78.3% (112/143) in the KIR genes mismatched group and 5.5% (4/73) in matched group. Of the 116 patients with detectable donor-specific KIR genes, 99.1% (115/116) patients had various donor-specific aKIR genes. Among 55 pairs of donors’ KIR-Bx genotype and patients’ KIR-AA genotype group, the most commonly observed genotypes were Bx1, Bx2, Bx3, Bx4, in which the donor-specific KIR genes were respectively KIR 3DS1, 2DL5A, 2DS5, 2DS1; KIR 3DS1, 2DL5A, 2DS3, 2DS1; KIR 2DS2, 2DL2; KIR 2DS2, 2DL2, 3DS1, 2DL5A, 2DS5, 2DS1. Of 44 pairs of donors’ KIR-AA genotype and patients’ KIR-Bx (AB) genotype group, 36.4% (16/44) recipients had donor-specific KIR2DS4 (FUL) gene. In 143 pairs of KIR mismatched group, the frequencies of donor-specific KIR genes were KIR2DS1 (35.7%) , KIR3DS1 (32.9%) , KIR2DS5 (29.4%) , KIR2DS4 (FUL) (25.9%) , KIR2DL2 (25.2%) , KIR2DS2 (24.5%) , KIR2DS3 (21.7%) and KIR3DL1 (8.4%) , respectively.@*Conclusion@#The donor-specific aKIR genes mainly existed in KIR mismatched group after unrelated allo-HSCT, and the different pairs of donors’ and patients’ KIR genotypes led to the diverse donor-specific aKIR. But there were higher specific aKIR genes in higher frequency of KIR AA, Bx1, Bx2, Bx3, Bx4 genotypes. All these can provide the experimental basis for studying the role of the donor-specific aKIR genes on the prognosis of HSCT.

MiR-20 Regulates Myocardiac Ischemia by Targeting KATP Subunit Kir6.1 / 华中科技大学学报（医学）（英德文版）

This study aimed to examine the functional role of microRNA-20 (miR-20) and its potential target,Kir6.1,in ischemic myocardiocytes.The expression of miR-20 was detected by real-time PCR.Myocardiocytes were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reagent for apoptosis evaluation.Western blotting was used to detect the Kit6.1 protein in ischemic myocardiocytes transfected with miR-20 mimics or inhibitors.Luciferase reporter gene assay was performed to confirm the targeting effect of miR-20 on KCNJ8.The results showed that miR-20 was remarkably down-regulated,while the KATP subunit Kir6.1 was significantly up-regulated,during myocardial ischemia.The miR-20 overexpression promoted the apoptosis of ischemic myocardiocytes,but showed no such effect on normal cells.Under ischemic condition,myocardiocytes transfected with miR-20 mimics expressed less Kir6.1.On the contrary,inhibiting miR-20 increased the expression of Kir6.1 in the cells.Co-transfection of miR-20 mimics with the KCNJ8 3’-UTR plasmid into HEK293 cells consistently produced less luciferase activity than transfection of the plasmid alone.It was concluded that miR-20 may regulate myocardiac ischemia by targeting KATP subunit Kir6.1 to accelerate the cell apoptosis.Therefore miR-20 may serve as a therapeutic target for myocardial ischemic disease.

Phenotypes and HIV-1-specific T cell responses of KIR3DL1 positive CD8 cells in patients with early HIV-1 infection / 中华微生物学和免疫学杂志

Objective To investigate the phenotypes and the HIV-1-specific T cell responses of KIR3DL1 positive CD8 cells in patients with early HIV-1 infection. Methods Fifty-six HIV-1 antibody negative individuals and thirty-two patients with early HIV-1 infection were enrolled in the study. Fluores-cence-activated cell sorting (FACS) was performed to detect the phenotypes of KIR3DL1 receptor expressed on the surface of CD8 cells. The levels of IFN-γwere measured by intracellular cytokine staining assay after the PBMCs were stimulated with an HIV-1 Gag peptide pool. Results The percentages of KIR3DL1+CD8 T cells in HIV-1 negative individuals and patients with early HIV-1 infection were 1. 45% (0. 12%-8. 4%) and 0. 82% (0. 14%-6. 14%), respectively, and there was no significant difference between them. The percentages of KIR3DL1+CD8 Temra cells in HIV-1 negative individuals and patients with early HIV-1 infec-tion were (4. 55±3. 84)% and (6. 71±8. 50)%, respectively, which were significantly higher than the per-centages of KIR3DL1+CD8 Tem cells, which were (0. 50±0. 59)% and (1. 18±1. 39)%, respectively (all P<0. 01). Moreover, the percentages of KIR3DL1+CD8 Tem cells in patients with early HIV-1 infection were higher than those in HIV-1 negative individuals (P=0. 001 2). The percentage of KIR3DL1+CD8 Temra cells was positively correlated with the HIV-1 viral load in patients with early HIV-1 infection ( rs=0. 576,P=0. 000 9). The percentages of KIR3DL1+CD8 Temra cells in HIV-1 patients, whose viral loads were larger than 4. 0log, were much higher than those in HIV-1 patients with viral loads less than 4. 0 log (P=0. 002). Additionally, the levels of IFN-γsecreted by KIR3DL1 positive CD8 cells were much lesser than those secreted by KIR3DL1 negative CD8 cells (P<0. 000 1). Conclusion The receptor of KIR3DL1 was mainly expressed on CD8 Temra cells in both HIV-1 negative subjects and patients with early HIV-1 infec-tion. High HIV-1 viremia was associated with the high percentage of KIR3DL1+CD8 Temra cells. The KIR3DL1 positive CD8 cells induced lower HIV-1-specific T cell responses.

Study on mechanism of regulating synthesis of β-sitosterol by SiSQS1 and SiSQS2 in Saussurea involucrata cells / 中草药

Objective: To explore the synthetic mechanism of β-sitosterol by comparing the locus mutation, prokaryotic expression, expression level of SiSQS1 and SiSQS2, and the content of β-sitosterol in three color types of cells. Methods: Firstly, we preformed the cloning and bioinformatic analysis of SiSQS1 and SiSQS2 which were key enzymes involved in the biosynthesis of β-sitosterol in Saussurea involucrata cells. Secondly, we compared the differences of prokaryotic expression between SiSQS1 and SiSQS2, then optimized the expression conditions. Finally, we compared the expression levels of SiSQS1 and SiSQS2 by Real-time PCR and the content of β-sitosterol by GC-MS in three color types of cells, and made the correlative analysis on the expression level and the content of β-sitosterol. Results: There was a locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2. The results of prokaryotic expression analysis and conditions optimization showed that both target proteins had been expressed successfully, but the optimal prokaryotic expression system was different. The results of expression level and quantitative analysis showed a positive correlation to the expression levels of SiSQS1 and SiSQS2 and the content of β-sitosterol, the correlation coefficients were 0.92 and 0.89, respectively. Conclusion: A locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2 may influence the expression of SiSQS, and there may exist the functional differences in catalytic activity and the accumulation of β-sitosterol. The study will provide technical support and lay a theoretical foundation for studying the accumulated mechanism of β-sitosterol regulated by SQS in S. involucrata cells.

Downregulation of inwardly rectifying potassium channel 5.1 expression in C57BL/6J cochlear lateral wall / 华中科技大学学报（医学）（英德文版）

Age-related hearing loss (AHL) is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1 (Kir5.1) plays a vital role in regulating cochlear K(+) circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The mRNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions (RT-PCR) and Western blotting respectively. Kir5.1 was detected in the type II and IV fibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 mRNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.

The role of KIR2DL3/HLA-C*0802 in Brazilian patients with rheumatoid vasculitis

OBJECTIVES: Rheumatoid arthritis is a polygenically controlled systemic autoimmune disease. Rheumatoid vasculitis is an important extra-articular phenotype of rheumatoid arthritis that can result in deep cutaneous ulcers. The objective of this study was to establish a correlation between the frequency of major histocompatibility complex class I/II alleles and killer immunoglobulin-like receptor genotypes in patients with cutaneous rheumatoid vasculitis. METHODS: Using the Scott & Bacon 1984 criteria to diagnose rheumatoid vasculitis and after excluding any other causes such as diabetes, atherosclerosis, adverse drug reactions, infection, and smoking, patients who met the criteria were selected. All of the selected rheumatoid vasculitis patients presented deep cutaneous ulcers. Identification of the major histocompatibility complex class I/II and killer immunoglobulin-like receptor genotypes was performed by polymerase chain reaction assays of samples collected from the 23 rheumatoid vasculitis patients as well as from 80 controls (40 non-rheumatoid vasculitis RA control patients and 40 healthy volunteers). RESULTS: An association between the presence of the HLA-DRB1*1402 and HLA-DRB1*0101 alleles and cutaneous lesions in rheumatoid vasculitis patients and a correlation between the inhibitor KIR2DL3 and the HLA-C*0802 ligand in rheumatoid vasculitis patients were found. CONCLUSION: An association was found between the presence of the HLA-DRB1*1402 and HLA-DRB1*0101 alleles and the development of cutaneous lesions in rheumatoid vasculitis patients. Additionally, the HLA-C*0802 ligand protects these individuals from developing cutaneous lesions. .

The Impact of HLA and KIR Ligand Mismatching on Unrelated Allogeneic Hematopoietic Stem Cell Transplantation in Korean Adult Patients

BACKGROUND: The impact of HLA and KIR ligand mismatching on the outcome of hematopoietic stem cell transplantation (HSCT) remains unclear. Previous reports have identified considerable ethnic differences in the impact of HLA and KIR ligand mismatches, as well as KIR ligand status, on HSCT; however, to date, no data has been acquired in Korean adult patients. METHODS: We investigated the association of high-resolution HLA matching on five loci (HLA-A, -B, -C, -DRB1, and -DQB1), KIR ligand mismatching, and KIR ligand status on the outcome of allogeneic HSCT from unrelated donors in 154 Korean adult patients treated at Seoul National University Hospital. RESULTS: In a multivariate analysis, less than 9/10 allelic matches in five HLA loci was an independent risk factor for acute graft-versus-host disease (GVHD) (grade II to IV) (P=0.019, odds ratio [OR]=2.7). In addition, HLA-A allele mismatching was increasingly prevalent in patients with acute GVHD compared to patients without (61.9% vs. 34.5%, P=0.06). For KIR ligand status, the patient and donor combination of both C1/C1 ligands showed better event-free and overall survival than combinations with C2 ligand patients or donors (P=0.048, P=0.034, respectively) by log-rank test. CONCLUSIONS: Korean adult transplant patients with less than 9 of 10 HLA allele matches in the HLA-A, -B, -C, -DRB1, and DQB1 loci have a higher likelihood of developing acute GVHD (grade II to IV). Impact of KIR ligand status on clinical outcome should be further studied in a larger patient population.

Effect of extract from callus of Saussurea involucrate on proliferation and differentiation of osteosarcoma cell MG-63 / 中草药

Objective: To investigate the effect of extract from callus of Saussurea involucrate (ESI) on the proliferation and differentiation of osteosarcoma cells MG-63 and explore its anti-osteoporosis and mechanism. Methods: Using human osteosarcoma cells MG-63 for the study, the effect of ESI on the proliferation was detected by MTT and LDH methods; The activity of alkaline phosphatase activity (ALP) and the level of hydroxyproline (Hyp) were investigated by related reagent kit; The effect of ESI on mineralized nodules was observed by Alizarin red staining and quantified by CPC, the expression of OPG/RANKL was measured by RT-PCR and Western blotting, and the proliferation, differentiation, mineralization, and OPG/RANKL expression levels of MG-63 were assayed when medium contains both p38 inhibitor SB203580 and ESI. Results: MTT and LDH assay showed that ESI (< 125 μg/mL) was non-toxic to MG-63, and it promoted the proliferation at the concentration of 31.25 and 62.5 μg/mL; ESI could significantly increase the ALP activity, Hyp content, and amount of mineralized nodules; At the mRNA level, ESI could significantly up-regulate OPG expression and down-regulate RANKL expression; At the protein level, ESI could significantly increase the ratio of OPG/RANKL; SB203580 could reverse the acceleration of ESI on the proliferation, differentiation, mineralization, and OPG/RANKL expression of MG-63. Conclusion: ESI has the facilitating effect on the osteoblast proliferation, differentiation, and mineralization; The mechanism may be associated with the expression of OPG and RANKL, as well as the signal transduction pathway of p38 Mitogen-activated potein kinase (MAPK).