| Chapter1Polymorphisms of KIR and HLA-C with Susceptibility to Tuberculosis InfectionBackgroundTuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosis (M.TB). According to a WHO report, there were8.7million new cases and nearly1.4million deaths annually around the globe. China is one of the22high-burden countries in the world. The fifth national epidemiological survey suggested that up to4.99million people over the age of15infected with active TB. The result, our country may have nearly50million infected people in the next10years unless measures are taken against it. Epidemiological studies also indicate that only5to10%of people infected with M. tuberculosis develop active TB. The immune system can prevent the progression of the active TB. Host susceptibility genes may play a very important role in determining outcomes of exposure and infection with MTB. They may affect the recognition, phagocytosis, and killing of mycobacterium, subsequently affect the development and result in infection. The immune system considered to be the most crucial link between tuberculosis and the host. The genetic factors of TB have been studied using many methods, including selection of candidate genes and genome-wide linkage studies.Killer cell immunoglobulin-like receptors (KIRs) are a highly diverse family of receptors expressed by natural killer cells and subsets of T lymphocytes. KIR molecule belongs to immuneglobulin superfamily and modulates cells function upon recognition of HLA class I molecules. KIR locus, containing a family of polymorphic and highly homologous genes, maps to chromosome19q13.42within the leukocyte receptor complex (LRC). Its encoding proteinum KIR receptor is transmembrane glycoprotein, according to the side of cytoplasmic domain, KIR divide into inhibitory receptors and activating receptors. Up to date, KIR family consists of8inhibitory genes (2DLI,2DL2,2DL3,2DL4,2DL5,3DLI,3DL2,3DL3),6activating genes (2DS1,2DS2,2DS3,2DS4,2DS5,3DS1),2pesudogenes KIR3DP1(including KIRX and KIRXv), and KIR1D. Upon interaction with HLA class I molecules expressed on the surface of target cells, KIR genes provide activating or suppressing signals to regulate the activation of NK cells and T cells, thereby playing an important role in antiviral and anti-tumor immunity. It has been reported that KIR genes present diversity in structure and function:①Different race and different individual have different frequencies;②Allelic polymorphism;③Different genotype frequencies;④Haplotype diversity. HLA class I is the most polymorphic region of the human genome. HLA-I is found at the A, B and C loci of chromosome6and has been shown to play an important role in controlling infection. HLA-C molecules are classified as either C1group (HLA-Cw*01, HLA-Cw*03, HLA-Cw*07, HLA-Cw*08, etc) or C2group (HLA-Cw*02, HLA-Cw*04, HLA-Cw*05, HLA-Cw*06, etc). KIR polymorphisms and allelic variation affect the KIR-HLA binding specificity and are linked to the outcome of diseases.Previous studies have demonstrated that KIR genes are involved in the pathogenesis of a variety of diseases, including rheumatoid arthritis, vasculitis, type1diabetes mellitus, and hepatitis B virus. A Mexico study revealed that the frequency of KIR2DL3was higher in TB patients compared with the control. However up to now, the role of KIR polymorphisms in patients with TB infection has not been investigated in China. HLA-Cw alleles also have been less well studied than their HLA-A and HLA-B counterparts. Therefore, we can infer that KIR gene polymorphisms may exert a crucial role in the mechanism of TB.Section1Polymorphisms of KIRsObjectiveTo explore whether killer cell immunoglobulin-like receptors (KIR) gene polymorphisms are associated with susceptibility to pulmonary tuberculosis infection. And provide a better understanding on the genetic diversity of KIR across patients with pulmonary tuberculosis.Methods1. Subjects:The400samples analyzed for this study comprised200samples from patients with PTB and200controls recruited from Shandong Provincial Hospital affiliated to Shandong University and Shandong Chest Hospital from2010to2012. Patients were diagnosed on’Tuberculosis diagnostic criteria for diagnosis and treatment’issued by Chinese medical association TB branch. The exclusion criterion for patients and controls were pneumonia, pneumoconiosis, lung cancer, chronic obstructive pulmonary disease, allergic asthma, diabetes mellitus, serious organic disease such as heart, liver and kidney, HIV, patients with weakened immune system. This study had ethical approval from the Hospital Ethics Committee and an informed consent was obtained from each individual.2. Genome DNA extraction:Genomic DNA sample was extracted from3mL thylene diamine tetraacetic acid (EDTA) anticoagulated peripheral blood with a TIANamp Blood DNA kit and stored at-20℃before use.3. KIR genotyping:Genotyping of KIR was conducted by SSP-PCR method, which was performed to detect the presence or absence of KIR genes. So KIR locus typing was performed to detect the presence or absence of6inhibitory KIR genes (2DLI,2DL2,2DL3,2DL5,3DL1,3DL3),6activating KIR genes (2DS1, DS2,2DS3,2DS4,2DS5,3DS1), the pseudo genes (3DPlx and3DPlv) and1D. All primers were validated and confirmed. PCR cycle reactions were performed on a Gene Amp PCR System9700.4. Electrophoresis and imaging:PCR products were subjected to electrophoresis to determine whether the required products were formed.5. Statistical analysis:Analysis were performed by Statistical Package for Social Sciences Version16.0(SPSS, Chicago, IL, USA). Phenotype frequency (pf%) of each gene was calculated as the percentage of positive numbers among all specimens. Genotype frequency (gf) of each locus was calculated using formula:gf=1-(?)(1-pf) Frequency differences between patients and controls were analyzed using chi-square test or Fisher test. P<0.05were considered statistically significant.Results1. In this study, all tested KIR genes were present in both patient groups and control group in different frequencies. The total carriage frequencies of KIR2DS1,2DS3, and3DS1in the patient groups were significantly higher than those in the control group (P<0.001, P<0.001, and P<0.001).2. The carriage frequencies of KIR2DS4and KIR2DS5were higher in sputum-negative patients than sputum-positive patients (P<0.001, P=0.001). Compared with the controls, the frequencies of KIR2DS1,2DL3, and2DS4were higher in sputum-negative patients (P=0.013, P<0.001, P<0.001). KIR2DS3was higher in sputum-positive patients compared with control group (P=0.011).3. The frequency of patients who had more than2activating KIRs were higher than those in the control group (P<0.001).4. The frequency of the genotype A/B (P<0.001)was increased in patients than controls but A/A(P<0.001) was decreased. There was no significant difference of B/B between the two groups (P=0.226).5. The frequency of haplotype A was higher in controls but haplotype B was higher in TB. But there was no significant difference (P=0.065, P=0.065).Conclusion1. It could be suggested that KIR2DS1,2DS3, and3DS1were TB-susceptive genes.2. The immune status determined the prognosis and outcome of mycobacterium tuberculosis infection.3. The imbalance between inhibitory receptors and activating receptors may be influence the immune status.Section2Polymorphisms of HLA-CObjectiveTo analyze the relationship between HLA-Cw polymorphisms and susceptibility to pulmonary tuberculosis, and to explore the susceptibility or resistant genes of tuberculosis infection.Methods1. Subjects:The400samples analyzed for this study comprised200samples from patients with PTB and200controls recruited from Shandong Provincial Hospital affiliated to Shandong University and Shandong Chest Hospital from2010to2012. Patients were diagnosed on’Tuberculosis diagnostic criteria for diagnosis and treatment’ issued by Chinese medical association TB branch. The exclusion criterion for patients and controls were pneumonia, pneumoconiosis, lung cancer, chronic obstructive pulmonary disease, allergic asthma, diabetes mellitus, serious organic disease such as heart, liver and kidney, HIV, patients with weakened immune system. This study had ethical approval from the Hospital Ethics Committee and an informed consent was obtained from each individual.2. Genome DNA extraction:Genomic DNA sample was extracted from3mL thylene diamine tetraacetic acid (EDTA) anticoagulated peripheral blood with a TIANamp Blood DNA kit and stored at-20°before use.3. HLA-Cw genotyping:Genotyping of HLA-Cw was also conducted by PCR-SSP method. KIR locus typing was performed to detect the presence or absence of8HLA-Cw allele genes:HLA-Cw*01, HLA-Cw*02, HLA-Cw*03, HLA-Cw*04, HLA-Cw*05, HLA-Cw*06, HLA-Cw*07, and HLA-Cw*08. All primers were validated and confirmed. PCR cycle reactions were performed on a Gene Amp PCR System9700.4. Electrophoresis and imaging:PCR products were subjected to electrophoresis to determine whether the required products were formed.6. Statistical analysis:Analysis were performed by Statistical Package for Social Sciences Version16.0(SPSS, Chicago, IL, USA). Frequency differences between patients and controls were analyzed using chi-square test or Fisher test. P<0.05were considered statistically significant.Results1. The carriage frequency of HLA-Cw*08was significantly higher in patients compared with the controls (P<0.001).2. The carriage frequency of HLA-Cw*04was higher in sputum-positive patients than sputum-negative patients (P=0.025), but HLA-Cw*08was lower (P=0.004).ConclusionHLA-Cw gene polymorphism is related to susceptibility to tuberculosis. HLA-Cw*08may be one of the susceptible genes for tuberculosis, and HLA-Cw*04and HLA-Cw*08may be related to MTB infectious status and clinical outcomes. Section3Relationship between KIR and HLA-C polymorphisms with susceptibility to tuberculosisObjectiveTo analyze the relationship between HLA-Cw polymorphism and susceptibility to pulmonary tuberculosis, and therefore to explore the susceptible or resistant genes of PTB.MethodsThis section was the same as Section1and2.Results1. The frequency of’2DL2/3with Cl’in patients was increased compared with control group (P<0.05).2. KIR2DS1was increased significantly in the patients group when HLA-C2alleles were absent.3. The frequency of’no KIR2DS3and no Cw*08’ was decreased compared with control group.4. The frequency of’Cw*04and no KIR2DS1’was decreased compared with control group.ConclusionThe results demonstrated the importance of KIR and HLA-Cw genes on susceptibility to tuberculosis infection. The mechanisms will be of help in treatment strategies. Additional genetic and functional studies will be necessary to clarify the involvement of the mechanism in tuberculosis infection. Chapter2Expression of KIR and HLA-C in patients with TuberculosisBackgroundAt present, many researchers at home and abroad have been screened more candidate susceptibility genes associated with tuberculosis. Many different explorations have been made between different nation, region, and race. To date, several candidate genes have been associated with the onset of tuberculosis. Therefore, many candidate genes have provided us the understanding of pulmonary tuberculosis (PTB) infection. KIR gene expression and regulation mainly depend on the transcription level, such as transcription of methylation. Study of KIR genotypes may be not enough to explore the mechanisms of the disease. More deeper study such as genetic susceptibility will be needed in the future study.Pander. R et al. made a statement in Nature that KIR molecules in cluster of differentiation (CD) was divided into CD158a, CD158bl, CD158b2, and CD158d. However, it is not clear in the function of CD158signal system in immune response. Research of the relevance between structure and function of CD158and tuberculosis are lacking in china. With the development of immune research related to tuberculosis, researchers will pay more and more attention in the expression of mRNA and protein level.Therefore, this chapter focused on the expression level of KIR genes of Chinese Han patients by using RT-PCR method and Real-time PCR. We also analyzed the characteristics of CDI58molecules in peripheral blood of patients with tuberculosis by using Flow cytometry. It will provide theory and molecular basis for improving the prevention and treatment of tuberculosis.Section1Expression of KIR mRNA in peripheral bloodObjectiveTo observe the mRNA transcription level of KIR inhibitory receptor in peripheral blood in patients with PTB. This section focused on the expression level of KIR genes of Chinese Han patients by using RT-PCR method and Real-time PCR.Methods1. Subjects:The60samples analyzed for this study comprised30samples from patients with PTB and30controls recruited from Shandong Provincial Hospital affiliated to Shandong University and Shandong Chest Hospital from2012to2013. Patients were diagnosed on’Tuberculosis diagnostic criteria for diagnosis and treatment’issued by Chinese medical association TB branch. This study had ethical approval from the Hospital Ethics Committee and an informed consent was obtained from each individual.2. Extraction of total RNA and identification:Total RNA was extracted from PBMCs by using RNAiso reagent. Quality and concentration of the extracted RNA were determined by measuring the absorbance of A260/A280.3. RT-PCR reverse transcription reaction:Transcription reaction was performed to detect the presence or absence of5inhibitory KIR genes (KIR2DL1,2DL2,2DL3,2DL4,3DL1). PCR reaction system is20μl, including template cDNA100ng, upstream and downstream primers (0.5μM)2μl, Buffer2μl, MgC121.6μl (25000μM)、 dNTP (10000μM)2μl, DNA polymerase0.125(5U/μ), add dH20supplement to20μ. The amplification of the framework P-actin is as the internal standards. Then all the products were run on a1.5%agarose gel prestained with ethidium bromide.4. Real-time PCR:Reactions were performed in a total volume of20μl, which included2μl of cDNA sample,0.8μOf0.2μM upstream and downstream primers, and10μl of SYBR Premix Ex Taq. PCR was on LightCycler480system. Finally, the PCR products were subjected to electrophoresis to determine whether the required products were formed.5. Statistical analysis:Analysis was performed by Statistical Package for Social Sciences Version16.0(SPSS, Chicago, IL, USA). If data conforms to normal distribution, the results presented as mean±standard deviation. If data do not accord with normal distribution, the results presented as median±standard deviation. P<0.05presented as significant difference.Results1. Based on the analysis of KIR genotyping, we found that some of the KIR phenotype is not consistent with its expression rate. This may be caused by different mRNA levels.2. Based on the results of RT-PCR, the expression level of KIR2DL3mRNA were higher in patients than controls (56.67%vs.30%, P=0.037).3. Based on the results of Real-time PCR, the expression level of KIR2DL3 were increased in patients compared with healthy controls (1.17±0.38vs.0.94±0.25, P<0.01).Conclusion1. Inhibitory KIR receptor2DL3may be affected by the regulation of peripheral blood lymphocytes.2. The different expression levels may be caused by the discrepancy of KIR genotype and phenotype.Section2Expression of CD158a and CD158b in peripheral bloodObjectiveWe also analyzed the characteristics of CD158molecules in peripheral blood of patients with tuberculosis by using Flow cytometry. It will provide theory and molecular basis for improving the prevention and treatment of tuberculosis.Methods1. Subjects:Refer to Section1of Chapter1.2. Flow cytometry:PBMCs were stained with10μl anti-human CD3,10μl anti-human CD16CD56,10μl anti-human CD158a/b to determin the expression of CD158a/b on NK cells. Isotype controls were used.3. Statistical analysis:Analysis was performed by Statistical Package for Social Sciences Version16.0(SPSS, Chicago, IL, USA). A t test was used for comparing quantitative variables of two different groups. P<0.05were considered statistically significant.Results1. CD158a and CD158b were expressed on CD3-CD56+NK cells of the two groups. The expression level of CD158a was similar in the two groups.2. However, the expression level of CD158b was decreased in patients compared with healthy controls (7.97±2.63%vs.9.58±2.92%, P=0.022).ConclusionInhibitive signal mediated by C158b may affect progression of tuberculosis. Chapter3Research on KIR genes in pulmonary tuberculosis patients with hepatitis BBackgroundAccording to the current epidemiological investigation, tuberculosis presents the rising trend in China. It is also one hundred and twenty million people infected with hepatitis B in China. Currently, there is no accurate statistics of TB patients with HBV.Whether there is a connection between these patients and KIR. To explore the role of immune factors in the disease, and to study the KIR expression feature of these patients, we used flow cytometry to analysis the expression of CD158f on their NK cells. Finally, NK cells toxicity experiment can better display effect on target cell killing effect of NK cells.Section1Polymorphisms of KIR in PTB patients with HBVObjectiveTo explore the association of KIR gene polymorphisms with susceptibility to PTB patients with HBV.Methods1. Subjects:The140samples analyzed for this study comprised40samples from PTB patients with HBV and100controls recruited from Shandong Provincial Hospital affiliated to Shandong University and Shandong Chest Hospital from2012to2013.2. Refer to Chapter1.ResultsThe carriage frequencies of KIR2DL5,2DS1,3DS1were higher in patients than controls. KIR2DL5:70%vs.50%, P=0.031;2DS1:65%vs.26%,P<0.001;2DS3:67.5%vs.48%,P=0.037。ConclusionIt could be suggested that KIR2DL5,2DS1,3DS1were susceptive genes for PTB patients with HBV. Section2Expression of CD158f in NK cellsObjectiveWe also analyzed the characteristics of CD158f molecules in peripheral blood NK cells of patients by using Flow cytometry.Methods1. Subjects:Refer to Section1of Chapter3. We selected KIR2DL5+subjects.2. Flow cytometry:NK cells were stained with10μl anti-human CD3,10μl anti-human CD16CD56,10μl anti-human CD158f to determin the expression of CD158f on NK cells. Isotype controls were used.3. LDH-Cytotoxicity Assay.4. Statistical analysis:Analysis was performed by Statistical Package for Social Sciences Version16.0(SPSS, Chicago, IL, USA). A t test was used for comparing quantitative variables of two different groups. P<0.05were considered statistically significant.ResultsCD158f were expressed on CD3-CD56+NK cells of the two groups. The expression level of CD158f was increased in patients compared with healthy controls.ConclusionCD158f were expressed on CD3-CD56+NK cells of the two groups. The expression level of CD158f was increased in patients compared with healthy controls. |
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