BACKGROUND: HLA-B27 typing has long been performed by the microlymphocytotoxicity method(MCT) but the flow cytometry method(FCM) was introduced several years ago. False positive results due to the HLA-B7 cross reactive groups(CREG) were the main drawback of the serologic method. The authors performed polymerase chain reaction-sequence specific primer(PCR-SSP) test for HLA-B27 to compare the results with serologic methods. METHODS: PCR-SSP test for HLA-B27 was performed on four hundred forty one samples. Three hundred twenty eight samples were tested by MCT and one hundred thirteen samples by FCM. PCR-SSP for HLA-B27 subtyping or Amplification Refractory Mutation System-PCR(ARMS-PCR) for HLA-B typing was performed on twenty four discrepant samples. RESULTS: The concordance rate between MCT and PCR-SSP was 92.9%(305/328) and the concordance rate between FCM and PCR-SSP was 99.1%(112/113). Twenty four(5.4%) out of four hundred forty one samples showed discrepancy between serologic methods and PCR-SSP method. Fourteen out of one hundred MCT positive samples and only one out of forty FCM positive samples showed negative by PCR-SSP. Nine samples showed PCR-SSP positive and MCT negative. CONCLUSIONS: The false positive rate of MCT was quite high and there were some false positive and negative results by PCR-SSP, too. From the above findings, we suggest that FCM is the most accurate method for HLA-B27 typing in those laboratory equipped with flow cytometry.
Flow Cytometry* ; HLA-B Antigens ; HLA-B27 Antigen* ; HLA-B7 Antigen

Antigen presenting is the initial step of the immune responses. In order to verify that human bronchial epithelial cells (HBECs) can express antigen presentation molecules, which can be modulated by intrapulmonary regulatory peptides, the present study was designed to examine the expressions of human leukocyte antigen DR (HLA-DR), CD80 and CD86 in resting or ozone-stressed HBECs by using immunocytochemistry and flow cytometry analysis. The results showed that HBECs expressed HLA-DR, CD80 and the expressions of HLA-DR and CD80 molecules were down-regulated under ozone stress. While VIP, P3513 and CGRP upregulated the expression of HLA-DR in resting or ozone-stressed HBECs, they had different effects on CD80 expression. VIP did not influence the expression of CD80 under resting state, but increased the expression of CD80 under ozone stress. CGRP decreased CD80 expression in resting HBECs, but increased CD80 expression in ozone-stressed HBECs. P3513 increased CD80 expression in resting HBECs, but decreased CD80 expression in ozone-stressed HBECs. The expression of CD86 was absent in resting or ozone-stressed HBECs. The results obtained demonstrate that HBECs have the capability to act as antigen presenting cells and the expression of HLA-DR and costimulatory molecules can be modulated by intrapulmonary regulatory peptides.
Antigen-Presenting Cells ; metabolism ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Bronchi ; cytology ; Calcitonin Gene-Related Peptide ; pharmacology ; Epithelial Cells ; metabolism ; HLA-DR Antigens ; metabolism ; Humans ; Ozone ; adverse effects ; Vasoactive Intestinal Peptide ; pharmacology

OBJECTIVETo investigate the effects of mobile phone 1800 MHz electromagnetic fields (EMF) on the surface markers and the functions of human dendritic cells (DC).

METHODSHuman DCs were exposed to intermittent 5 min on/10 min off EMF with specific absorption rates (SAR) 4 W/kg for 0 h, 1 h, 12 h or 24 h, respectively. FACS analysis was used to detect the positive percentage of DC surface markers including HLA-DR and co-stimulatory molecules such as CD80, CD86, CD40 and CD11c. CCK-8 kit was adopted to examine the function of allo-mixed lymphocyte reaction (allo-MLR) of DC, and enzyme linked immunosorbent assay (ELISA) to identify the levels of IL-12p70 and TNF-alpha secreted by DC.

RESULTCompared with the sham radiation group, after exposure to the electromagnetic fields for 1 h, 12 h, or 24 h, HLA-DR, CD80,CD86 and CD40 were all declined except CD11c. The ability of DC allo-MLR in each exposure group was decreased significantly (P<0.05), especially in the 24 h exposure group. However, the secreted levels of IL-12p70 and TNF-alpha of DC in each exposure group remained no changed.

CONCLUSIONThe study showed that EMF exposure could down-regulate the surface molecules and stimulation ability of human DC.

B7-1 Antigen ; B7-2 Antigen ; immunology ; Biomarkers ; analysis ; CD11c Antigen ; immunology ; Cell Phone ; Cells, Cultured ; Dendrites ; pathology ; Dendritic Cells ; metabolism ; physiology ; radiation effects ; Electromagnetic Fields ; HLA-DR Antigens ; analysis ; Humans ; Interleukin-12 ; immunology

Adult ; Antigens, CD ; analysis ; B7-1 Antigen ; analysis ; B7-2 Antigen ; Dendritic Cells ; immunology ; Female ; HLA-DR Antigens ; analysis ; Hepatitis B, Chronic ; drug therapy ; immunology ; Humans ; Immunophenotyping ; Intercellular Adhesion Molecule-1 ; analysis ; Interferon-alpha ; therapeutic use ; Male ; Membrane Glycoproteins ; analysis

OBJECTIVE: To explore the effect of unmethylated CpG motif containing oligodeoxynucleotides (CpG ODN) on the function of dendritic cells (DCs) in patients with chronic hepatitis B (CHB). METHODS: DCs were obtained from peripheral blood mononuclear cells (PBMCs) of 15 CHB patients, 12 hepatitis B virus (HBV) carriers, and 10 healthy controls. The expressions of HLA-DR, CD80, and CD86 on DCs were determined by fluorescence activated cell sorting (FACS). The IL-12 level in supernatant of the culture medium was measured by ELISA, and the morphological changes of DCs were observed under transmission electron microscope. RESULTS: Compared with the controls, DCs stimulated with CpG ODN represented enrichment in cell surface protrusions and rough endoplasmic reticulum, decreased or disappeared vacuole. The expressions of HLA-DR, CD86, and CD80 were much higher in DCs stimulated with CpG ODN than those in complete medium control (P<0.05). When culturing in complete medium, the expressions of HLA-DR, CD86, and CD80 were much lower in CHB patients and HBV carriers than healthy controls (P<0.05). The expressions of HLA-DR and CD86 stimulated with CpG ODN were much lower in CHB patients than HBV carriers and healthy controls (P<0.05). The expressions of CD80 were much lower in CHB patients and HBV carriers than healthy controls (P<0.05). The levels of IL-12 were much higher in DCs stimulated with CpG ODN than that in complete medium controls (P<0.05). The levels of IL-12 in complete medium or medium added with CpG ODN were much lower in CHB patients and HBV carriers than in healthy controls (P<0.05). CONCLUSION CpG ODN could significantly promote the maturation of dendritic cells in peripheral blood in CHB patients.
Adult ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Carrier State ; immunology ; Case-Control Studies ; CpG Islands ; Dendritic Cells ; drug effects ; immunology ; Female ; HLA-DR Antigens ; metabolism ; Hepatitis B, Chronic ; immunology ; Humans ; Male ; Oligodeoxyribonucleotides ; pharmacology

BACKGROUND: Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. METHODS: We compared four storage methods: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. RESULTS: Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. CONCLUSIONS: The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.
Blood Platelets/metabolism ; Erythrocytes/metabolism ; *Flow Cytometry ; Freezing ; HLA-B27 Antigen/*blood ; HLA-B7 Antigen/blood ; Histocompatibility Testing ; Humans ; Leukocytes, Mononuclear/metabolism ; Real-Time Polymerase Chain Reaction ; Spondylarthropathies/diagnosis ; Temperature

BACKGROUND: Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. METHODS: We compared four storage methods: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. RESULTS: Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. CONCLUSIONS: The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.
Blood Platelets/metabolism ; Erythrocytes/metabolism ; *Flow Cytometry ; Freezing ; HLA-B27 Antigen/*blood ; HLA-B7 Antigen/blood ; Histocompatibility Testing ; Humans ; Leukocytes, Mononuclear/metabolism ; Real-Time Polymerase Chain Reaction ; Spondylarthropathies/diagnosis ; Temperature

BACKGROUND: HLA-B27 is associated with an increased incidence of specific spondyloarthropathies(SpA), most notably ankylosing spondylitis(AS). I evaluated the cases referred for HLA-B27 antigen using flowcytometry (FCM) to find the clinical characteristics of the patients and the diagnostic utilities of median fluorescence intensity (MFI) in HLA-B27 program. METHODS: I evaluated 443 subjects of HLA-B27 cases using FACScan flowcytometry, consisted with software for automated calibration and analysis, calibration beads, and the anti-HLA- B27 fluorescein isothiocyanate (FITC)/anti-CD3 phycoerythrin (PE) monoclonal antibodies (all from Becton Dickinson, San Jose, CA). RESULTS: Of the total 443 cases, the positive rate in male cases was 44% (132/300) and it was higher than that of female cases (22.4%, 32/143). The gating procedure was failed in one sample of 443 (0.23%). The positive rates in each diagnostic criteria were as follows; AS 61.6%, gout 20.0%, herniated intervertebral disc 20%, lower back pain 25.6%, polyarthritis 16.0%, psoriatic arthritis 20.0%, rheumatoid arthritis 28.3%, reactive arthritis 26.9%, SpA, undifferentiated 31.8% and uveitis/iritis 23.8%. In AS group, 89 cases (95.7%) showed MFI values higher than 150. CONCLUSION: About 62% of AS group showed HLA-B27 positivity using FCM and the positive rates of other diseases group in SpA categories were around 20-30%. If we considered MFI value 150 as differential value, about 95% of HLA-B27 positive AS cases might not need further confirmatory study to differentiate HLA-B7.
Antibodies, Monoclonal ; Arthritis ; Arthritis, Psoriatic ; Arthritis, Reactive ; Arthritis, Rheumatoid ; Calibration ; Female ; Fluorescein ; Fluorescence ; Gout ; HLA-B27 Antigen* ; HLA-B7 Antigen ; Humans ; Incidence ; Intervertebral Disc ; Low Back Pain ; Male ; Phycoerythrin ; Spondylarthropathies ; Spondylitis, Ankylosing

In order to study how to induce tolerogenic dendritic cells in vitro and its mechanism, the K562 cells transduced with HLA-G construct were used to co-culture with DC. Then their related immunological changes, such as membrane molecules CD80, CD86, ILT3 and ILT4 expression levels were detected by flow cytometry. Allogeneic proliferation of peripheral blood mononuclear cells (PBMNC) was detected by mixed lymphocyte reaction. The results showed that CD80 and CD86 expressions on DC were downregulated, while ILT3 and ILT4 expressions were upregulated after co-culturing with K562-HLA-G cells. The DCs were less able to stimulate the allogenic PBMNC. It is concluded that the membrane-bound HLA-G can upregulate expression of inhibitory receptors ILT3 and ILT4, inducing tolerogenic DC in vitro, which may provide a novel strategy for transplant tolerance induction.
B7-1 Antigen ; biosynthesis ; B7-2 Antigen ; biosynthesis ; Coculture Techniques ; Dendritic Cells ; cytology ; immunology ; HLA Antigens ; immunology ; HLA-G Antigens ; Histocompatibility Antigens Class I ; immunology ; Humans ; Immune Tolerance ; immunology ; K562 Cells ; Lymphocyte Culture Test, Mixed ; Membrane Glycoproteins ; biosynthesis ; Receptors, Cell Surface ; biosynthesis ; Receptors, Immunologic ; biosynthesis ; T-Lymphocytes ; immunology ; Transfection

OBJECTIVETo define roles of B7-1 co-stimulation factor expressed in human malignant cell lines in mediating anti-tumor T cell immune responses.

METHODSExamining human leucocyte antigen (HLA) and B7 expressions on 8 human blood malignancies cell lines by flow cytometry. Transfecting B7-1 gene to B7-1 negative (B7(-)) Raji and B7(-) Jurkat cell lines by liposome, and comparing the potencies of blood malignant cell lines in the induction of T cell activation by examination of T cell cytokine mRNAs before and after transfection using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).

RESULTSHigh level of HLA I and II molecules were expressed in most human blood malignant cell lines examined, and the co-stimulatory factor B7-2 was also highly expressed. In contrast, another member of B7 family: B7-1 was either not expressed or very limitedly expressed in most of these hematopoietic malignant cell lines. Most importantly, transfection of B7-1 gene to B7(-). Raji and B7(-). Jurkat cell lines made these cell lines better antigen presenting cells for stimulation of anti-tumor T cell activation, which was demonstrated by up regulation of expression of T cell cytokines IL-2, IL-4 and INF-gamma mRNAs after incubation of these tumor cells with T cells for 24 h.

CONCLUSIONSB7 co-stimulation plays an important role in anti-tumor immunity. Transfection of B7-1 gene to the human hematopoietic malignant cell lines that are deficient in the B7-1 expression empowers their antigen presentation potency for activation of anti-tumor T cells. Our results suggested that repairing the deficiency of B7-1 co-stimulatory pathway in tumor cells might be a novel immunotherapeutic approach for human hematopoietic malignancies.

Antigen Presentation ; Antigens, CD ; analysis ; B7-1 Antigen ; analysis ; physiology ; B7-2 Antigen ; Cytokines ; genetics ; Flow Cytometry ; HLA-DR7 Antigen ; physiology ; Hematologic Neoplasms ; immunology ; Humans ; K562 Cells ; Lymphocyte Activation ; Membrane Glycoproteins ; analysis ; RNA, Messenger ; analysis ; T-Lymphocytes ; immunology ; Transfection ; U937 Cells