Peripheral Blood Of Normal Controls Research Articles

Abstract 18 Developing a Potency Assay for Cord Tissue MSCs

IntroductionThe potency of mesenchymal stromal cells (MSCs) can be assessed by their ability to suppress proliferation of third party stimulated peripheral blood T cells. This correlates with decreased cellular production of IL-2Ra.ObjectiveWe optimized a potency assay for human cord tissue MSCs (hCT-MSC) in clinical trials for subjects with neuroinflammation. Originally, this T cell suppression assay required radioactive tritiated thymidine, rendering the assay difficult to use in a quality control laboratory for product release. Accordingly, we are developing a modified assay that eliminates the tritiated thymidine and instead tests for production of IL-2Rα in harvested supernatants.MethodsThe modified assay utilizes freshly thawed hCT-MSCs plated in a 96-well Corning CellBIND plate in Fuji Prime-XV XSFM MSC expansion medium at three concentrations. The hCT-MSCs are incubated 1-6 days at 37°C with humidified 5% CO2 until 80%-100% confluent. At this point, three different, previously qualified, normal control peripheral blood mononuclear cells (PBMCs) are suspended in RPMI 1640 with HEPES medium and 10% fetal bovine serum (FBS) with and without Dynabead (Human T-Activator CD3/CD28). After 4 additional days in culture, 75 µL of supernatant is removed and frozen at –80°C for future assaying of IL-10, TNFα, IFNg, and IL-2Ra using the Ella (Bio-Techne). IL-2Ra was determined to have the best correlation with the original method of suppression of proliferation. In the original radioactivity method after 4 days, the cells were pulsed with tritiated thymidine, harvested 6-10 hours after pulsing, and counted in the MicroBeta2 counter.ResultsSeventeen hCT-MSC lines were then tested in both assays. The passing criteria was >70% suppression in the radioactivity assays or >70% decrease in IL-2Ra production in the Ella assay. All 17 hCT-MSC lots passed in the radioactivity-based assay, while 16/17 lines passed using the IL-2Rα assay. Subsequently, the number of PBMCs plated per well and the number of days the PBMCs were in culture were optimized to maximize production of IL-2Rα.DiscussionWe developed a potency assay for MSCs that eliminates the use of radioactive reagents and usable as a release assay in manufacturing of hCT-MSC.

Identification of autophagy-related genes as potential biomarkers for type 1 diabetes mellitus.

BackgroundType 1 diabetes mellitus (T1DM) is a metabolic disease in which the autoimmune destruction of pancreatic islet β-cells occurs. This study sought to investigate the role of autophagy-related genes and immune cells in the development of T1DM.MethodsWe acquired the raw gene expression profiles of 302 T1DM and 422 normal control peripheral blood samples from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified using the Limma package, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. The Search Tool for the Retrieval of Interacting Genes/Proteins (https://string-db.org/) and Cytoscape autophagy genes were intersected with the DEGs for the immune cell analysis and the correlation analysis.ResultsA total of 568 DEGs were identified in the T1DM and normal samples, of which 301 were upregulated and 267 were downregulated. The results of the functional and pathway enrichment analyses showed that the DEGs were closely associated with autophagy and immunity. Member RAS oncogene family (RAB11A), protein tyrosine phosphatase non-receptor type 11, lamin A/C, heat shock protein70, heat shock protein family A member 4, cluster of differentiation 8A, caspase 3 (CASP3), exportin 1, proto-oncogene, non-receptor tyrosine kinase, SMAD family member 4, and sirtuin 1 (SIRT1) were located at the center of the protein-protein interaction network as the core genes. The peripheral blood T cells were more elevated in the T1DM subjects than the normal subjects. RAB11A, CASP3, and SIRT1 are autophagy-associated genes. RAB11A and CASP3 were positively correlated with most immune cells, while SIRT1 was negatively correlated with most immune cells.ConclusionsAutophagy-related genes (i.e., RAB11A, CASP3, and SIRT1) and immune cells (i.e., T and B cells) may play important regulatory roles in the development of T1DM. Our findings provide novel insights into and potential targets for T1DM prediction and treatment.

The differentially expressed circular ribonucleic acids of primary hepatic carcinoma following liver transplantation as new diagnostic biomarkers for primary hepatic carcinoma.

Recent studies have shown that circular ribonucleic acids have differential expression in some diseases. This study compared the expression levels of five circular ribonucleic acids between patients of primary hepatic carcinoma following liver transplantation and healthy individuals for searching a new diagnostic biomarker about primary hepatic carcinoma. We chose differentially expressed targeted circular ribonucleic acids according to fold change ≥2.0 or ≤-2.0 between circular ribonucleic acids microarray of perioperative liver transplantation and normal controls. Then we used the Arraystar home-made micro-ribonucleic acid target prediction software based on TargetScan and miRanda to predict circular ribonucleic acid/micro-ribonucleic acid interactions. And we assess the expression levels of hsa_circ_100571, hsa_circ_400031, hsa_circ_102032, hsa_circ_103096, and hsa_circ_102347 in the peripheral blood of normal controls and liver transplantation patients before transplantation and on the first, third, and seventh days after transplantation by real-time quantitative polymerase chain reaction. We chose five circular ribonucleic acids, two of which have been correlated with micro-ribonucleic acid-related carcinoma recurrence after liver transplantation, hepatocellular carcinoma and analyzed their expression with 2-△△Ct method. The expression level of hsa_circ_100571 and hsa_circ_400031 on day 1 after liver transplantation was higher than pre-transplantation (p < 0.01), and these levels showed a declining trend on post-transplantation. The expression level of hsa_circ_102032 and hsa_circ_103096 on day 1 after liver transplantation was lower than pre-transplantation (p < 0.01) and decreased on post-transplantation. There were the significantly different expressions between the post-transplantation day 7 and normal control (p < 0.01). The expression level of hsa_circ_102347 on day 1 after liver transplantation was lower than pre-transplantation (p < 0.01). This expression showed a declining trend on post-transplantation, and the postoperative day 7 level was similar to normal control (p > 0.05). Five types of circular ribonucleic acid-related micro-ribonucleic acids had varying degrees of upregulation and downregulation between perioperative transplantation of primary hepatic carcinoma patients and normal controls; the hsa_circ_102347 is most likely to have association with primary hepatic carcinoma.

Increased regulatory T cells in acute lymphoblastic leukaemia patients

Introduction: Regulation in adaptive immune response balances a fine line that prevents instigation of self-damage or fall into unresponsiveness permitting abnormal cell growth. Mechanisms that keep this balance in check include regulatory T cells (Tregs). Tregs consist of a small but heterogeneous population, which may be identified by the phenotype, CD3+CD4+CD25+CD127−. The role of Tregs in pathogenesis of cancers is thus far supported by evidence of increased Tregs in various cancers and may contribute to poorer prognosis. Tregs may also be important in acute leukaemias.Objective: A review of the literature on Tregs in acute leukaemias was conducted and Tregs were determined in B-cell acute lymphoblastic leukaemias (ALLs).Results: Studies on Tregs in B-cell ALL are few and controversial. We observed a significantly increased percentage of Tregs (mean±SD, 9.72 ± 3.79% vs. 7.05 ± 1.74%; P = 0.047) in the bone marrow/peripheral blood of ALL (n = 17) compared to peripheral blood of normal controls (n = 35). A positive trend between Tregs and age (R = 0.474, P = 0.055, n = 17) implicates this factor of poor prognosis in B-cell ALL.Discussion: Tregs in cancer are particularly significant in immunotherapy. The manipulation of the immune system to treat cancer has for a long time ignored regulatory mechanisms inducible or in place. In lymphoma studies, tumour-specific mechanisms that are unlike conventional methods in the induction of Tregs have been hypothesized. In addition, tumour-infiltrating Tregs may present different profiles from peripheral blood pictures. Tregs will continue to be dissected to reveal its mysteries and their impact on clinical significance.

Increased regulatory T cells in acute lymphoblastic leukemia patients

IntroductionRegulation in adaptive immune response balances a fine line that prevents instigation of self-damage or fall into unresponsiveness permitting abnormal cell growth. Mechanisms that keep this balance in check include regulatory T cells (Tregs). Tregs consist of a small but heterogeneous population which may be identified by the phenotype, CD3+CD4+CD25+CD127−. Role of Tregs in pathogenesis of cancers is thus far supported by evidence of increased Tregs in various cancers and may contribute to poorer prognosis. Tregs may also be important in acute leukemias.ObjectiveA review of the literature on Tregs in acute leukemias was conducted and Tregs were determined in B-cell acute lymphoblastic leukemias (ALLs).ResultsStudies on Tregs in B-cell ALL are few and controversial. We observed a significantly increased percentage of Tregs (mean ± SD, 9.72 ± 3.79% vs. 7.05 ± 1.74%; P = 0.047) in the bone marrow/peripheral blood of ALL (n = 17) compared to peripheral blood of normal controls (n = 35). A positive trend between Tregs and age (R = 0.474, P = 0.055, n = 17) implicates this factor of poor prognosis in B-cell ALL.DiscussionTregs in cancer are particularly significant in immunotherapy. The manipulation of the immune system to treat cancer has for a long time ignored regulatory mechanisms inducible or in place. In lymphoma studies tumor-specific mechanisms that are unlike conventional methods in the induction of Tregs have been hypothesized. In addition, tumor-infiltrating Tregs may present different profiles from peripheral blood pictures. Tregs will continue to be dissected to reveal their mysteries and their impact on clinical significance.

Expression and significance of vitamin D and its receptor mRNA in the peripheral blood of initial immune thrombocytopenic patients

To investigate the effect of vitamin D in pathogenesis and clinical treatment of patients with immune thrombocytopenic (ITP), ELISA was used to detect the level of 25-hydroxylate vitamin D3[25(OH)D3] and 1,25-dihydroxyvitamin D3[1,25(OH)2D3] in peripheral blood from 45 ITP patients and 30 normal controls. Vitamin D receptor (VDR) mRNA expression was detected by RT-PCR. The results showed that the levels of 25(OH)D3 (10.6 ± 7.7 ng/ml) and 1,25(OH)2D3 (69.9 ± 29.0 pg/ml) in peripheral blood of the initial ITP patients were lower than those in peripheral blood of normal controls (13.7 ± 9.1 ng/ml, 87.3 ± 19.9 pg/ml) (P < 0.05). The expression of VDR gene obviously increased in peripheral blood of initial ITP patients (1.588 ± 0.127), as compared with that in peripheral blood of normal controls (1.055 ± 0.734) (P < 0.05). It is concluded that vitamin D level and its receptor expression may play an important role in ITP, and vitamin D and its similarities may be a new agent to treat patients with ITP.

CD161 Expression on Peripheral Blood CD4+CD39+ T Cell Populations is Associated With Immune Deviation in IBD and TH17 Expansion

Background The balance between T regulatory cells (Treg) and T helper type 17 (Th17) cells appears important in modulating immune responses in inflammatory bowel disease. CD39 is an immune cell ectonucleotidase, expressed in lipid rafts, which generates immune suppressive adenosine. Genetic polymorphisms of CD39 are noted in IBD and the ectoenzyme has been validated as a marker of memory type Treg. CD161 (killer cell lectin subfamily B, member 1 or NKR-P1A) is a relatively specific surface marker for human Th17 cells and is linked to activation of acid sphingomyelinase that might in turn impact CD39 bioactivity. It is unknown whether there are T cell populations in IBD that express both markers and the putative relevance is unexplored. Aims: To identify dual expression of CD39 and CD161 in blood T cells from patients with IBD, and explore the role of CD161 in regulating immune deviation of CD4+CD39+ T cell populations. Methods The percentage of CD39+CD161+ CD4 T cells in peripheral blood of patients with active or inactive Crohn's disease was determined by FACS. Four phenotypic subtypes of peripheral blood CD4 T cells including CD39+CD161+, CD39+CD161-, CD39-CD161+, and CD39-CD161from healthy donors were sorted by FACS, and stimulated respectively by anti-CD3/CD28 antibodies with with TGF-beta and IL-2 (for Treg cell expansion) or IL-6, IL-23, IL-1beta and TGFbeta (for Th17 cell expansion) for 3 days, and intracellular IL-17 and INF-gamma or FOXP3 were detected by FACS. These four subgroups were treated by ATP or NAD+ for 1 hour, and stained by Annexin V to detect early events of P2X7-mediated apoptosis. Result The percentage of CD39+CD161+ CD4 T cells in peripheral blood of normal controls, active and inactive Crohn's disease patients was 1.26±0.73, 4.55±2.39 and 0.95±0.52%, respectively (p=0.01, for active disease vs. both controls and inactive Crohn's disease). After 3 days, Treg expansion, of FOXP3-positive cells within the CD39+CD161+ group, was less than from the CD39+CD161group (p=0.01). After 3 days In Vitro expansion, the percentages of intracellular IL-17-positive and/or IFN-gamma-positive cells in CD4+CD39+CD161+ populations were higher than other three groups (p=0.01). However, CD39+CD161+ CD4 T cells also exhibited highest levels of NAD+-induced apoptosis (p=0.05 vs. other groups). Conclusions Peripheral blood CD4+CD39+CD161+ T cells are increased in active Crohn's disease. CD161 expression impacts the reciprocal balance of Treg and Th17 cells generated from CD4+CD39+ cells.

The Nuclear Protein HMGB1 is Increased in the Stools of IBD Patients and Appears to Be a Novel Marker of Intestinal Inflammation

Background The balance between T regulatory cells (Treg) and T helper type 17 (Th17) cells appears important in modulating immune responses in inflammatory bowel disease. CD39 is an immune cell ectonucleotidase, expressed in lipid rafts, which generates immune suppressive adenosine. Genetic polymorphisms of CD39 are noted in IBD and the ectoenzyme has been validated as a marker of memory type Treg. CD161 (killer cell lectin subfamily B, member 1 or NKR-P1A) is a relatively specific surface marker for human Th17 cells and is linked to activation of acid sphingomyelinase that might in turn impact CD39 bioactivity. It is unknown whether there are T cell populations in IBD that express both markers and the putative relevance is unexplored. Aims: To identify dual expression of CD39 and CD161 in blood T cells from patients with IBD, and explore the role of CD161 in regulating immune deviation of CD4+CD39+ T cell populations. Methods The percentage of CD39+CD161+ CD4 T cells in peripheral blood of patients with active or inactive Crohn's disease was determined by FACS. Four phenotypic subtypes of peripheral blood CD4 T cells including CD39+CD161+, CD39+CD161-, CD39-CD161+, and CD39-CD161from healthy donors were sorted by FACS, and stimulated respectively by anti-CD3/CD28 antibodies with with TGF-beta and IL-2 (for Treg cell expansion) or IL-6, IL-23, IL-1beta and TGFbeta (for Th17 cell expansion) for 3 days, and intracellular IL-17 and INF-gamma or FOXP3 were detected by FACS. These four subgroups were treated by ATP or NAD+ for 1 hour, and stained by Annexin V to detect early events of P2X7-mediated apoptosis. Result The percentage of CD39+CD161+ CD4 T cells in peripheral blood of normal controls, active and inactive Crohn's disease patients was 1.26±0.73, 4.55±2.39 and 0.95±0.52%, respectively (p=0.01, for active disease vs. both controls and inactive Crohn's disease). After 3 days, Treg expansion, of FOXP3-positive cells within the CD39+CD161+ group, was less than from the CD39+CD161group (p=0.01). After 3 days In Vitro expansion, the percentages of intracellular IL-17-positive and/or IFN-gamma-positive cells in CD4+CD39+CD161+ populations were higher than other three groups (p=0.01). However, CD39+CD161+ CD4 T cells also exhibited highest levels of NAD+-induced apoptosis (p=0.05 vs. other groups). Conclusions Peripheral blood CD4+CD39+CD161+ T cells are increased in active Crohn's disease. CD161 expression impacts the reciprocal balance of Treg and Th17 cells generated from CD4+CD39+ cells.

Cytogenetics and spectral Karyotyping analysis in CLL Patients: Correlation with FISH and ZAP70 Expression

AbstractAbstract 4844Cytogenetic abnormalities play an important role as prognostic factors in CLL. However, due the low mitotic index of CLL B cells in vitro, analysis of a set of subjects for the most commonly known aberrations is usually done by FISH on interphase cell. The objectives of this investigation were the use of the oligonucleotide DSP30 in combination with IL-2, as a B-cell mitogen for cytogenetic investigation in CLL and correlation among the karyotype analysis obtained (G-banding + SKY), FISH profile from unstimulated cells, ZAP70 expression and stratification status for each patient. For metaphase induction, peripheral blood mononuclear cells were cultured in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and IL-2. Additionally, one set of cell culture was performed for each patient without any stimulant agent, for FISH analysis. The FISH panel included probes for the detection of +12, and deletions of 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). The cut off levels for trissomy 12 (>2%), del(13q) (>2.4%), del(11q23.3) (>2.5%), del(17p13.1) (>3%) were established according to the iFISH patterns observed in a group of 4 age and sex-matched normal control peripheral blood samples studied with the same probes. Spectral karyotype analysis (SKY) was performed, according manufactures' instruction. The ZAP70 profile was obtained by flow cytometry analysis. In concordance with literature, the cut off value adopted for ZAP70 was 20%. In a group of 64 subjects studied, the cytogenetic analysis showed chromosomal aberrations in 52 patients (81.25%). The profile of abnormalities observed were del(6)(q24), +8(x2), del(11)(q13~q23), +12, +15(x2), del(12)(p13), -17, +21, +19, +18, del(13)(q31), del(14)(q24), del(17)(p13), +21, +4, +5, +11, t(1;12)(q31;p13), t(11;13)(q23;q12), t(15;18)(q11.1;q11), t(1;10)(p22;p14), t(14;22)(q32;q11), t(17;18)(q10;q10), t(9;13)(q21;q22), t(10;13)(q26;q14), t(9;12)(q12;p11), t(X;12)(p11.2;q24). Twelve patients exhibited normal karyotype (18.75%). All subjects presenting chromosomal abnormalities, by using G-banding analysis, were confirmed by SKY. In patients with normal cytogenetic, SKY analysis did not identified any criptic abnormality. Cells without any stimulant agent showed concordance with the cytogenetic profile obtained (FISH analysis). The ZAP70 expression did not show any relationship between the group of patients with chromosomal abnormalities and the group with normal karyotype. The use of the immunostimulatory oligonucleotide DSP30 in combination with IL-2 showed to be effective to induce cell cycle progression of CLL cells in vitro than others mitogens. Cytogenetic aberrations detected by G-banding in addition to FISH analysis were heterogeneous. The limited spectrum of chromosomal abnormalities seen by FISH analysis may contribute to underestimate the prognostic value, where others abnormalities may be present in patient's karyotype. These results indicate that classical cytogenetic analysis can contribute to the stratification of different subsets of CLL patients with complex karyotype associated with poor prognosis.Financial support: FAPESP (Proc. 07/52462-7). Disclosures:No relevant conflicts of interest to declare.

Tumor-Derived Microvesicles From Sera of Patients with Acute Myeloid Leukemia Suppresses NK Cell Cytotoxicity Via Transforming Growth Factor Beta-1.

Abstract 2678Poster Board II-654Natural killer (NK) cell cytotoxicity in patients with acute myeloid leukemia (AML) is significantly decreased relative to that in normal controls (NC). However, the mechanisms responsible for low NK cell activity in AML are not known. We considered the possibility that tumor-cell-derived microvesicles (MV) mediate suppression of NK cells. MV originate from the endosomal compartment of activated normal and neoplastic cells. Evidence suggests that tumor-derived MV exert detrimental effects on cells of the immune system and may play a role in tumor progression. To determine their contribution to immune suppression in AML, MV were isolated from sera of patients newly diagnosed with AML prior to any treatment and used to evaluate MV-mediated NK cell suppression. The protein content of MV isolated using exclusion chromatography and ultracentrifugation from sera of 19 AML patients was significantly higher than that of MV isolated from sera of 25 NC (75μg±12/mL vs 1.2μg±0.4/mL, p<0.001 ). MV from AML patients were positive for membrane-associated TGFb-1 and FasL in Western blots, whereas no TGFb-1 or FasL was detected in MV from NC. For functional assays, NK cells sorted from peripheral blood of NC were cultured with MV isolated from sera of the AML patients. A significant decrease in NK cell cytotoxicity was observed after co-incubation with MV (2412 LU before vs 1640 LU after, p<0.002). Concomitantly, a decrease in the expression of the NK cell activating receptor, NKG2D, was observed (57% before vs 38% after, p<0.001). The addition of TGFb1-neutralizing antibody abrogated the effects of MV on the NK cell cytotoxicity and receptor expression. The increased levels in sera of AML patients of MV mediating potent NK cell suppression is likely to compromise anti-tumor immune responses. Therefore, modulation of the levels and functions of MV might provide new immunotherapeutic approaches in AML. Disclosures:No relevant conflicts of interest to declare.

Spectral Karyotyping (SKY) ANALYSIS APPLIED to CHRONIC LYMPHOCYTIC LEUKEMIA CELLS Immunostimulated with the COMBINATION DSP30/IL-2.

Abstract 4692Cytogenetic abnormalities play an important role as prognostic factors in CLL. The immunostimulatory oligonucleotide DSP30 in combination with IL-2 is an easy and efficient stimulus in metaphase generation for chromosomal banding. This technique allows a more comprehensive chromosome analysis compared to FISH. On the other hand, spectral karyotyping (SKY) analysis, a recent molecular cytogenetic tool for the screening of the entire genome, has been shown to provide additional chromosome information. By using a combination of molecular cytogenetics strategies, the goal of this investigation was to use the SKY to identify masked chromosomal abnormalities in CLL cells stimulated by the combination of DSP30 and IL-2. In addition we compared the cytogenetic profile obtained (DSP30/IL-2) with FISH analysis from unstimulated cells and ZAP70 expression for each patient. For metaphase induction, peripheral blood mononuclear cells were cultured in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and IL-2. One extra set of cell culture without any stimulant agent for iFISH analysis was performed for each patient. The iFISH panel included probes for the detection of +12, and deletions of 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). The cut off levels for trissomy 12 (>2%), del(13q) (>2.4%), del(11q23.3) (>.5%), del(17p13.1) (>3%) were established according to the iFISH patterns observed in a group of 4 age and sex-matched normal control peripheral blood samples studied with the same probes. Spectral karyotype analysis (SKY) was performed, according manufactures' instruction, in all patients. The ZAP70 expression was determined by flow cytometry analysis and the cut off value was 20%. In a group of 35 subjects studied, the cytogenetic analysis with DSP30/IL-2 showed chromosomal aberrations in 27. The following abnormalities were observed: +4, +5, +8(x2), +11, +12, +15(x2), -17, +18, +19, +21, del(6)(q24), del(11)(q13∼q23), del(12)(p13), del(13)(q31), del(14)(q24), del(17)(p13), t(1;12)(q31;p13), t(11;13)(q23;q12), t(15;18)(q11.1;q11), t(1;10)(p22;p14), t(14;22)(q32;q11), t(17;18)(q10;q10), t(9;13)(q21;q22), t(10;13)(q26;q14), t(9;12)(q12;p11), t(X;12)(p11.2;q24). Eight patients exhibited normal karyotype. The SKY analysis confirmed the abnormalities previously seen by G-banding (DSP30/IL-2), however, did not identify any new abnormality in subjects with normal karyotype. The iFISH analysis agreed with the cytogenetic profile obtained with DSP30/IL-2. The ZAP70 expression did not show any relationship between the group of patients with chromosomal abnormalities and the group with normal karyotype. The use of the immunostimulatory oligonucleotide DSP30 in combination with IL-2 showed to be effective to induce cell cycle progression of CLL. Cytogenetic aberrations detected by G-banding in addition to FISH were heterogeneous. The limited panel used for iFISH analysis may contribute to underestimate the prognostic value, since others abnormalities may be present in patient's karyotype. In conclusion, SKY analysis did not reveal any masked abnormality beyond those showed by G-banding resolution. These results indicate that G-banding analysis (DSP30/IL-2) can contribute to the stratification of different subsets of CLL patients with complex karyotype associated with poor prognosis. Disclosures:No relevant conflicts of interest to declare.

Study on Dendritic Cells in Peripheral Blood of the Patients with Autoimmune Hemolytic Anemia/Evans Syndromes.

Objective To study the alteration of the numbers and the subsets of DCs in perpheral blood of the patients with AIHA/Evans syndromes and also the costimulation molecules on the surface of DCs.Methods Labeled with three-color immune monoclonal antibodies, total DC, pDC and mDC in the peripheral blood (PB) of 14 cases with AIHA and 21 normal controls were detected by Facs. The expressions of CD80, CD86 andCD40 on DCs in the PB of 14 cases with AIHA/Evans syndomes and 6 normal controls were detected.Results The percentage of the total DCs in the PB (DC/PBMNC) of AIHA/Evans syndromes cases((11.41±3.91)%)was significantly higher than that of in the normal controls((1.95±0.66)%) (t=2.384, p=0.032).mDC in the PB of the AIHA/Evans syndromes patients ((10.29±3.90)%) was significantly higher than that of controls((1.26±0.46)%)(t=2.297, p=0.038). But pDC in the PB of AIHA/Evans cases((1.12±0.68)%)was not different from that of controls((0.68±0.23)%)(t=0.612, p=0.55). The proportion of pDC to DC in the PB of the patients((21.26±5.99)%)was significant lower than that of normal controls((40±3.14)%)(t=2.77, p=0.012). Whereas, the proportion of mDC to DC in the PB of the patients((75.93±6.79)%)was higher than that of normal controls((59.99±3.23)%)(t=2.77, p=0.012). mDC in the PB of normal controls((1.26±0.46)%) was not different from their pDC((0.68±0.23)%). mDC of the patient with AIHA/Evans cases((10.29±3.90)%) was higher than their pDC((1.12±0.68)%)(t=2.316, p=0.036). CD80, CD86 and CD40 expressions on DCs in PB of AIHA/Evans syndromes patients were (7.63±4.71)%, (13.55±4.57)% and (1.68±1.04)% respectively. Those of normal controls were (1.24±0.70)%, (3.21±1.55)% and (0.28±0.16)% respectively. There was a significant difference of the expression of CD86 between the patients and control groups (t=2.142, p=0.048).Conclusions DCs, particularly mDCs, in the PB of AIHA/Evans syndromes patients significantly increased. The patients with AIHA/Evans syndromes have more activated DCs in their PB, on which costimulatory molecules, mainly CD86 are over expressed.

Identification of autoantigens in psoriatic plaques using expression cloning.

To search for autoantigens in psoriatic plaques, we screened cDNA libraries of plaque epidermis with psoriatic serum samples. This approach has been highly successful in identifying tumor antigens, but has not been widely applied to autoimmune disease. We identified 11 autoantigens including three with prominent reactivity and plausible disease relevance. These are keratin 13 (K13), heterogeneous nuclear ribonucleoprotein-A1 (hnRNP-A1), and a previously uncharacterized protein, FLJ00294. Serum antibody screening for these demonstrated reactivity in 40%, 38%, and 27% of psoriasis patients, respectively. Most positive samples reacted with all three, and we found that this was due to cross-reactivity among them. Enzyme-linked immunospot assay (ELISPOT) analysis of psoriatic peripheral blood T cells confirmed that these autoantigens are also recognized by T cells. This demonstrates that this is a feasible method to identify autoantigens in an autoimmune target tissue, and suggests that these antigens warrant further study in psoriasis. Furthermore, but peripheral blood of normal controls reacted to these autoantigens with essentially the same frequencies as patients, suggesting that psoriatics may have not only an immune system which is capable of reacting to certain autoantigens, but also to a skin immunoregulatory alteration which allows this normal reactivity to develop into abnormal inflammation.

Lunx Is a Superior Molecular Marker for Detection of Non-Small Lung Cell Cancer in Peripheral Blood

The clinical management of non-small cell lung cancer (NSCLC) would benefit greatly by a test that was able to detect small amounts of NSCLC in the peripheral blood. In this report, we used a novel strategy to enrich tumor cells from the peripheral blood of 24 stage I to IV NSCLC patients and determined expression levels for six cancer-associated genes (lunx, muc1, KS1/4, CEA, CK19, and PSE). Using thresholds established at three standard deviations above the mean observed in 15 normal controls, we observed that lunx (10 of 24, 42%), muc1 (5 of 24, 21%), and CK19 (5 of 24, 21%) were overexpressed in 14 of 24 (58%) peripheral blood samples obtained from NSCLC patients. Patients who overexpressed either KS1/4 (n = 2) or PSE (n = 1) also overexpressed either lunx or muc1. Of patients with presumed curable and resectable stage I to II disease (n = 7), at least one marker was overexpressed in three (43%) patients. In advanced stage III to IV patients (n = 17), at least one marker was overexpressed in 11 patients (65%). These results provide evidence that circulating tumor cells can be detected in NSCLC patients by a high throughput molecular technique. Further studies are needed to determine the clinical relevance of gene overexpression.

Amplification of mitochondrial DNA in acute myeloid leukaemia.

There is a long-standing interest in the possible role of mitochondria in malignancy. We sought to discover whether amplification of mitochondrial DNA (mtDNA) occurred in leukaemia, and found it was often remarkably amplified in the blast cells of acute myeloid leukaemia (AML). We used gene dosage experiments to quantify the amount of mtDNA relative to nuclear DNA. DNA extracted from peripheral blood leucocytes or bone marrow of healthy individuals or patients was simultaneously hybridized with a probe for the mitochondrial genome and a control probe for the renin gene on human chromosome 1. Comparative densitometric ratios of approximately 1 were obtained between the two signals in 20 normal control peripheral blood samples. In contrast, comparative ratios in the range of 2-50 were observed in 25 AML samples and 13 of these showed 8-fold or greater amplification of mtDNA relative to normal peripheral blood controls. An additional four cases of AML were investigated at both presentation and remission and showed 3-10-fold amplification of mtDNA at presentation, but no amplification when in clinical remission. 18 cases of chronic granulocytic leukaemia (CGL) were also studied in chronic phase and showed mtDNA dosage levels equivalent to normal peripheral blood controls. However, 8/9 CGL patients showed mtDNA amplification during transformation from chronic phase. We conclude that amplification of mtDNA is an invariable feature of acute myeloid leukaemia and that it may be a useful marker for detecting transformation of CGL.

Tγδ Cells in Juvenile Rheumatoid Arthritis and Rheumatoid Arthritis

Using the anti-TcR gamma/delta-1 monoclonal antibody and flow cytometry, we examined the number of T gamma delta cells in paired samples of peripheral blood and synovial fluid or tissue from 24 children with juvenile rheumatoid arthritis (JRA), five adult patients with JRA, and 14 patients with rheumatoid arthritis (RA). No significant difference was found in the synovial compartment T gamma delta values compared with the blood in JRA, adult JRA, or RA patients. Nor was any significant difference found in the peripheral blood or synovial compartment T gamma delta values in any of the three patient groups compared with the peripheral blood of normal controls. However, seven of the children with JRA had very high T gamma delta values in the synovial compartment while none of the normal children had high T gamma delta values in the blood (P = 0.02, Fisher's exact test). This may indicate a possible separate JRA patient group with high T gamma delta levels in the synovial compartment. In six JRA patients further analysed for T gamma delta subpopulations, a significant predominance of V delta 1+ cells was found in the synovial compartment compared with the corresponding peripheral blood samples (P less than 0.05, Wilcoxon's signed test) and with peripheral blood of child controls (P less than 0.05, Mann-Whitney U test). In these six patients, the T gamma delta-cell expression of the very early activation antigen CD69 were significantly higher (P less than 0.05, Wilcoxon's signed test) in the synovial compartment compared with the peripheral blood. Synovial T gamma delta cells expressing HLA-DR and interleukin 2 receptors could also be detected, in contrast to the peripheral blood in which no T gamma delta cells expressing these antigens could be found. These data suggest that the synovial T gamma delta cells had been activated in vivo.

Interleukin 2 responsive T cell clones from rheumatoid and normal subjects: Proliferative responses to connective tissue elements

In vivo-activated interleukin 2 responsive T cell clones were generated from peripheral blood (PB) and synovial fluid (SF) of rheumatoid arthritis (RA) patients and from normal control PB. The specificity of these clones was assessed by measuring proliferation induced by the connective tissue elements (CTE) collagen types I and II, native and denatured, proteoglycans, and irrelevant control antigens. The cloned T cells from RA patients but not from normal subjects responded in vitro with proliferation to all CTE but not to control antigens purified protein derivative, ovalbumin, or lysozyme. Proliferation occurred in the presence and absence of accessory cells (AC), but the responses were consistently higher in the presence of AC. Antibodies to HLA-DR abrogated the proliferative response to CTE suggesting that DR expression was necessary for the induction of proliferation. These findings demonstrate the existence of clonable T cells responsive to CTE in PB and SF of RA patients. Expression of reactivity to CTE may contribute to the chronicity of the inflammation in RA.

Inflammatory Synovial T Cells in Different Activity Subgroups of Patients with Rheumatoid Arthritis and Juvenile Rheumatoid Arthritis

Mononuclear cells were eluted from synovial membranes of 39 patients with rheumatoid arthritis and 12 patients with juvenile rheumatoid arthritis. A considerable cell loss, about 50% or more, was seen during the various isolation steps. The CD4/CD8 ratio just after enzyme treatment (stage I) was significantly higher than at later stages, i.e. after removal of adherent cells (stage II, p less than 0.05) and after Isopaque Ficoll gradient centrifugation (stage III, p less than 0.01). This indicates a selective loss of CD4+ cells during isolation. In addition, stages I and II had higher CD4/CD8 ratios than peripheral blood of normal controls (p less than 0.01 and p less than 0.03), but not significantly higher than in peripheral blood of patients (p greater than 0.05). The CD4/CD8 ratio in eluted synovial membrane cells did not differ between patients with high and patients with low disease activity (p greater than 0.05). No correlation was found between any of the CD4/CD8 ratios and individual disease activity variables. Furthermore, a laboratory activity index and a disease outcome index were determined for each patient and no correlation was found between these indices and the CD4/CD8 ratios.

Analysis of Density Changes and Chemotactic Receptors of Leukocytes from Chronic Hemodialysis and Peritoneal Dialysis Patients

Analysis of standard Ficoll-Hypaque (density = 1.077 g/ml) separation profiles of peripheral white blood cells (WBC) from patients undergoing hemodialysis (HD) demonstrated that dialysis caused a marked decrease in the density of polymorphonuclear leukocytes (PMN) resulting in about 50% of these cells separating with the mononuclear cells. In vitro exposure of normal control peripheral blood to HD membranes as well as to the purified chemotactic factors C5a, C5ades-Arg, and formyl-Met-Leu-Phe (fMLP) also resulted in PMN density changes which altered the Ficoll-Hypaque separation profiles of WBC. Therefore, these results imply that C5a generation, resulting from complement activation by the HD membrane, induced the density changes in the PMN from HD patients. Further studies using flow cytometry and fluorescein-labeled chemotactic factors (C5a, formyl-Met-Leu-Phe-Lys [fMLPL] and casein) indicated that HD patients had a significant reduction in the ability of their PMN and monocytes to bind C5a. This contrasted with the findings of no significant difference in the percentage or fluorescence intensity of HD patients' PMN or monocytes binding casein or fMLPL. Functional studies to analyze chemotactic-factor-mediated responses indicated that there was a decreased ability of HD patients' PMN and monocytes to generate superoxide anion, produce H2O2 and release myeloperoxidase in response to both C5a and fMLP. Additional studies evaluated the binding of chemotactic factors to PMN and monocytes from normal blood following passage through a hemodialyzer and from patients undergoing HD. Analysis of receptor binding by control cells passed through the dialyzer showed that there was a progressive decrease in the percentage of C5a-receptor-positive PMN and monocytes but no change with casein or fMLPL. In contrast, peripheral PMN and monocytes from chronic renal failure patients on HD showed no difference in C5a, casein or fMLPL receptors during the course of HD as compared to the predialysis period. This appears to be attributable to a difference in the regulation of the C5a that is generated as a result of the dialysis-membrane-induced activation of the complement system. Although C5a has been shown to be continuously generated during the course of HD, these patients show no modulation of their C5a receptors during the course of HD or when their whole blood is exposed to dialysis membrane fibers. These findings suggest that there are mechanisms functioning in chronically dialyzed patients to protect them from the effects of excessive C5a generation during HD.(ABSTRACT TRUNCATED AT 400 WORDS)

Lymph node anti-tumour effector cell mechanisms in colorectal carcinoma.

Eighty lymph nodes from 61 cases of colorectal carcinoma were studied by in vitro microcytotoxicity assay. It was found that 25 nodes (31%) from 22 of these cases (36%) contained lymphoid cells which were cytotoxic against autologous carcinoma cells in vitro. Lymph node cells (LNC) were not cytotoxic against 51Cr chicken red blood cells (CRBC). This assay was used as an indicator of natural killer cell (NK) activity. Comparably, normal control peripheral blood lymphocytes (PBL) were cytotoxic against CRBC but not against colonic target cells obtained from surgical biopsy specimens. Fc receptor-bearing cells, monitored by cytotoxicity against antibody-coated CRBC, were detected in 50 and 60% of cytotoxic and non-cytotoxic LNC populations. The varying effects of E- and EAC-rosette fractionation and iron filing treatment of effector cells indicate that regional LNC cytotoxicity in colorectal carcinoma is a complex phenomenon, which, however, is predominantly a function of E-rosetting lymphocytes. Lymphocytes obtained from all regional nodes were capable of responding to phytohaemagglutinin (PHA). The relative proportion of T lymphocytes (E-rosetting cells) was significantly higher in those lymph node suspensions which were cytotoxic against colon carcinoma cells.