Chronic obstructive pulmonary disease is characterized by reduced levels and defective suppressive function of regulatory B cells in peripheral blood.

Chronic obstructive pulmonary disease (COPD) is characterized by a progressive, persistent immune response to cigarette smoke, and it has been suggested that immune dysregulation is involved in its pathogenesis. A subset of regulatory B cells (Bregs) with high levels of the surface markers CD24 and CD38 (CD24hiCD38hi) has previously been shown to exert an immunosuppressive function. This study investigated the levels and activity of CD24hiCD38hi Bregs in stable COPD (sCOPD). Testing the peripheral blood from 65 patients with sCOPD and 39 control subjects for CD24hiCD38hi Breg subsets by flow cytometry showed that the patients with sCOPD had significantly lower levels of CD24hiCD38hi Bregs and IL-10+ B cells. The patients with sCOPD had lower serum interleukin-10 levels than the controls. The patients with most severe sCOPD had the lowest levels of CD24hiCD38hi Bregs. Spearman correlation analysis showed that the levels of CD24hiCD38hi Bregs in the patients with sCOPD positively correlated with serum interleukin-10 concentrations but not with levels of C-reactive protein. Compared to healthy controls, functional studies showed that Breg cells from patients with sCOPD exhibit a decreased suppressive function. We conclude that sCOPD is characterized by the exhaustion of CD24hiCD38hi regulatory B cells compartment. Therefore, CD24hiCD38hi Bregs may contribute to the pathogenesis of sCOPD.

Circulating CD19+CD24hiCD38hi regulatory B cells as biomarkers of response to methotrexate in early rheumatoid arthritis.

OBJECTIVE: The protagonism of regulatory B cells seems to vary along the course of the disease in murine models of inflammatory conditions. Decreased numbers of circulating regulatory CD19+CD24hiCD38hi transitional (cTr) B cells have been described in patients with long-standing RA, thus our objective was to examine the frequency and evolution of cTr B cells in the peripheral blood of early RA (ERA) patients. METHODS: Freshly isolated peripheral blood mononuclear cells from 48 steroid- and DMARD-naïve ERA patients with a disease duration of <24 weeks and 48 healthy controls (HCs) were examined by flow cytometry. Co-cultures of isolated memory B cells were established with autologous T cells in the absence or presence of Tr B cells. RESULTS: As compared with HCs, ERA patients demonstrated an increased frequency of cTr B cells. cTr B cells of ERA patients and HCs displayed an anti-inflammatory cytokine profile and were able to downregulate T cell IFN-γ and IL-21 production, together with ACPA secretion in autologous B/T cell co-cultures. Basal frequencies of cTr B cells above the median value observed in HCs were associated with a good EULAR response to MTX at 12 months [relative risk 2.91 (95% CI 1.37, 6.47)]. A significant reduction of cTr B cells was observed 12 months after initiating MTX, when the cTr B cell frequency was no longer elevated but decreased, and this was independent of the degree of clinical response or the intake of prednisone. CONCLUSION: An increased frequency of regulatory cTr B cells is apparent in untreated ERA and the baseline cTr B cell frequency is associated with the clinical response to MTX at 12 months.

Integrated Microfluidic Device for Accurate Extracellular Vesicle Quantification and Protein Markers Analysis Directly from Human Whole Blood.

Extracellular vesicles (EVs) have the potential to be utilized as disease-specific biomarkers. Although strategies for on-chip isolation and detection of EVs have recently been developed, they need preprocessing of clinical samples and are not accurate enough for disease diagnosis just judging by EVs concentration. Here, we designed an integrated microfluidic device named a plasma separation and EV detection (PS-ED) chip for plasma separation, quantification, and high-throughput protein analysis of EVs directly from clinical whole blood samples. The device included two modules (PS and ED module): the PS module was a six-loop microchannel for rapid separation of plasma from clinical whole blood samples under inertial force; the amount of EVs in the separated plasma kept the same value as in the initial blood samples. The module reduced the mechanical damage to the blood cells and thus reduced the interference of debris or cellular contents from damaged cells during EVs detection; the ED module contained four S-channels for quantification and high-throughput protein analysis of EVs; a wide detection range from 2.5 × 102 to 2.5 × 108 particles/µL with a detection limit of 95 particles/µL was obtained. Through simultanously monitoring three proteins (CD81, CD24, and EpCAM) of EVs, the cancer type can be accurately confirmed. Furthermore, clinical blood sample analysis verified that the proposed device could be used for accurate diagnosis and therapy monitoring of ovarian cancer.

CD24hiCD38hi and CD24hiCD27+ Human Regulatory B Cells Display Common and Distinct Functional Characteristics.

Although IL-10-producing regulatory B cells (Bregs) play important roles in immune regulation, their surface phenotypes and functional characteristics have not been fully investigated. In this study, we report that the frequency of IL-10-producing Bregs in human peripheral blood, spleens, and tonsils is similar, but they display heterogenous surface phenotypes. Nonetheless, CD24hiCD38hi transitional B cells (TBs) and CD24hiCD27+ B cells (human equivalent of murine B10 cells) are the major IL-10-producing B cells. They both suppress CD4+ T cell proliferation as well as IFN-γ/IL-17 expression. However, CD24hiCD27+ B cells were more efficient than TBs at suppressing CD4+ T cell proliferation and IFN-γ/IL-17 expression, whereas they both coexpress IL-10 and TNF-α. TGF-ß1 and granzyme B expression were also enriched within CD24hiCD27+ B cells, when compared with TBs. Additionally, CD24hiCD27+ B cells expressed increased levels of surface integrins (CD11a, CD11b, α1, α4, and ß1) and CD39 (an ecto-ATPase), suggesting that the in vivo mechanisms of action of the two Breg subsets are not the same. Lastly, we also report that liver allograft recipients with plasma cell hepatitis had significant decreases of both Breg subsets.

Cancer stem cell markers ALDH1 and CD44+/CD24- phenotype and their prognosis impact in invasive ductal carcinoma.

Breast cancer is a very heterogeneous disease. The intrinsic molecular subtypes can explain the intertumoral heterogeneity and the cancer stem cell (CSC) hypothesis can explain the intratumoral heterogeneity of this kind of tumor. CD44+/CD24- phenotype and ALDH1 expression are the major CSC markers described in invasive breast cancer. In the present study, 144 samples of invasive breast carcinoma, no special type were distributed in 15 tissue microarrays (TMA) and then evaluated for expression of the CD44+/CD24- phenotype and ALDH1 to understand the importance of these CSC markers and the clinical aspects of breast cancer. The samples were classified into four molecular subtypes according to clinicopathological criteria: Luminal A, Luminal B, HER2, and Basal-like. A statistical association was found between the molecular subtypes and the CSC markers, with HER2 the most frequent subtype for both markers. ALDH1 was also associated with other poor prognostic variables, such as a high histological grade and larger tumors, but it was not associated with the patients' prognosis in this sample and nor was the CD44+/CD24- phenotype in a multivariate analysis. There are still many controversies about the role of these markers in breast cancer molecular subtypes. The identification of these populations of cells, through immunohistochemical markers, can help to better understand the CSC theory in clinical practice and, in the near future, contribute to developing new target therapies.

Killer (FASL regulatory) B cells are present during latent TB and are induced by BCG stimulation in participants with and without latent tuberculosis.

Regulatory B cells (Bregs) have been shown to be present during several disease states. The phenotype of the cells is not completely defined and the function of these cells differ between disease. The presence of FASL expressing (killer) B cells during latent and successfully treated TB disease have been shown but whether these cells are similar to regulatory B cells remain unclear. We assessed the receptor expression of FASL/IL5 (killer B cells), CD24/CD38 (regulatory B cells) on whole peripheral blood of participants with untreated active TB and healthy controls. We then isolated B cells from a second cohort of M.tb exposed (Quantiferon (QFN) positive) and unexposed (Quantiferon negative) HIV negative participants, and evaluated the frequency of killer B cells induced following stimulation with BCG and/or CD40 and IL5. Our data reveal no difference in the expression on CD24 and CD38 between participants with active TB and the controls. There was also no difference in the frequency of regulatory B cells measured in the peripheral blood mononuclear cells (PBMC) fraction between latent TB and uninfected controls. We did however notice that regulatory B cells (CD24hiCD38hi) population express the FASL receptor. The expression of killer B cell phenotype (CD178+IL5RA+) was significantly higher in controls compared to those with active TB disease (1,06% vs 0,455%). Furthermore, we found that BCG restimulation significantly induced the FASL/IL5RA B cells but this was only evident in the QFN positive group. Our data suggest that both regulatory and killer B cells are present during latent and active TB disease but that the frequency of these populations are increased during latent disease. We also show that the FASL+IL5RA+ B killer B cells are induced in latent TB infection following BCG restimulation but whether these cells are indicative of protection remains unclear.

Marked elevation of circulating CD19+CD38hiCD24hi transitional B cells give protection against neonatal sepsis.

BACKGROUND: Adequate functions of immunoregulation, mediated by regulatory cells such as IL-10 producing CD19+CD38hiCD24hi transitional B cells (Trans), play an important role in control of excessive inflammatory response. Yet, the role of Trans in neonatal sepsis is incompletely understood. We investigated the role of Trans in late-onset sepsis (LOS). METHODS: We used multicolor flow cytometry to analyse the phenotypes of B cells drawn from a cohort of 16 neonatal late-onset sepsis (LOS) (12 survivors and 4 non-survivors) and 20 healthy neonates over time. RESULTS: Patients undergone a serious decline of lymphocytes at the beginning of sepsis and then noticeable elevation during one week of follow-up had a good prognosis. Intriguingly, peripheral blood B cells, especially Trans, were the marked increase lymphocyte subset and maintained a high level of producing IL-10 during the 7 days of follow-up. CONCLUSION: The level of IL-10 producing Trans was significantly elevated in peripheral blood of good prognosis newborns with LOS and might contribute to the successful immunoprotective state of the disease.

[Isolation and characterization of exosomes from blood plasma of breast cancer and colorectal cancer patients]. / Vydelenie i kharakterizatsiia ékzosom plazmy krovi bol'nykh rakom molochnoi zhelezy i kolorektal'nym rakom.

A simple approach for isolation of exosomes from the blood plasma, which allows to obtain highly purified preparations of microvesicles no larger than 100 nm has been proposed. The presence of different subpopulations of exosomes in the blood plasma of healthy donors and cancer patients has been recognized. We found the presence of the universal markers CD9, CD24 and CD81 on exosomes isolated from blood plasma that can be used to their routine typing.

Annexin A10 is a candidate marker associated with the progression of pancreatic precursor lesions to adenocarcinoma.

Annexins are a multigene family of calcium and phospholipid-binding proteins that play important roles in calcium signaling, cell motility, differentiation and proliferation. Our previous mass spectrometry-based proteomics study revealed that annexin A10 (ANXA10) was uniquely overexpressed in pancreatic CD24+ adenocarcinoma cells that were dissected from clinical PDAC tissues but was absent in CD24- adjacent normal cells. The correlation between ANXA10 expression and the progression of pancreatic cancer remains unknown. In this study, we performed an immunostaining assay to evaluate ANXA10 expression in 155 primary human tissue specimens, including normal pancreas, chronic pancreatitis (CP), pancreatic adenocarcinoma (PDAC), pancreatic intraepithelial neoplasia (PanIN, the most important precursor of PDAC), and intraductal papillary mucinous neoplasm (IPMN). The immunostaining result showed that ANXA10 was significantly overexpressed in PanINs, IPMNs, and PDACs but negative in normal pancreas and the majority of chronic pancreatitis tissues. Statistical analysis revealed that ANXA10 expression was significantly associated with PDAC and its precursor lesions (p<0.0001). Abundant ANXA10 expression was predominantly present in pancreatic ductal epithelial cells of PanINs, IPMNs, and tumor cells of PDACs. Since PDAC develops through a series of PanINs which in turn arise from pancreatic ducts, the consistent overexpression of ANXA10 in ductal epithelial cells in PanINs and PDACs but negative in normal pancreatic ducts suggests that ANXA10 could serve as a potential marker indicating the presence of PDAC at its earliest precancerous stages. Double immunostaining of ANXA10 and CD24 showed that there was a large overlap between these two markers in PDAC and high-grade neoplasia lesions. The statistical analysis showed that the coexpression of ANXA10 and CD24 was significantly correlated with the progression of pancreatic precursor lesions towards PDACs.

Reduction in Peripheral CD19+CD24hCD27+ B Cell Frequency Predicts Favourable Clinical Course in XELOX-Treated Patients with Advanced Gastric Cancer.

BACKGROUND: Accumulating evidence suggests that regulatory B cells (Bregs) play an important role in modulating the immune response to tumours. Our previous study indicated that a small percentage of peripheral CD19+CD24hCD27+ Breg cells slowed gastric cancer progression in XELOX-treated patients. Here, we further investigated the relationship between dynamic changes in circulating Breg cells and the clinical course in XELOX-treated gastric cancer patients. METHODS: A total of 52 patients with advanced gastric cancer were enrolled in this study. The frequencies of CD19+CD24hCD27+ cells in peripheral blood were tested before (as a baseline) and 9 weeks after administration of oxaliplatin and capecitabine (XELOX). The primary endpoint of the study was progression-free survival time (PFS) of the patients. The overall survival (OS) and adverse events of chemotherapy were also recorded. RESULTS: The median PFS of patients was 6 months (95% CI, 5.27-6.73) with effective rate of 46.2%. The percentage of CD19+CD24hCD27+ cells in lymphocytes ranged from 0.007% to 1.94%, with a median value of 0.45%. The median percentage of CD19+CD24hCD27+ lymphocytes was 0.59% (0.01%-6.02%) 9 weeks after treatment. There were no significant differences for this index. However, the patients with decreased Breg frequencies after XELOX treatment had a longer PFS time (7.0 months vs. 5.0 months, p=0.01) than those with increased Breg frequencies. CONCLUSION: Patients with downtrend of CD19+CD24hCD27+ B lymphocytes during early stages of chemotherapy relative to their initial values had longer PFS times, and this could be used to predict the efficacy of chemotherapy and help physicians adjust treatments accordingly.

Percentage of Peripheral CD19+CD24hiCD38hi Regulatory B Cells in Neonatal Sepsis Patients and Its Functional Implication.

BACKGROUND As a major cause of mortality in neonates, neonatal sepsis is often accompanied by immune dysfunctions, which are frequently caused by dysregulated T cell sub-populations. The role of regulatory B cells in neonatal sepsis, however, remains unknown. Therefore, this study investigated the percentage and functional variation of CD19+CD24hiCD38hi regulatory B cells in peripheral blood of neonatal sepsis patients in an attempt to elucidate the role of these regulatory B cells in pathogenesis of sepsis. MATERIAL AND METHODS Flow cytometry was used to quantify the percentage of CD19+CD24hiCD38hi regulatory B cells from peripheral blood samples. The correlation between B cell percentage and C reactive protein (CRP) level was analyzed. Secretion level of interleukin-10 (IL-10) and effects on the proliferation of naïve CD4+ T cells were further analyzed. RESULTS The percentage of CD19+CD24hiCD38hi regulatory B cells in neonatal sepsis patients was significantly higher compared to healthy controls (p<0.05), and was positively correlated with serum CRP level. The percentage of IL-10+ CD19+CD24hiCD38hi regulatory B cells was also higher in sepsis patients, and also had more potent inhibition on naïve CD4+ T cells (p<0.01). CONCLUSIONS The elevation of CD19+CD24hiCD38hi regulatory B cells in neonatal sepsis can inhibit body immune function and thus may participate in the pathogenesis of sepsis.

Characterization of B cells in healthy pregnant women from late pregnancy to post-partum: a prospective observational study.

BACKGROUND: B cells play a role in pregnancy due to their humoral and regulatory activities. To our knowledge, different maturational stages (from transitional to memory) of circulating B cell subsets have not yet been characterized (cell quantification and phenotype identification) in healthy pregnant women. Thus, the objective of our study was to characterize these subsets (as well as regulatory B cells) from late pregnancy to post-partum and to compare them with the circulating B cells of non-pregnant women. METHODS: In all of the enrolled women, flow cytometry was used to characterize the circulating B cell subsets according to the expression of IgD and CD38 (Bm1-Bm5 classification system). Regulatory B cells were characterized based on the expression of surface antigens (CD24, CD27, and CD38) and the production of IL-10 after lipopolysaccharide stimulation. RESULTS: Compared to the absolute counts of B cells in the non-pregnant women (n = 35), those in the pregnant women (n = 43) were significantly lower (p < 0.05) during the 3rd trimester of pregnancy and on delivery day (immediately after delivery). The percentages of these cells on delivery day and at post-partum were significantly lower than those in the non-pregnant women. In general, the absolute counts and percentages of the majority of the B cell subsets were significantly lower in the 3rd trimester of pregnancy and on delivery day than in the non-pregnant women. However, these counts and percentages did not differ significantly between the post-partum and the non-pregnant women. The most notable exceptions to the above were the percentages of naïve B cells (which were significantly higher in the 3rd trimester and on delivery day than in the non-pregnant women) and of CD24(hi)CD38(hi) regulatory B cells (which were significantly higher in the post-partum than in the non-pregnant women). CONCLUSION: According to our study, the peripheral B cell compartment undergoes quantitative changes during normal late pregnancy and post-partum. Such findings may allow us to better understand immunomodulation during human pregnancy and provide evidence that could aid in the development of new strategies to diagnose and treat pregnancy-associated disturbances. Our findings could also be useful for studies of the mechanisms of maternal responses to vaccination and infection.

Predictive Levels of CD24 in Peripheral Blood Leukocytes for the Early Detection of Colorectal Adenomas and Adenocarcinomas.

CD24 is expressed in 90% of colorectal adenomas and adenocarcinomas. Colorectal cancer (CRC) can be mostly prevented but average risk population screening by stool testing or colonoscopy faces many hurdles. Blood testing is clinically needed. We aimed to evaluate the utility of CD24 expression in peripheral blood leukocytes (PBLs). Two independent case studies were conducted in eligible individuals undergoing colonoscopy. Protein extracted from PBLs was subjected to immunoblotting using anti-CD24 monoclonal antibodies. CD24 sensitivity and specificity were determined using receiver operating characteristic (ROC) analysis. Initially, 150 subjects were examined: 63 had CRC, 19 had adenomas, and 68 had normal colonoscopies. The sensitivity and specificity of CD24 for distinguishing CRC from normal subjects were 70.5% (95% CI, 54.8-83.2%) and 83.8% (95% CI, 74.6-92.7%) and for adenomas 84.2% (95% CI, 60.4-96.4%) and 73.5% (95% CI, 61.4-83.5%), respectively. In the second trial (n = 149), a similar specificity but higher sensitivity was achieved: 80.0% (95% CI, 63.1-91.6%) for CRC and 89.2% (95% CI, 74.6-97%) for adenomas. A simple noninvasive blood test evaluating CD24 levels has high sensitivity and specificity for detecting colorectal adenomas and cancer in patients undergoing colonoscopy at an urban medical center. Larger multicenter studies are warranted to establish the potential of this promising test.

A new tumor biomarker, serum protein peak at 3,144 m/z, in patients with node-positive breast cancer.

PURPOSE: To explore the association between the 3,144 m/z protein peak and the clinicopathological features and prognosis in breast cancer. METHODS: Using SELDI-TOF MS, we analyzed serum protein peak at 3,144 m/z in 283 patients with node-positive breast cancer, its relationship with clinicopathological features and their prognosis evaluating value of survival. RESULTS: 3,144 m/z positive rate was higher in elderly patients (42.8 % in ≥50-year-old vs. 31.2 % in <50, P = 0.04). However, no correlation was observed between 3,144 m/z and other clinicopathological features (body mass index, menstrual status, family history, TNM, molecular subtypes, vascular invasion, neural invasion, p53 and CA15-3). However, the positive rate of 3,144 m/z was higher than that of CA15-3 (35.5 vs. 11.4 %, McNemar χ (2) test, p < 0.001). 3,144 m/z-negative patients (n = 177) had a better 3-year overall survival (OS) than 3,144 m/z-positive patients (n = 106) (89.8 vs. 81.2 %, P = 0.045). Younger patients (P = 0.016), postmenopausal status (P = 0.019), small tumor (P < 0.001), less positive nodes (P < 0.001), early stage (P < 0.001), favorable molecular subtype (P = 0.007), normal CA15-3 (P = 0.003) and neoadjuvant chemotherapy (P = 0.001) predicted better survival. Cox analysis showed that T3-4 (95 % CI 1.419-8.057, P = 0.006), lymph node metastasis (95 % CI 1.242-3.632, P = 0.006) and p53 mutation (95 % CI 1.088-6.378, P = 0.032) were independent adverse prognostic factors. But childbirth ≥2 (95 % CI 0.163-0.986, P = 0.046), adjuvant chemotherapy (95 % CI 0.062-0.921, P = 0.038) and adjuvant radiotherapy (95 % CI 0.148-0.928, P = 0.034) were the independent factors in reducing risk of death in breast cancer patients. Combination testing of 3,144 m/z and CA15-3 will improve the prognosis value of 3-year survival (P = 0.011); patients with CA153-/3144- were characterized by the longest survival (89.8 %) and the CA153+/3144+ patients by the shortest. CONCLUSIONS: Serum protein peak at 3,144 m/z is a new biomarker for breast cancer diagnosis and prognosis and showed a higher positive rate than serum CA15-3. Combining 3,144 m/z and CA15-3 testing may improve prognosis of longer survival in breast cancer patients.

Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production.

The role of CD19+ CD24high CD38high and CD19+ CD24high CD27+ regulatory B cells in patients with type 1 autoimmune pancreatitis.

BACKGROUND: Patients with type 1 autoimmune pancreatitis (AIP) have several immunologic and histologic abnormalities. It is known that depletion of B cells by rituximab is effective for treatment of IgG4-related disease (IgG4-RD) such as type 1 AIP, suggesting that B cells may be a key player in IgG4-RD. However, the role of regulatory B cells (Bregs) in type 1 AIP is unclear, and the objective of this paper is to clarify the role of Bregs in the pathophysiology of type 1 AIP by analyzing circulating Bregs. METHOD: We recruited 21 patients with type 1 AIP as determined by the International Consensus Diagnostic Criteria for AIP (ICDC). No patients received corticosteroid treatments. For comparison, we recruited 14 patients with chronic pancreatitis (CP), 20 patients with pancreatic cancer, and 25 healthy subjects as controls. We analyzed Bregs as CD19+ CD24high CD38high and CD19+ CD24high CD27+ from peripheral blood by flow cytometry. RESULTS: In peripheral blood, CD19+ CD24high CD38high Bregs were significantly increased in type 1 AIP patients compared with CP, pancreatic cancer, and healthy controls. Although not significant different, CD19+ CD24high CD27+ Bregs of type 1 AIP were decreased compared to those of other groups. IL-10(+) B cells were not significantly different from type 1 AIP patients and healthy controls. In untreated type 1 AIP patients, the number of CD19+ CD24high CD38high Bregs and IgG4 were not correlated. CONCLUSIONS: Our data suggested that CD19+ CD24high CD38high Bregs seemed to increase reactively to suppress the disease activity, and are consistent with the hypothesis that CD19+ CD24high CD27+ Bregs might be involved in the development of type 1 AIP, although it still remains unclear whether the decrease of CD19+ CD24high CD27+ cells is cause or effect of AIP.

Laboratory tests for paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder that is often suspected in a patient presenting with non-immune hemolytic anemia associated with pancytopenia or venous thrombosis. This disorder is a consequence of acquired somatic mutations in the phosphatidylinositol glycan class A (PIG-A) gene in the hematopoietic stem cells (HSC) of patients. The presence of these mutations leads to production of blood cells with decreased glycosyl phosphatidylinositol-anchored cell surface proteins, making red blood cells derived from the clone more sensitive to complement mediated hemolysis. The diagnosis of PNH may be difficult in some cases due a low proportion of PNH cells in the blood and occasionally due to difficulties in selecting the most appropriate diagnostic studies. The latest generation of tests allow for detection of very small populations of PNH cells, for following the natural course and response to therapy of the disease, and for helping to decide when to initiate therapy with monoclonal antibody targeting the terminal complement protein C5 (Eculizumab), anticoagulation, and in some cases allogeneic HSC transplant. In this article, we review the different diagnostic tests available to clinicians for PNH diagnosis.

Peripheral CD24hi CD27+ CD19+ B cells subset as a potential biomarker in naïve systemic lupus erythematosus.

AIM: B cells are likely to play critical roles in the pathogenesis of systemic lupus erythematosus (SLE). Our aim was to investigate the role of peripheral CD24(hi) CD27(+) CD19(+) B cells in Chinese patients with new-onset SLE. METHOD: Peripheral CD24(hi) CD27(+) CD19(+) B cells were analyzed in 55 new-onset lupus and 36 healthy controls by flow cytometry. All SLE cases were treated with prednisolone and hydroxychloroquine during a 1-year follow-up. Thirteen cases were added with cyclophosphamide or mycophenolate mofetil. The CD24(hi) CD27(+) CD19(+) B cells were analyzed at days 0, 7, 14 and months 1, 3, 6, 9 and 12. Interleukin-10 (IL-10)-producing B cell was detected in eight naïve lupus and 10 healthy controls. RESULTS: Compared to healthy controls, the frequency and number of primary circulating CD24(hi) CD27(+) CD19(+) B cells was significantly reduced in SLE cases (8.22 ± 3.48% vs. 31.67 ± 5.53%, P < 0.0001; 4.04 ± 2.85 vs. 38.66 ± 10.22 10(3) cells/mL, P = 0.0001) before treatment; IL-10(+) CD19(+) B cells and IL-10(+) CD24(hi) CD27(+) CD19(+) B cells also decreased in SLE. Interestingly, primary CD24(hi) CD27(+) CD19(+) B cells inversely correlated with SLE disease activity index (SLEDAI) score. Patients with arthritis and hematologic disorders had a lower primary CD24(hi) CD27(+) CD19(+) B cells. In 48 SLE cases who finished the 1-year follow-up, the frequency and number of CD24(hi) CD27(+) CD19(+) B cells increased from 8.26 ± 3.61% to 25.51 ± 4.56%; 3.99 ± 2.86 to 28.64 ± 11.81 10(3) cells/mm(3) (P < 0.0001), accompanied by a significantly decreased SLEDAI score. Of note, CD24(hi) CD27(+) CD19(+) B cells decreased in some flare cases with an elevated SLEDAI score. CONCLUSION: These results demonstrate that a lower primary CD24(hi) CD27(+) CD19(+) B cells may be an immunologic aspect of new-onset SLE. CD24(hi) CD27(+) CD19(+) B cells may be a useful tool to evaluate lupus activity and monitor the response to therapy.

Plasma CD24 as a novel useful biomarker in differentiating hepatocellular carcinoma patients from normal individuals.

BACKGROUND/AIMS: CD24 is reported to be up-regulated in the tissues of HCC patients when compared with normal liver tissues. We aim to determine whether CD24 protein is also overexpressed in the plasma of HCC patients, and its diagnostic value for HCC. METHODOLOGY: Plasma levels of CD24 protein and AFP were measured by enzyme linked immunosorbent assay (ELISA) in the plasma of 90 patients with hepatocellular carcinoma and 30 healthy controls. The sensitivity and specificity were calculated and the relationship between the expression of CD24 and clinical pathological parameters was analyzed. RESULTS: Both plasma CD24 protein and AFP levels in HCC patients were higher than those in healthy controls (p<0.05). There was no correlation between plasma levels of AFP and CD24 in 90 patients with HCC (r=-0.084, p=0.430). The best cut-off value of CD24 was 3.31ng/mL, which yielded a sensitivity and specificity of 83.3% and 93.3%, respectively, for screening HCC; and plasma CD24 level was not associated with gender, age, hepatitis infection status, tumor size and histological differentiation and TNM stage (p>0.05). CONCLUSIONS: Plasma CD24 protein might serve as a novel tumor marker in differentiating HCC patients from normal individuals as well as monitor HCC status in AFP negative HCC patients.