Hyperferritinemia Correlated with Activated Population of Natural Killer Cells in Pediatric Major β-Thalassemia Patients

Natural killer (NK) cells act both as cytotoxic and cytokine producers in the innate immune response. Hyperferritinemia resulting from a routine blood transfusion as a specific treatment in major β-thalassemia patients may disturb the cellular immune system’s harmony. This study aims to investigate the correlation between hyperferritinemia and the NK cell subsets in major β-thalassemia settings. Pediatric major β-thalassemia patients who routinely received a blood transfusion at Dr. Hasan Sadikin General Hospital in 2016 were included in this cross-sectional study. Blood samples were treated with the monoclonal antibody of CD3, CD56, and CD16 to count the NK cells subsets as CD56bright, CD56dim, and CD16+ using flowcytometry. CD69+ used as an activation marker. The median fluorescence intensity (MFI) of CD56, CD16, and CD69 was measured. Total iron-binding capacity (TiBC), ferritin, and serum iron level examined as iron status. A Spearman correlation test was used for statistical analysis. Fifty-five blood samples were obtained for analysis. This study reveals that the percentage of CD3− lymphocyte population was correlated with the ferritin levels (r=−0.45, p=0.0009). Positive correlation was revealed between activated population (CD69+) of CD56bright and CD56dim NK cell subsets and hyperferritinemia [(r=0.353, p=0.008) and (r=0.355, p=0.008)]. The activated CD56bright cells was associated with ferritin level (r=0.353, p=0.008) and TiBC (r=0.334, p=0.018). Hyperferritinemia in pediatric major β-thalassemia patients may influence NK cell subsets' balance population, particularly the CD56bright and CD56dim NK cell subsets, then alter their immune response to pathogens. KORELASI ANTARA HIPERFERITINEMIA DAN SEL NATURAL KILLER TERAKTIVASI PADA ANAK DENGAN TALASEMIA BETA MAYORSel-sel natural killer (NK) telah diketahui memiliki peran sitotoksik dan dalam produksi sitokin pada respons imun bawaan. Hiperferitinemia merupakan hasil dari transfusi darah rutin yang dijalani sebagai terapi utama pada talasemia mayor. Penelitian ini bertujuan mempelajari hubungan hiperferitinemia dan sel NK pada talasemia beta mayor. Penelitian potong lintang ini melibatkan anak dengan talasemia beta mayor yang secara rutin menerima transfusi darah di RSUP Dr. Hasan Sadikin selama tahun 2016. Sampel darah diberi marker CD3, CD56, dan CD16 untuk menghitung subset sel NK sebagai CD56bright, CD56dim, dan CD16+ menggunakan flowcytometry. CD69+ digunakan sebagai penanda aktivasi. Median fluorescence intensity (MFI) CD56, CD16, dan CD69 diukur. Kadar TiBC, ferritin, dan Fe serum diperiksa sebagai status besi. Uji korelasi Spearman digunakan pada analisis statistik. Analisis dilakukan terhadap 55 sampel darah anak dengan talasemia. Penelitian ini mendapatkan populasi limfosit CD3 berkorelasi dengan kadar feritin (r=−0,45; p=0,0009). Korelasi positif didapatkan pada populasi teraktivasi (CD69+) dari subset sel CD56bright dan CD56dim NK serta hiperferitinemia [(r=0,353; p=0,008) dan (r=0,355; p=0,008)]. Sel CD56bright teraktivasi berkorelasi dengan kadar feritin (r=0,353; p=0,008) dan TiBC (r=0,334; p=0,018). Hiperferitinemia pada anak dengan talasemia mayor dapat memengaruhi populasi sel NK, khususnya pada subset CD56bright dan CD56dim sehingga berpengaruh pada respons imun terhadap patogen.

Flow cytometry expression pattern of CD44 and CD18 markers on feline leukocytes

The paucity of specific feline antibodies for flow cytometry (FC) is an ongoing challenge. Flow cytometrists must extrapolate information from relatively few markers. We evaluated the expression pattern of the panleukocyte markers CD18 and CD44 on leukocyte (white blood cell, WBC) subclasses in the peripheral blood (PB) of 14 healthy cats. The degree of expression of CD18 and CD44 was calculated as the ratio between the median fluorescence intensity (MFI) value of antibody-stained cells and autofluorescence. All samples were acquired with the same cytometer with constant photomultiplier setting and compensation matrices. Both molecules were expressed at higher levels on monocytes, intermediate levels on polymorphonuclear cells (PMNs), and lower levels on lymphocytes. CD18-MFI discriminated well among the 3 populations, whereas CD44-MFI mostly overlapped between monocytes and PMNs. However, CD44-MFI had a lower intra-population variability. Evaluation of CD18 and CD44, together with morphologic parameters, was useful for discriminating among WBC subclasses in healthy cats. This information may be helpful for future studies given that an increase in CD18-MFI may indicate reactive changes, whereas fluctuations in CD44-MFI may suggest neoplasia.

ROS Production Triggers Anti-Leukemic Effects of Green Tea

Beneficial effects of green tea (GT) consumption have been described, including the ability to reduce cancer development. Polyphenols are the main chemical constituents of GT extract and have been identified as the most effective substances that can inhibit tumorigenesis. Acute myeloid leukemia is an aggressive hematologic malignancy and there is no sufficient evidence that supports a protective role of tea intake on its development. In this concern, the aim of this study was to investigate GT effects in acute promyelocytic leukemia (APL) mice. A total of 1 × 106 leukemic cells obtained from hCG-PML-RARa transgenic mice were injected in the tail vein of 12- to 16-week-old NOD.CB17-Prkdcscid/J mice, after 4-6 h of sublethal cobalt irradiation with 2 Gy. The hematologic counts were monitored weekly, and the following criteria were used for the diagnosis of leukemia: presence of at least 1% of blast in peripheral blood associated with leukocytosis above 30 000 cells/L, hemoglobin levels below 10 g/dL, and thrombocytopenia below 500 × 103 cells/L (He et al, 1997). Twelve days after transplantation, mice were then submitted to daily oral treatment (gavage) with 250 mg/kg/day GT or vehicle only (water) for 5 consecutive days and were sacrificed; bone marrow (BM) and spleens were collected to the assays. Treatment with GT significantly increased the mean number of apoptotic cells in the BM (29.4 ± 5.2 vs untreated 21.0 ± 2.1 %, P < 0.05) and spleen (13.9 ± 3.1 vs untreated 9.2 ± 1.9 % P < 0.05) of mice, evaluated by Annexin V-FITC/PI. GT induced an increase in the median fluorescence intensity (MFI) of cleaved caspase-3 in the BM (83.9 ± 3.6 vs untreated 72.6 ± 4.7, P < 0.05) and in the spleen (75.5 ± 28.2 vs untreated 55.8 ± 7.3, P < 0.01); cleaved caspase-8 in the BM (117.3 ± 9.9 vs untreated 89.1 ± 12.3, P < 0.005) and in the spleen (118.0 ± 31.5 vs untreated 81.5 ± 14.8, P < 0.001); and cleaved caspase-9 in the BM (138.2 ± 52.4 vs untreated 85.8 ± 12.9, P < 0.001) and in the spleen (121.7 ± 49.2 vs untreated 76.5 ± 21.9, P < 0.001) of leukemic mice. Moreover, GT treatment reduced the percentage of CD34+ hematopoietic progenitor cells (32.4 ± 2.3 vs untreated 41.0 ± 0.5 %) as well as of CD117+ cells (33.4 ± 3.7 vs untreated 44.2 ± 1.8 %). We then evaluated the phenotype of cells infiltrated in the spleen. Interestingly, we found that GT induces a decrease in the percentage of CD117+ (40.7 ± 0.3 vs leukemic 44.6 ± 0.9 %) and Gr-1 cells (60.8 ± 0.2 vs untreated 65.6 ± 0.5 %) present in the spleen. We then analyzed the effects of GT in the production of intracellular ROS in the BM subpopulations of CD34+, CD117+ and Gr-1+ cells from leukemic mice. Significant increases in the median fluorescence intensity (MFI) of intracellular ROS production by Gr-1 cells of GT-treated mice were observed (670 ± 103 vs untreated 428.5 ± 5.2). Interestingly, GT induced a reduction of MFI of intracellular ROS production in the CD34+ (167.5 ± 27.1 vs untreated 405.5 ± 73.3) and CD117+ (360 ± 142 vs untreated 1635 ± 40.4) cells. We then studied the expression and localization of CXCR4 and HIF-1α proteins. Studies have shown that ROS increases expression of CXCR4 in cancer and immune cells (Li et al, 2009; Lin et al, 2011; Chetram et al, 2011; 2013) through nuclear translocation of HIF-1α (Lee et al, 2002; Salmeen et al, 2003). Our results showed that GT decreased the MFI of CXCR4 in the leukemic mice (9028 ± 1367 vs untreated 4196 ± 970). Reduction of the nuclear translocation of HIF-1α in GT-treated mice was also observed, using the ImageStream imaging flow. In conclusion, GT treatment in APL mice induces apoptosis of cells in the BM and spleen, confirmed by activation of caspase-3, -8 and -9, probably by modulating the production of intracellular ROS in the leukemic cells. Although GT and its polyphenols are well known as antioxidants, there is evidence that some of the effects of these compounds may be related to induction of oxidative stress in immune cells, which might be responsible for the induction of apoptosis of tumor cells. These pro-oxidant properties may also induce endogenous antioxidant systems in normal tissues that offer protection against cancer. On the other hand, it is possible that, in leukemic cells, which has excessive ROS, the antioxidant effect of GT become more evident. Several potential mechanisms have been proposed including both antioxidant and pro-oxidant effects to polyphenol compounds, but questions remain concerning the relevance of these mechanisms to cancer prevention. Disclosures Torello: Fundação de Amparo à Pesquisa do Estado de São Paulo - Fapesp: Research Funding; Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq: Research Funding. Shiraishi:University of Campinas: Employment.

Effects of conventional CPB and mini-CPB on neutrophils CD162, CD166 and CD195 expression

Objective: Cardiac surgery is known to trigger a systemic inflammatory response. While the use of conventional cardiopulmonary bypass (CPB) results in profound inflammation, modified mini-CPB is considered less harmful. We evaluated the impact of cardiac surgery on the expression of CD162, CD166, CD195 molecules and their association with the type of CPB used. Methods and Results: Twenty-four patients were enrolled in our study. Twelve of them were operated using conventional CPB while the other twelve patients underwent surgery with mini-CPB. Blood samples were analysed by flow cytometry. We observed a significant increase in median fluorescence intensity of CD162 and CD195 that peaked instantly after surgery and normalized to the baseline value on the 1st day post surgery, whereas CD166 was initially down-regulated and its median fluorescence intensity (MFI) value increased to the baseline in the next few days. Conclusion: We observed immediate changes in the expression of CD162, CD166, and CD195 molecules on the neutrophils after surgery in both study groups of patients. The intensity of the observed changes was significantly greater in the group of patients who underwent conventional CPB compared to patients who underwent mini-CPB cardiac surgery.

Expression of CD14 and toll-like receptors 2 and 4 by milk neutrophils in bovine mammary glands infected with Corynebacterium bovis

This study evaluated the expression of CD14, toll-like receptor (TLR) 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x105 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters.

Hydroxycarbamide Is Related with Higher Expression of Band-3 and CD59 in Sickle Erythrocytes

BACKGROUND: Sickle Cell Anemia (SCA) presents extra and intravascular hemolysis. The eryptosis depends on phagocytosis signs like CD47, which is known as a "don't eat me" signal. Other mechanisms associated to hemolysis, such as complement activation mediated by CD59, are poorly understood. One of the effects of hydroxycarbamide (HU) is the reduction of hemolysis, but little is known about the action of HU in these mechanisms. OBJECTIVES: To evaluate markers related to hemolysis in patients with SCA at steady state, with and without HU. METHODS: We studied 40 adult patients with SCA (n=36) or Sβ0-Thalassemia (n=4), 70% females, median age 30 years (range 18-70y). The patients were followed at the Outpatient Clinic of EPM /UNIFESP. Inclusion criteria: patients in use of HU in maximum tolerated dose for more than 1 year (G1, n=20); or without HU (G2, n=20). Exclusion criteria: pregnant women and chronic transfusion. Laboratory evaluation: CBC, reticulocyte (Ret), fetal hemoglobin (HbF), indirect bilirubin, lactate dehydrogenase (LDH), LDH isoforms, free Hb (free-Hb), haptoglobin (Hp), hemopexin, phosphatidylserine, microvesicles (Mcv), Howell-Jolly bodies (in erythrocytes and Ret), and expression of band-3, CD47 (in erythrocyte and Ret) and CD59 (in erythrocytes and Mcv). The last parameters were evaluated by flow cytometry (FACS Calibur®, BDB, San Jose, CA), using specific antibodies according to the manufacturer instructions. Data were analyzed by CellQuest® software (BDB), and the results expressed in median fluorescence intensity or percentage of positivity. Statistical analysis: Mann-Whitney test and Pearson's correlation, with significance level of 5%. RESULTS: Themean corpuscular volume and HbF were higher in G1 than G2 (p=0.033, p=0.0001, respectively) (Table 1). Free-Hb, LDH and isoforms LDH-1, -2, and -3 were lower in G1 (p=0.023, p=0.003, p=0.005, p=0.001, p=0.002, respectively). There were correlation between free-Hb and LDH-1 (r=0.45, p=0.003), LDH-2 (r=0.80, p=0.001), and LDH-3 (r=0.71, p=0.001). As expected, Hp was diminished in all patients, however G1 patients showed higher values (p=0.040). The expression of band 3 and of CD59, in erythrocytes and Mcv, were higher in G1 (p=0.001, p<0.0001 and p<0.0001, respectively). Other variables showed no statistical difference. DISCUSSION AND CONCLUSIONS: The beneficial effects of HU include decrease hemolysis, although the associated mechanisms are still unknown. Total LDH and isoforms levels were decreased in patients using HU. LDH-2 and LDH-3 showed an important correlation with free-Hb. Despite the fact that LDH-3 has been considered a nonspecific marker of tissue damage and frequently associated with lung injury, our results suggest that LDH-3 may be a valuable marker of hemolysis. The use of HU was also associated with higher levels of Hp, reinforcing its possible role on intravascular hemolysis. The higher band-3 and CD59 expression in HU patients support the protective action of this drug in sickle erythrocytes, since lower expression of these proteins have been associated with erythrocyte aging. These data present new and important insights on the effects of HU in sickle erythrocyte survival. This work was supported by FAPESP (2011/17349-0) and CAPES-SUS. Table 1: Laboratorial data. G1with HU G2without HU p Mean corpuscular volume (fL) 111.0 (86.6 - 125.0) 86.8 (71.9 - 106.6) < 0.0001 Fetal Hemoglobin (HbF, %) 12.6 (1.5 - 46.6) 6.9 (0.8 - 27.1) 0.0337 LDH (U/L) 351 (214 - 728) 526 (174 – 1,112) 0.0035 LDH -1 (U/L) 154.9 (72.1 - 361.0) 225.7 (54.1 -3,248) 0.0054 LDH -2 (U/L) 132.5 (86.5 - 361.4) 203.2 (71.9 - 421.4 ) 0.0013 LDH -3 (U/L) 33.7 (25.7 - 69.9) 56.0 (28.0 - 111.2) 0.0025 Free Hemoglobin (free-Hb, mcg/dL) 51.2 (15.1 - 248.1) 104.6 (24.9 - 286.1) 0.0239 Haptoglobin (Hp, mcg/dL) 0.6 (0 - 90.8) 0.1 (0 - 56.59) 0.0406 Microvesicles (Mcv, /µL) 21,643 (5,322 – 51,726) 32,185 (8,708 – 114,384) 0.0639 Howell Jolly in erytrocytes (x106) 5,350 (1,800 – 164,850) 2,750 (100 -75,850) 0.0829 Band-3 (MFI) 396.0 (291.6 - 461.5) 342.2 (217.7 - 388.9) 0.0010 CD47 in Ret (MFI) 164.7 (95.6 - 230.8) 146.6 (96.4 - 226.7) 0.2913 CD59 in erytrocytes (MFI) 1,039 (749.9 – 1,202) 808.0 (523.5 -1,032) <0.0001 CD59 in Mcv (MFI) 830.7 (636.4 - 1.023) 604.8 (495.9 - 802.2) <0.0001 HU = hydroxycarbamide; Ret= reticulocytes; LDH= Lactate Dehydrogenase ; MFI= median fluorescence intensity Disclosures No relevant conflicts of interest to declare.