The role of transferrin receptor CD71 as a diagnostic and prognostic biomarker in sepsis / 中华危重病急救医学

Objective:To find new biomarkers for the diagnosis and prognosis of sepsis through analyzing the differential expression protein in sepsis by proteomics and bioinformatics analysis and enzyme linked immunosorbent assay (ELISA).Methods:Patients with sepsis admitted to the emergency department of the Affiliated Hospital of Southwest Medical University from January to December 2019 were enrolled. And meanwhile, healthy volunteers who had normal physical examinations were included as the control group. Blood samples from two groups were collected. The samples were randomly selected for the protein concentration by data independent acquisition (DIA). Bioinformatics method was used in differentially expressed proteins by gene ontology (GO) pathway, enrichment analyses, groups meta-analysis and survival curves construction. ELISA method was used to verified marker screened. Then the data of transferrin receptor CD71 and the clinical data of procalcitonin (PCT), C-reactive protein (CRP) and blood lactic acid (Lac) were collected to construct receiver operator characteristic curve (ROC curve), and biomarker was screened for diagnostic and prognostic of sepsis.Results:The result of DIA showed that 71 differentially expressed proteins were screened out from sepsis group, 6 proteins were down-regulated and 65 proteins were up-regulated. Those differentially expressed proteins were enriched in the inflammatory response, response to stress, leukocyte migration in the GO pathway and enrichment analyses. The meta-analysis showed that the expression level of CD71 was higher in sepsis group than normal control group [standardized mean difference ( SMD) = -0.47, 95% confidence interval (95% CI) was -0.93 to 0.00, P < 0.01], the expression level of CD71 was higher in non-survivor group than survivor group ( SMD = -0.44, 95% CI was -0.70 to -0.18, P = 0.63). Survival curve showed that the expression of CD71 was inversely correlated to survival rates, the patients with a lower expression had higher survival rates ( P = 0.000 34); the ELISA showed that the level of CD71 was higher in sepsis group than normal control group (nmol/L: 156.83±84.71 vs. 87.99±47.89, P < 0.05), the level of CD71 was higher in non-survivor group than survivor group (nmol/L: 219.63±125.59 vs. 130.97±40.45, P < 0.05). The area under the ROC curve (AUC) of CD71 in diagnostic performance of sepsis was 0.790 (sensitivity was 65.1%, specificity was 90.0%), the AUC of CD71 in prognostic performance of sepsis was 0.744 (sensitivity was 57.1%, specificity was 94.1%); CD71 had a better prognostic performance than PCT (AUC = 0.547, sensitivity was 64.3%, specificity was 55.9%), CRP (AUC = 0.594, sensitivity was 64.3%, specificity was 61.8%), Lac (AUC = 0.540, sensitivity was 42.9%, specificity was 82.4%). Conclusion:CD71 had a great value of diagnostic and prognostic performance in sepsis, and it was expected to be a potential biomarker for sepsis.

Role of CD71 in acute leukemia– An immunophenotypic marker for erythroid lineage or proliferation?

Background: CD71 or Transferrin receptor is expressed on the surface of erythroid lineage cells. CD71 expression has been found to be significantly increased in rapidly proliferating cells. Methods: This cross-sectional study included 37 bone marrow samples of acute leukemia cases diagnosed between October 2016 to April 2018. The samples were analysed on BD FACS Canto II. We evaluated the expression of CD71 on leukemic blasts and compared median fluorescent intensities (MFI) of blasts in different types of acute leukemias. Results: The 37 cases comprised of 21 Acute Myeloid Leukemia (AML), 13 B-Acute Lymphoblastic Leukemia (B-ALL), 2 T- Acute Lymphoblastic Leukemia (T-ALL) and 1 mixed phenotypic acute leukemia (MPAL), T/Myeloid. CD 71 expression was noted in 70.3% (n= 26/37) of acute leukemia cases. CD71 expression was most commonly observed in AML (n= 15/21;71.4%), followed by B-ALL (n= 9/13;69.2%) and T-ALL (n= 1/2;50%). Single case of MPAL revealed blasts positive for CD71. MFI of leukemic blasts of single CD71 positive T-ALL was found to be highest, followed by AML, MPAL (T/Myeloid) and least in B ALL. Of the AML cases, the blasts of AML-M6, acute promyelocytic leukemia and AML-M1 showed higher CD71 expression in terms of MFI. Conclusions: Surface CD71 expression is not only found in erythroid lineage cells, but also in proliferating cells. CD71 MFI is highest in T lymphoblasts followed by leukemic erythroblasts, myeloblasts and least in B lymphoblasts.

Effect of siwutang on recovery of anemia induced by chemotherapy in mice and its mechanism / 吉林大学学报(医学版)

Objective: To study the recovery effect of Siwutang on the anemia induced by chemotherapy in the mice, and to clarify its mechanism. Methods: A total of 24 mice were randomly divided into control group (n=8, not given any drugs), model group (n= 8, given 5-fluorouracil), Siwutang group (n= 8, given 5-fluorouracil + Siwutang). After administration for 15 d, the peripheral blood of mice was taken, and the morphology and the number of red cells were observed under microscope. The number of peripheral blood red cells and the levels of hemoglobin of the mice in various groups were calculated by full-automatic hemocytometer; Double staining was used to mark the surface antigens of red cells, then antibody binding and staining were performed. Flow cytometry was used to detect the number of peripheral blood-labelled and spleen-labelled red cells. Results: Compared with control group, the number of red cells in peripheral blood of the mice in model group was significantly decreased (P<0. 05), and the cells shrinkaged. Compared with model group, the number of red cells in peripheral blood of the mice in Siwutang group was significantly increased (P<0. 05) and the cells showed rounding. Compared with control group, the level of hemoglobin in peripheral blood of the mice in model group was significantly decreased (P<0. 05); compared with model group, the level of hemoglobin in peripheral blood of the mice in Siwutang group was significantly increased (P<0. 01). The flow cytometry results showed that compared with control group, the expression level of CD71/Terll9 in peripheral blood of the mice in model group was significantly decreased (P<0. 01), and the number of red cells in various quadrants was decreased (P<0. 01); compared with model group, the expression level of CD71/Terll9 in peripheral blood of the mice in Siwutang group was significantly increased (P<0. 01), and the number of red cells in various quadrants was increased (P<0. 01); compared with control group, the expression level of CD71/Terll9 in spleen blood of the mice in model group was significantly decreased (P< 0. 01), and the number of cells in various quadrants was decreased (P<0. 01); compared with model group, the expression level of CD71/Terll9 in spleen blood of the mice in Siwutang group was significantly increased (P<0.01), the number of red cells in UL quadrant was decreased (P<0. 01), and the number of red cells in UR and LR quadrants was increased (P<0.01). Conclusion: Siwutang can promote the recovery of anemia induced by chemotherapy in the mice, increase the number of red cells, improve the morphology of red blood cells, increase the level of hemoglobin in peripheral blood, and increase the expression levels of marker antigens on the surface of red cells in the peripheral blood and spleen.

Influence of CD96 and CD71 expressions in surface of leukemia stem cells in therapeutic effects and prognosis of children with acute leukemia / 吉林大学学报(医学版)

Objective:To observe the influence of the CD96 and CD71 expressions in the surface of leukemia stem cells (LSC) in the therapeutic effects and prognosis of the children with acute leukemia,and to clarify the relationships between the molecular biological characteristics of LSC in the children with leukemia and their therapeutic effects and prognosis.Methods:Eighty children with acute leukemia were selected as the subjects.Among them, 39 cases were acute lymphoblastic leukemia (ALL) and 41 cases were acute myeloid leukemia (AML).The CD96 and CD71 expressions on the surface of LSC were detected with flow cytometry;the therapeutic effects of the first cycle chemotherapy, the survival rate of 5-year, the incidence of infection after chemotherapy, the recurrence rate after chemotherapy, and the incidence of extramedullary infiltration of the children were observed and compared.Results: The CD96 expression on the surface of LSC was positive in 38 cases (47.5%) and the CD71 expression on the surface of LSC was positive in 45 cases (56.3%);the difference of positive expression rates of CD96 and CD71 was not significant (χ2=1.227, P=0.268).The positive expression rates of CD96 and CD71 on the surface of LSC of the children with AML were significantly higher than those in the children with ALL, and the differences were statistically significant (χ2=22.225, χ2=34.028, P<0.01).The distribution of therapeutic effects and the clinical efficiency of the first cycle chemotherap, in the children with negative CD96 expression on the surface of LSC were superior to those with positive CD96 expression on the surface of LSC;the distribution of therapeutic effects and the clinical efficiency of the first cycle chemotherapy in the children with negative CD71 expression on the surface of LSC were superior than those with positive CD71 expression on the surface of LSC;the differences between two groups were statistically significant (χ2=11.323, χ2=16.589, P<0.05;U=2.939, U=2.291, P<0.05).The survival rate of 5-year in the children with negative CD96 expression on the surface of LSC was higher than those with positive CD96 expression;the incidence of infection after chemotherapy,the recurrence rate after chemotherapy and the incidence of extramedullary infiltration were lower than those with positive CD96 expression.The incidence of infection after chemotherapy and the recurrence rate after chemotherapy in the children with negative CD71 expression on the surface of LSC were lower than those with positive CD71 expression,and the differences between two groups were statistically significant (χ2=5.051,χ2=13.288, P<0.05).Conclusion: The expressions of CD96 and CD71 on the surface of LSC in the children with acute leukemia has relationship with the subtypes of the disease and the therapeutic effects of the first cycle chemotherapy, which can be used as markers for the diagnosis and evaluation of therapeutic effects.The expression level of CD96 is related to the prognosis of the patients, which can be used as an indicator for predicting the prognosis of patients.

Expression of CD71 by flow cytometry in acute leukemias: More often seen in acute myeloid leukemia.

Background: CD71 is a marker that has been usually used for identifying dysplasia in the erythroid series. We have tried to evaluate the expression of CD71 in various types of acute leukemias. Materials and Methods: We studied 48 patients of acute leukemia, of which 25 were acute myeloid leukemia (AML), 13 were precursor B‑acute lymphoblastic leukemia (B‑ALL), 8 were T‑ALL, and 2 were mixed phenotype acute leukemia (T/myeloid) as per the WHO classification. Results: We found that the expression of CD71 was most prevalent in AMLs (84%), followed by T‑ALL (50%) and least in B‑ALL (30%). Conclusion: This finding clearly shows the higher expression of CD71 in AMLs compared to other common type of leukemias, such as B‑ and T‑ALL. We suggest that the high expression of CD71 in AMLs could be used as a diagnostic marker and may also be used for minimal residual disease analysis after further studies in posttreatment scenario. This study is the first of its kind in the South Asian population.

Avaliação da expressão dos antígenos Ki-67 e CD71 em células blásticas CD34 positivas nas leucemias agudas / Determination of Ki-67 and CD71 antigens expression on CD34-positive blastic cells in acute leukaemias

Foram avaliadas a expressão de marcadores Ki-67 e CD71 nas células imaturas CD34+ em pacientes com leucemias agudas (LA) e a associação entre esses marcadores. Foram selecionados 54 pacientes com LA atendidos no Centro Oncológico de Referência do estado do Maranhão, de dezembro/2008 a novembro/2009. A expressão de Ki-67 e CD71 foi determinada por citometria de fluxo, em células positivas para o marcador de células imaturas CD34. Dos 54 pacientes, 34 (63,0%) eram portadores de leucemias linfoides agudas (LLA), destes, 73,5% eram do tipo B (LLAB) e 26,5% do tipo T (LLAT). A maior expressão de Ki-67 em medula óssea (MO) e sangue periférico (SP) foi detectada na LLAB; e, nas amostras de MO, o CD71 apresentou maior expressão na LLAT. Em SP, houve maior expressão do CD71nas LMA. Houve associação entre o Ki-67 e o CD71 em MO na LMA e, em SP, na LLAB. A expressão do Ki-67 nas leucemias agudas não diferiu nas amostras estudadas; contudo, diferenças na expressão do CD71 foram maiores na LLAT em MO e em SP na LMA. Esses achados serão úteis no diagnóstico e nomonitoramento de pacientes leucêmicos quanto à agressividade neoplásica pela detecção dos marcadoresde proliferação celular.

Childhood acute erythtoleukemia diagnosis by flow cytometry.

Acute erythroid leukemia in children is very. Here is a case of erythroleukemia in a child of a age 1.5 years, which was diagnosed on peripheral smear, bone marrow examination, cytochemistry but was confimed on immunophenotyping. CD45 versus side scatter demonstrated blast population (29%) expressing CD45 of variable intensity (dim to negative). The myeloid nature of blast population showed bright expression of CD13, CD33. These blasts also showed bright positivity of CD71 which showed erythroid nature of blasts. Flow cytometry can be comprehensive enough to completely subtype cases of leukemias/myelodysplastic syndromes, polycythemia rubra vera, non-neoplastic conditions like reactive erythroid hyperplasia following immunosuppressive therapy or viral infections or nutritional deficiencies, unlyzed RBCs or thrombocytosis which may mimic acute erythroid leukemia on flow cytometry.

pMHC/scFv fusion protein mediates killing of tumor cells by viral specific cytotoxic T cells / 中华微生物学和免疫学杂志

Objective To study whether the HBC-A2/scFv fusion protein mediates killing of tumor cells by viral specific cytotoxic T cells. Methods The fusion protein was attached to the CD71-expressing, HLA class Ⅰ negative tumor cells. And then, cytolysis by viral peptide-specific CTLs which were generated by co-culture of peripheral blood lymphocytes from HLA-A2 positive donors with inactivated T2 cells pulsed with the viral peptide were tested by lactate dehydrogenase (LDH) releasing. Results The fusion protein can attach the active viral peptide/HLA-A2 complex to K562, HepG2 and U937 cells through binding of CD71 scFv to CD71 (37.30% ±8.25%, 27.20% ±3.88%, 21.80% ±6.49% ) and mediate cytotoxicity of viral peptide-specific CTLs against those cells in vitro ( K562: 42.08% ± 1.14% vs 8.07%± 1.39%; HepG2: 49.72% ± 1.59% vs 12.46% ± 1.26%; U937: 39.72% ± 3.26% vs 7.13% ±1.48% ). Conclusion This viral peptide/HLA-A2 complex targeted by CD71 scFv is able to redirect viral peptide-specific T-cell mediated immune responses against tumor cells.

Prenatal diagnosis by isolation of fetal nucleated RBCs in maternal peripheral blood / 대한산부인과학회지

OBJECTIVE: To identify prenatal fetal sex and chromosomal aneuploidies by FISH using isolation of fetal nucleated RBCs. METHODS: peripheral blood samples was collected from 37 women between 11 and 24 weeks of gestation. we tried to enrich nucleated RBCs morphologically by Kleihaur-Betke staining after double gradient centrifugation and magnetic activating cell sorting (MACS) from maternal blood. Fluorescence in situ hybridization (FISH) analyses with CEP X and CEP Y probes for K-B positive nucleated RBCs were performed to detect whether fetal cells were existed among nucleated RBCs by observation of sex chromosomes. RESULTS: The average number of K-B positive nucleated RBCs separated from 10ml of maternal blood was 17.3 (+/-17.2) and the maximum number of nucleated RBCs was 54. We observed FISH signals in nucleated RBCs separated from 18 pregnant women, and Y probe signals were observed in 67.3% of nucleated RBCs separated from 10 pregnant women. CONCLUSION: We confirmed that separated nucleated fetal RBCs can be used to identify fetal sex and chromosomal aneuploidies by FISH. Since nucleated RBCs from maternal origin were not excluded, further studies are needed to overcome this limitation.

Receptor de transferrina (cd71) como indicador ara el diagnostico de anemia ferropénica: estudio comparativo / Transferrin receptor (cd71) as an indicator for the diagnosis of iron deficiency anemia: comparative study

El diagnóstico de la anemia por deficiencia de hierro clásicamente se realiza cuando la ferritina sérica está disminuida o cuando la administración de hierro induce un ascenso de hemoglobina. Esta deficiencia, a nivel celular se asocia con el incremento de la expresión de receptores para la transferrina en la superficie de células inmaduras eritroides o linfocitarias que responden a la demanda de hierro intracelular por sobre regulación de estos receptores. Nuestro objetivo fue estudiar la sensibilidad y especificidad del receptor de transferrina (CD71) presente en linfocitos T, frente a indicadores tradicionales utilizados para diagnosticar anemia ferropénica. Se estudiaron 1000 niños escolares de ambos sexos, de 6 a 10 años de edad matriculados en escuelas fiscales periurbanas de Cochabamba. El grupo de estudio fue conformado por 101 niños anémicos y 35 niños sanos. Se determinaron los siguientes parámetros bioquímicos: hemoglobina, hematocrito, hierro sérico, ferritina, transferrina y receptores de transferrina (CD71). El análisis de nuestros resultados reveló que la transferrina y los receptores CD71, son los únicos indicadores que discriminan exactamente a los niños anémicos por deficiencia de hierro y niños sanos. La hemoglobina tiene igual comportamiento porque fue utilizada como punto de corte para seleccionar el grupo de estudio. A diferencia de los indicadores tradicionales, la transferrina y los receptores CD71, presentaron una especificidad del 100% y sensibilidad de 88% y 99% respectivamente, lo cual significa 100% de probabilidad para el diagnóstico de niños anémicos, siendo el CD71 el indicador más sensible y específico para apreciar reservas fisiológicas de hierro y evidenciar una carencia asociada a la deficiencia del mismo.

The diagnosis of anaemia due to iron deficiency classically is made when the séric ferritin is diminished or when the iron administration induces an increase of serie haemoglobin. This deficiency is associated with increase of transferrine receptors expression at eritroid or lymphocyte immature cells surface that respond to the demand of intracellular iron by increase of receptors turnover. Our objective was to study the sensitivity and specifícity of the transferrina receptors (CD71) present in T lymphocytes, in contrast to traditional indicators used to diagnose ferropénic anemia. One thousand of scholar children of both sexes were studied, whose ages were between 6to 10 years oíd registered in periurbans public schools of Cochabamba. The study group was conformed by 101 anaemically children and 35 healthy children. The following biochemical parameters were determined: hemoglobina, hematocrito, séric iron, ferritine, transferrine and transferrine receptors (CD71). The analysis of our results revealed that the transferrine receptors CD71, are the only indicators that precisely discriminate between anaemic children due to iron deficiency and healthy children. The hemoglobine has similar behaviour because it was used as cut point to select the studied group. Unlike the traditional indicators, the transferrine and CD71 receptors respectively displayed a specifícity of 100% and sensitivity of 88% and 99%, which means 100% of probability for the diagnosis of anaemic children, being the CD71 the most sensible and specific indicator to appreciate physiological iron reserves and to demónstrate a deficiency associated to the deficiency of it.

Comparison of anti-gamma hemoglobin antibody and CD71 antibodies in isolation of fetal nucleated erythrocytes from maternal blood / 대한산부인과학회지

OBJECTIVE: In an attempt to further maximize the potential of genetic analysis from fetal cells isolation, fetal nucleated red blood cell (FNRBC) recovery with direct anti-gamma hemoglobin staining after density gradient and depletion was compared with three different whole blood magnetic separations (1-step and 2-step ferrofluid, 2-step Dynal beads). METHODS: In model systems such as quantitatively defined spikes of fetal into adult blood, as well as blood samples after surgical termination procedures, fetal cell yield and purity through the results of fluorescence in situ hybridization (FISH), quantitative real time polymerase chain reaction (PCR), and fluorescence-activated cell sorting (FACS) were calculated. RESULTS: The yield of total number of cells with a XY signal after FISH was the highest on direct anti-gamma hemoglobin staining. After normalizing the results of each experiment to the corresponding result from anti-gamma hemoglobin staining (1), ratio is 0.42 in 1-step ferrofluid, 0.33 in 2-step ferrofluid, and 0.76 in 2-step dynal beads. The fetal cell purity is clearly better in direct anti-gamma hemoglobin staining than those of the magnetic separations from whole blood. The median ratio is 56.3% in anti-gamma hemoglobin staining, 7.7% in 1-step ferrofluid, 6.5% in 2-step ferrofluid, and 31.4% in 2-step dynal beads. CONCLUSION: This study shows that the direct anti-gamma staining is the best fetal cell recovery system and it is very useful to isolate fetal nucleated red blood cells as a non-invasive genetic source.

Construction and expression of an anti-CD71 mouse/human chimeric antibody (D2C) / 中国免疫学杂志

Objective:To construct the expression vectors of VH and VL from an anti -CD71 monoclonal antibody (line7579) , and express an anti-CD71 mouse/human chimeric antibody (D2C) in vitro.Methods:The variable regions of heavy and light chains from an anti -CD71 monoclonal antibody (line7579) cloned in the pGEM-T vectors were constructed respectively into the expression vectors,p?-Expr(including human IgG1 constant region) and p?-Expr(including human Ig? constant region).The expression plasmid vectors (Chi7579-p?-Expr and Chi7579- p?-Expr) ,confirmed by a clone-PCR and a further sequence analysis, were co-transfected by electroporation into the non-Ig-producing murine hybridomas SP2/0.Results:Screening for transfectomas secreting chimeric antibody was done by cross sandwich ELISA, which identified the transfectomas to secrete a chimeric antibody (D2C) with the human heavy constant region(C?) and light constant region(C?). FCM analysis of culture supernatants of the transfectomas demonstrated that the binding capacity of the chimeric Ab (D2C) to the antigen (CD71) was retained.Conclusion:The construction of the V H and V L expression vectors and the expression of an anti-CD71 mouse/human chimeric antibody (D2C) were accomplished successfully.

Detection of fetal erythroid cells from maternal blood using fluorescence in situ hybridization and liquid culture

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis. However, the estimated ratio of fetal to maternal cells is extremely small. In order to enrich these cells, we performed direct culture using a two-phase liquid system. Mononuclear cells were obtained from maternal blood samples at 8-10(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the nonadherent cells were harvested and recultured with erythropoietin in the second phase for another 3-5 days. We examined cellular morphology, and counted the number of benzidine- positive cells and the percentage of glycophorin A/CD71 positive erythroid cells. We also did Kleihauer-Betke stain for Hb F, polymerase chain reaction (PCR) for SRY/DYZ1, chromosome analysis, and fluorescence in situ hybridization (FISH). The number of total erythroid cells reached about 0.1x10(6)-1.0x10(6)/mL with a purity of 84.0-97.3%. Hb F stain showed total erythroid cells of approximately 0.4x10(4)-9.8x10(4)/mL. Male DNA was detected in one case by PCR. In this case, the XY karyotype was confirmed by FISH and amniocentesis. This approach provides enriched source of fetal cells for further prenatal genetic analysis without complicated separation or sorting procedures.

Effect of heterogenic corneal stroma transplantation on peripheral T cells of rats / 中国病理生理杂志

0.05), the expression of CD4+ was increased in rabbit corneal stroma (P