Development and functional verification of CAR-T cells targeting CLL-1 / 中华血液学杂志

Objective: To explore the development of a CAR-T cells targeting CLL-1 and verify its function. Methods: The expression levels of CLL-1 targets in cell lines and primary cells were detected by flow cytometry. A CLL-1 CAR vector was constructed, and the corresponding lentivirus was prepared. After infection and activation of T cells, CAR-T cells targeting CLL-1 were produced and their function was verified in vitro and in vivo. Results: CLL-1 was expressed in acute myeloid leukemia (AML) cell lines and primary AML cells. The transduction rate of the prepared CAR T cells was 77.82%. In AML cell lines and AML primary cells, CLL-1-targeting CAR-T cells significantly and specifically killed CLL-1-expressing cells. Compared to untransduced T cells, CAR-T cells killed target cells and secreted inflammatory cytokines, such as interleukin-6 and interferon-γ, at significantly higher levels (P<0.001) . In an in vivo human xenograft mouse model of AML, CLL-1 CAR-T cells also exhibited potent antileukemic activity and induced prolonged mouse survival compared with untransduced T cells [not reached vs 22 days (95%CI 19-24 days) , P=0.002]. Conclusion: CAR-T cells targeting CLL-1 have been successfully produced and have excellent functions.

Evaluación de la respuesta inmunológica del Apis Mellifica homeopatizada en cultivos de células mononucleares en sangre periférica / Evaluation of the immune response of the homeopathized Apis Mellifica in cultures of mononuclear cells in peripheral blood

Introducción: La Apis Mellifica, uno de los medicamentos homeopáticos utilizado por la homeopatía que tiene efectos sobre al sistema inmune, por ejemplo: actúa sobre la basófilos, provocando una inhibición en la liberación de histamina. El objetivo de este estudio es evaluar la respuesta de las células mononucleares frente al Apis Mellifica homeopatizada. Materiales y Métodos: La medición de los niveles de las citoquinas IL-1ß, IL- 2, IL-4, IL-6, IL-8 e IL-10, se determinaron cuando las células mononucleares en sangre periférica humana se expusieron al Apis Mellifica homeopatizada a la potencia 30 CH, mediante el ensayo "Cytokine Human Panel" de Invitrogen®. Resultados: Las células mononucleares crecieron un 18% en comparación con el grupo control, mostrando efectos mitogénicos del Apis Mellifica homeopatizada sobre las células mononucleares. Los resultados de los niveles de citoquinas IL-1ß,IL- 2, IL-4, IL-6, IL-8 e IL-10, medidos en el equipo de Luminex®, no evidencio modificación estadísticamente significativa de las mismas por parte del Apis Mellifica homeopatizada. Conclusiones: Los resultados del estudio mostraron un efecto estimulante de proliferación de las células mononucleares expuestas al Apis Mellifica homeopatizada, este estudio, deja la puerta abierta, para la realización de nuevos estudios.

Expression of discoidin domain receptor 2 in different phases of alcoholic liver fibrosis in a rat model / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the expressions of discoidin domain receptor 2 (DDR2) in different phases of alcoholic liver fibrosis (ALF) in a rat model and to study the possible association between DDR2 and collagen deposition in ALF.</p><p><b>METHODS</b>After an ALF rat model was established by alcohol gastrogavage and an olive oil diet, the liver histopathology was observed in different phases of the development of fibrosis. The expressions of DDR2 mRNA and protein were also detected by RT-PCR and Western blot respectively to make a dependability analysis with the index of ALF.</p><p><b>RESULTS</b>(1) The expressions of DDR2 mRNA and protein increased gradually along with ALF aggravation. In the normal control group, they were respectively 1.023+/-0.132 and 0.321+/-0.027; in the model 1 group (week 12) they were 3.644+/-1.686, 0.476+/-0.046; in the model 2 group (week 16) they were 8.337+/-2.387, 0.738+/-0.057; and in the model 3 group (week 20) they were 15.730+/-4.569, 0.997+/-0.049. The differences of DDR2 mRNA (F = 21.74, P less than 0.01) and protein (F = 10.38, P less than 0.01) among these four groups were significant. (2) The expressions of DDR2 had a positive correlation with collagen type I, III, IV contents and the serum index of ALF, especially with type III and IV collagen and serum hexadecenoic acid.</p><p><b>CONCLUSION</b>The expression of DDR2 in this ALF model correlates closely with collagen deposition in the liver, suggesting that it may play an important role in ALF pathogenesis.</p>

Expression of Ca2+ -activated K+ Channels and Their Role in Proliferation of Rat Cardiac Fibroblasts

Cardiac fibroblasts constitute one of the largest cell populations in the heart, and contribute to structural, biochemical, mechanical and electrical properties of the myocardium. Nonetheless, their cardiac functions, especially electrophysiological properties, have often been disregarded in studies. Ca2+-activated K+(KCa) channels can control Ca2+influx as well as a number of Ca2+-dependent physiological processes. We, therefore, attempted to identify and characterize KCa channels in rat Cardiac fibroblasts. First, we showed that the cells cultured from the rat ventricle were cardiac fibroblasts by immunostaining for discoidin domain receptor 2 (DDR-2), a specific fibroblast marker. Secondly, we detected the expression of various KCa channels by reverse transcription polymerase chain reaction (RT-PCR), and found all three family members of KCa channels, including large conductance KCa (BK-alpha 1- and -beta 1~4 subunits), intermediate conductance KCa (IK), and small conductance KCa (SK1~4 subunits) channels. Thirdly, we recorded BK, IK, and SK channels by whole cell mode patch clamp technique using their specific blockers. Finally, we performed cell proliferation assay to evaluate the effects of the channels on cell proliferation, and found that the inhibition of IK channel increased the cell proliferation. These results showed the existence of BK, IK, and SK channels in rat ventricular fibroblasts and involvement of IK channel in cell proliferation.

Discoidin domain receptors (DDRs) expression in pathological scar fibroblast formation / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the role of discoidin domain receptors (DDRs) in the formation of the keloid.</p><p><b>METHODS</b>The real-time quantitative PCR was used to compare the DDRs expression in the keloids and normal fibroblasts.</p><p><b>RESULTS</b>The level of DDR1 expression was significantly higher in keloid than in normal fibroblast (20.98 vs 4.2, P <0.01; 7.9 vs 4.23, P <0.05). The level of DDR1 expression in keloid was also higher significantly than that in hypertropic scar (20.98 vs 7.9, P < 0.01). However, the level of DDR2 expression was somewhat higher in keloid than in normal fibroblasts, the difference seemed not to be significantly in probability (358, 332 vs 278, P > 0.05).</p><p><b>CONCLUSIONS</b>DDRs may exert effect on keloid cell behaviours.</p>

Regular expression of discoidin domain receptor 2 in the improved adjuvant-induced animal model for rheumatoid arthritis / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the expression of discoidin domain receptor 2 (DDR2) of fibroblast-like synovial cells in improved adjuvant-induced animal (AIA) model for rheumatoid arthritis (RA) and to provide evidence for DDR2's antagonist use clinically.</p><p><b>METHODS</b>AIA was modified by administrating 0.1 mL of complete Freund's adjuvant (CFA, mixed with 5 mg Bacillus Calmette-Guerin vaccine/mL) into rats' right hind paws and 0.125 mL tumor necrosis factor-alpha (2 U/mL) into right ankles and subpatellar fatty tissue. The expression of DDR2 in fibroblast-like synovial cells was assessed using immunohistochemistry, immunofluorescence histochemistry, and in situ hybridization methods. Levels of anti-collagen II antibody were measured using enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>Given the terms mentioned above, we found a more practical rat model, apparently decreasing immunization time (average 3-5 days). DDR2 can be detected upon the 15th day of immunization; expression gradually increased with time going on, and reaching a peak 35 days after immunization before gradually decreasing. Serum anti-collagen II antibody showed similar expression patterns as DDR2, but reached peak later than DDR2, about 40 days after immunization.</p><p><b>CONCLUSION</b>Regular expression of DDR2 in animal models infers its important role in the pathological process of RA.</p>

Frecuencia de la variante aint del antígeno A / Frecuency of aint antigen

Se definen como A intermedio (Aint) los hematíes que comparten caracteres con A1 y A2. Existen diferencias cualitativas y cuantitativas en los epitopes A. Los hematíes A, son aglutinados por la lectina de Dolichus biflorus (anti-A1), los A2 por la lectina Ulex europaeus (anti-H), en tanto los hematíes Aint son variablemente aglutinados por ambas de manera inesperada. Se realizó una búsqueda de individuos Aint en una población hospitalaria de acuerdo con la reacción con las lectinas mencionadas evaluando el proceso de aglutinación producida mediante una técnica cinética. Se caracterizó la actividad aglutinante de la lectina de Amaranthus hypochondriacus (específica para N-acetil-D-galactosamina) antes y después de la incubación con saliva A y frente a eritrocitos A1, Aint y A2. Se estudiaron 458 muestras, resultando 242 O (52,8 por ciento), 170 A (37,1 por ciento), 38 B (8,3 por ciento), 5 A1B (1,1 por ciento) y 3 A2B (0,7 por ciento). Las muestras A se subdividieron en 138 A1 (81,2 por ciento), 23 A2 (13,5 por ciento) y 9 Aint (5,3 por ciento). La comparación de las curvas de cinética obtenidos permite diferenciar los hematíes de grupos A1, Aint y A2. Nuestros resultados concuerdan con observaciones previas sobre la marcada heterogeneidad de los hematíes Aint. El reconocimiento de variantes débiles del grupo A reviste importancia cuando se presentan reacciones transfusionales hemolíticas y en la práctica forense. Este estudio es relevante, en inmunogenética poblacional, por su contribución al conocimiento del mestizaje con poblaciones negras.

Differential binding properties of Gal/GalNAc specific lectins available for characterization of glycoreceptors.

Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E. coli agglutinin, Shiga toxin and Mistletoe toxic lectin-I (ML-I) and abrin-a.

Bauhinia purpurea agglutinin (BPA) binding sites in human gastrointestinal tract.

The binding of biotinylated BPA to parraffin sections of 18 normal gastrointestinal tract mucosa, 5 nonneoplastic polyps (NNP), 12 adenomas, and 59 carcinomas was studied by using avidinbiotin peroxidase complex (ABC) technique. In normal mucosa BPA appeared to bind both mucus and nonmucus glycoproteins but goblet cell mucus showed a decrease in binding and increase in binding of nonmucus glycoproteins as the cells lose their differentiation. BPA showed characteristic binding patterns in adenoma and carcinoma that differed from the pattern in normal mucosa. In normal mucosa linear binding to the apical cytoplasm in the columnar cells of the surface epithelium was observed, whereas in adenomas and carcinomas, in addition to the linear binding to the apical cytoplasm, diffuse cytoplasmic and granular deposits in the supranuclear, paranuclear or infranuclear zones were seen. Our findings suggest that BPA binding patterns in normal and neoplastic mucosa are related to the degree of cellular differentiation. In the process of malignant transformation the carbohydrate distribution undergoes progressive changes through the adenoma carcinoma sequence. These changes are related to the degree of dysplasia in adenomas and to the degree of differentiation in carcinomas.

Phagocytosis of enteropathogenic Escherichia coli and Candida albicans by lectin-like receptors

1. Ingestion of enteropathogenic Escherichia coli or Candida albicans by thioglycollate-elicited macrophages and polymorphonuclear leukocytes was investigated in vitro, 2. Goat antiserum against mannose receptors caused about 50% inhibition of E. coli phagocytosis and about 90% inhibition of C. albicans phagocytosis. 3. E. coli and C. albicans uptake was inhibited by about 60% and 98%, respectively, by plating the macrophages onto substrates coated with poly-L-lysine-mannan. Further addition of 50 mM mannose to the medium significantly increased the inhibition of phagocytosis of E. coli by macrophages from 60.7 +/- 1.5 to 79.8 +/- 13.1 and by polymorphonuclear cells from 58.9 +/- 3.7 to 88.7 +/- 4.9. 4. Preincubation of phagocytic cells with antiserum against substance A of human erythrocytes reduced E. coli ingestion by 95%, but this inhibition was not observed when the antiserum was incubated with N-acetylgalactosamine (50 mM) before being added to the phagocytes. The phagocytosis of C. albicans was not inhibited by anti-substance A antiserum. 5. The phagocytosis of E. coli was inhibited by about 25% by the addition of 7.8 micrograms/ml soluble mannan to the medium, and by about 50% by the addition of 50 mMN-acetylgalactosamine; when both substances were added to the medium, an additive inhibition of about 75% was observed. 6. These results indicate that mannose receptors on the surface of phagocytic cells mediate E. coli or Candida albicans uptake and that the binding of bacteria to N-acetylgalactosamine residues from the membrane of phagocytes is also involved in the phagocytosis of E. coli

Lectin receptors distribution in the surface membrane of Trypanosoma cruzi blood forms collected from mice submitted to specific treatment

The author investigated the distribution of lectin receptors on Trypanosoma cruzi blood forms collected from mice inoculated with, respectively, the drug-resistant and drug-sensitive strains VL-10 and CL, and treated with the two standard active nitroheterocyclic compounds nifurtimox and benznidazole used for treatment of human Chagas' disease. Blood trypomastigotes purified in Fycoll-Hypaque were incubated with fluorescein-labelled lectins Con A, WGA, EE, WFA, TPA and PNA and then microscopically examined. Neither qualitative or quantitative differences in the fluorescence intensity could be detected between parasites from VL-10 and CL strains submitted or not to treatment. The results suggest that both strains do not differ in their surface membrane carbohydrate moieties. Moreover, the rapid clearance of blood forms the drug-sensitive strain in animals treated with singlo doses of both compounds is not likely to depend on membrane alterations expressed by changes in the carbohydrate components. furthermore, resistance or sensitivity to drugs is not apparently related to carbohydrate distribution on T. cruzi blood forms

Plant lectins--histochemical and cytochemical applications in oncology.

Lectins are sugar binding proteins or glycoproteins of non-immune origin derived from various plants or animals with specific sugar binding capacity. This property of lectins can be used to identify structural differences between normal and malignant cells. Malignant transformation is accompanied by several changes in cell membrane. Studies have shown that the lectin binding pattern of these cells may indicate the invasive potential of tumours. Lectins can also be used as carriers. Lectins conjugated to chemotherapeutic agents has been found to be more useful in the treatment of tumours induced in animals.

Native plasma kallikrein is cleared at similar rates by mammalian and avian livers

Rat plasma kallikrein (RPK) is a serine protease that circulates as an inactive precursor, prokallikrein, and once activated is efficiently cleared by the liver by a carbohydrate-dependent, Ca2+ - independent mecanism. Seven hepatic lectin systems have been described for mammals but not all of these animal lectins are expressed in the avian liver. Using a liver perfusion system we compared the plasmakallikrein clearance of rats (N=10) and pigeons (N+4). Our results show that the lectin responsible for the hepatic clearance of plasma kallikrein is also present in pigeon liver and that this organ clears the enzyme with an efficiency (11.4 ñ 1.3 pmol/g,20 min) similar to that of the rat liver (10.0 ñ 0.7 pmolg, 20 min)_

Receptor de interleucina-2 (segunda parte) / Interleukin-2 receptor (second part)

Como todo factor de crecimiento, la interleucina-2 (IL-2) necesita unirse a un receptor específico, el IL-2 (IL-2R), para mediar su actividad. Este receptor se diferencia de los estudiados en endocrinología en que un factor no específico (IL-2) promueve una expansión clonal antígeno específica, sin inducir los IL-2R hasta que el receptor de antígenos es activado. El sistema se complica al descubrirse dos tipos de receptores para el IL-2, los de alta y baja afinidad. La explicación molecular, genética e importancia biológica del IL-2R se describe en el presente artículo, así como la trascendencia del empleo de las técnicas de anticuerpos monoclonales y de recombinación genética en la caracterización del IL-2R. Y teniendo como base dicha caracterización explicar algunos de los mecanismos que se proponen para la transducción de la señal y que culminan con la inducción de nuevos receptores para IL-2 y una proliferación ceclular

Receptor de interleucina-2 (primera parte) / Receptor of interleukin-1 (first part)

Como todo factor de crecimiento, la interleucina-2 (IL-2) necesita unirse a un receptor específico, el IL-2 (IL-2R), para mediar su actividad. Este receptor se diferencia de los estudiados en endocrinología en que un factor no específico (IL-2) promueve una expansión clonal antígeno específica, sin inducir los IL-2R hasta que el receptor de antígeno es activado. El sistema se complica al descubrise dos tipos de receptores para el IL-2, los de alta y baja afinidad. La explicación molecular, genética e importancia biológica del IL-2R se describe en el presente artículo, así como la trascendencia del empleo de las técnicas de anticuerpos monoclonales y de recombinación genética en la caracterización del IL-2R. Y teniendo como base dicha caracterización explicar algunos de los mecanismos que se proponen para la transducción de la señal y que culminan con la inducción de nuevos receptores para IL-2 y una proliferación celular

An enzyme-linked lectin assay for the study of lectin receptors of Leishmania

An enzyme-linked lectin assay (ELLA) based on the ELISA assay, using intact formalin-fixed promastigotes to coat poly-L-lysine-treated microtiter plates is described. The assay was used to study the lectin receptors of Leishmania donovani chagasi, L. donovani donovani and L. mexicana amazonensis. ConA, RCA, WGA, and PNA receptors were found in the three parasites. SBA receptors were found to be as frequent as the other receptors in L. donovani chagasi but not in the other two parasites which showed little SBA binding. Trypsin treatment of the two L. donovani subspecies did not remove any of the lectin receptors studied