TWO PATHWAYS OF SHEDDING OF L-SELECTIN AND CD23 FROM HUMAN B-LYMPHOCYTES

Lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) express large numbers of P2X7 receptors for extracellular adenosine triphosphate (ATP). Activation of P2X7 receptors induces multiple downstream effects, of which the best documented is the opening of an ionic channel that is selective for divalent cations. Another effect of ATP is to induce the shedding of L-selectin (CD62L), a molecule which is involved in the adhesive interactions of lymphocytes on endothelial cells. High levels of soluble L-selectin and CD23 are found in the serum of patients with B-CLL, although the mechanisms involved in their production are poorly characterized. Because extracellular ATP causes shedding of L-selectin, we studied the effect of ATP on shedding of CD23, an adhesion molecule expressed on the surface of B-CLL lymphocytes. ATP induced the shedding of CD23 at an initial rate of 12% of that for L-selectin, while the EC50 of ATP (35 uM) and BzATP (10 uM) was identical for shedding of both molecules. Inactivation of the P2X7 receptor by pre-incubation with OxATP, an irreversible inhibitor of P2X7 purinoceptor, abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2X7 receptor inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nM). Ro 31-9790, a membrane permeant zinc chelator which inhibits the phorbol-ester stimulated shedding of L-selectin also inhibited shedding of CD23 from B-CLL lymphocytes, but the IC50 was different for the two shed molecules (25 versus 1 ug/ml respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester no CD23 was lost under these conditions. Also, Ca2+ inhibits ATP-induced CD23 shedding but not L-selectin shedding. Since soluble CD23 and L-selectin are found in the serum of normal subjects and B-CLL patients, the expression of these two adhesion molecules on lymphocytes before and after transendothelial migration was studied in an in vitro model of this process. In normal and B-CLL subjects, 71±5% of L-selectin from both T and B cells and 90% of CD23 from B cells was lost following transmigration, while the expression of a range of other adhesion molecules such as VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with OxATP retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Ro 31-9790, which can protect ATP-induced both L-selectin and CD23 shedding, had no effect on inhibiting L-selectin and CD23 lost during transmigration. These data show the presence of a second pathway for the downregulation of L-selectin and CD23 from the lymphocyte surface. Data in vivo from 'knock-out' mice show that L-selectin is essential for the emigration of lymphocytes through high endothelial venules into lymph nodes. The migration of normal and B-CLL lymphocytes across confluent human umbilical vein endothelial monolayers was studied in an in vitro model of this process. Lymphocytes treated with ATP or BzATP showed 56±25% or 67±16% loss of L-selectin on the surface and 36±24% or 64±19% decrease of transmigration, respectively, while OxATP, which does not alter the L-selectin level, had no effect on lymphocyte transmigration. Further experiments examined this correlation between L-selectin expression and lymphocyte transendothelial migration in this model system. A quantitative assay for cell surface L-selectin showed that expression of L-selectin was lower on B-CLL lymphocytes (8,880±5,700 molecules/cell) than on normal lymphocytes (29,500±7,500 molecules/cell, p less than 0.001). Also the rate of transmigration of B-CLL lymphocytes (1.5±0.9 migrated cells/HUVEC) was lower than normal peripheral lymphocytes (2.4±0.9 migrated cells/HUVEC, p=0.04). Incubation of lymphocytes in complete medium for 24 hrs increased the expression of L-selectin on B-CLL lymphocytes by 1.5 to 2 fold while the normal lymphocyte L-selectin remained at the initial level. This upregulation of B-CLL L-selectin correlated with a 2 fold increased rate of transendothelial migration. A correlation was found between L-selectin expression on lymphocytes and their ability for transendothelial migration (r^2=0.6). This study shows that the adhesion molecules L-selectin and CD23 can be lost from lymphocytes by two different physiological pathways. One is by P2X7 receptor activation by extracellular ATP while the second is activated by transendothelial migration of these cells. A second finding is that B-CLL lymphocytes have lower level of L-selectin expression and an impaired ability for transendothelial migration compared with normal peripheral blood lymphocytes. Do these results explain the high serum levels of soluble L-selectin and CD23 observed in B-CLL? Although B-CLL lymphocytes do not recirculate as rapidly as normal peripheral blood lymphocytes, the greatly increased number of leukaemic cells in B-CLL ensures that much more soluble L-selectin and CD23 is generated during the recirculation of these cells through the body

THE P2X7 RECEPTOR OF HUMAN LEUKOCYTES

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms

THE P2X7 RECEPTOR OF HUMAN LEUKOCYTES

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms

Tropical mid-tropospheric CO_2 variability driven by the Madden–Julian oscillation

Carbon dioxide (CO_2) is the most important anthropogenic greenhouse gas in the present-day climate. Most of the community focuses on its long-term (decadal to centennial) behaviors that are relevant to climate change, but there are relatively few discussions of its higher-frequency forms of variability, and none regarding its subseasonal distribution. In this work, we report a large-scale intraseasonal variation in the Atmospheric Infrared Sounder CO_2 data in the global tropical region associated with the Madden–Julian oscillation (MJO). The peak-to-peak amplitude of the composite MJO modulation is ~1 ppmv, with a standard error of the composite mean < 0.1 ppmv. The correlation structure between CO2 and rainfall and vertical velocity indicate positive (negative) anomalies in CO_2 arise due to upward (downward) large-scale vertical motions in the lower troposphere associated with the MJO. These findings can help elucidate how faster processes can organize, transport, and mix CO_2 and provide a robustness test for coupled carbon–climate models

The Double-ITCZ Bias in CMIP3, CMIP5 and CMIP6 Models Based on Annual Mean Precipitation

The double‐intertropical convergence zone (ITCZ) bias is one of the most outstanding errors in all previous generations of climate models. Here, the annual double‐ITCZ bias and the associated precipitation bias in the latest climate models for Coupled Model Intercomparison Project (CMIP) Phase 6 (CMIP6) are examined in comparison to their previous generations (CMIP Phase 3 [CMIP3] and CMIP Phase 5 [CMIP5]). All three generations of CMIP models share similar systematic annual multi‐model ensemble mean precipitation errors in the tropics. The notorious double‐ITCZ bias and its big inter‐model spread persist in CMIP3, CMIP5, and CMIP6 models. Based on several tropical precipitation bias indices, the double‐ITCZ bias is slightly reduced from CMIP3 or CMIP5 to CMIP6. In addition, the annual equatorial Pacific cold tongue persists in all three generations of CMIP models, but its inter‐model spread is reduced from CMIP3 to CMIP5 and from CMIP5 to CMIP6

The Double-ITCZ Bias in CMIP3, CMIP5 and CMIP6 Models Based on Annual Mean Precipitation

The double‐intertropical convergence zone (ITCZ) bias is one of the most outstanding errors in all previous generations of climate models. Here, the annual double‐ITCZ bias and the associated precipitation bias in the latest climate models for Coupled Model Intercomparison Project (CMIP) Phase 6 (CMIP6) are examined in comparison to their previous generations (CMIP Phase 3 [CMIP3] and CMIP Phase 5 [CMIP5]). All three generations of CMIP models share similar systematic annual multi‐model ensemble mean precipitation errors in the tropics. The notorious double‐ITCZ bias and its big inter‐model spread persist in CMIP3, CMIP5, and CMIP6 models. Based on several tropical precipitation bias indices, the double‐ITCZ bias is slightly reduced from CMIP3 or CMIP5 to CMIP6. In addition, the annual equatorial Pacific cold tongue persists in all three generations of CMIP models, but its inter‐model spread is reduced from CMIP3 to CMIP5 and from CMIP5 to CMIP6

Vertical Moist Thermodynamic Structure and Spatial–Temporal Evolution of the MJO in AIRS Observations

The atmospheric moisture and temperature profiles from the Atmospheric Infrared Sounder (AIRS)/Advanced Microwave Sounding Unit on the NASA Aqua mission, in combination with the precipitation from the Tropical Rainfall Measuring Mission (TRMM), are employed to study the vertical moist thermodynamic structure and spatial–temporal evolution of the Madden–Julian oscillation (MJO). The AIRS data indicate that, in the Indian Ocean and western Pacific, the temperature anomaly exhibits a trimodal vertical structure: a warm (cold) anomaly in the free troposphere (800–250 hPa) and a cold (warm) anomaly near the tropopause (above 250 hPa) and in the lower troposphere (below 800 hPa) associated with enhanced (suppressed) convection. The AIRS moisture anomaly also shows markedly different vertical structures as a function of longitude and the strength of convection anomaly. Most significantly, the AIRS data demonstrate that, over the Indian Ocean and western Pacific, the enhanced (suppressed) convection is generally preceded in both time and space by a low-level warm and moist (cold and dry) anomaly and followed by a low-level cold and dry (warm and moist) anomaly. The MJO vertical moist thermodynamic structure from the AIRS data is in general agreement, particularly in the free troposphere, with previous studies based on global reanalysis and limited radiosonde data. However, major differences in the lower-troposphere moisture and temperature structure between the AIRS observations and the NCEP reanalysis are found over the Indian and Pacific Oceans, where there are very few conventional data to constrain the reanalysis. Specifically, the anomalous lower-troposphere temperature structure is much less well defined in NCEP than in AIRS for the western Pacific, and even has the opposite sign anomalies compared to AIRS relative to the wet/dry phase of the MJO in the Indian Ocean. Moreover, there are well-defined eastward-tilting variations of moisture with height in AIRS over the central and eastern Pacific that are less well defined, and in some cases absent, in NCEP. In addition, the correlation between MJO-related midtropospheric water vapor anomalies and TRMM precipitation anomalies is considerably more robust in AIRS than in NCEP, especially over the Indian Ocean. Overall, the AIRS results are quite consistent with those predicted by the frictional Kelvin–Rossby wave/conditional instability of the second kind (CISK) theory for the MJO

Vertical Moist Thermodynamic Structure of the Madden–Julian Oscillation in Atmospheric Infrared Sounder Retrievals: An Update and a Comparison to ECMWF Interim Re-Analysis

The large-scale vertical moist thermodynamic structure of the Madden–Julian oscillation (MJO) was documented using the first 2.5 yr (2002–05) of version 4 atmospheric specific humidity and temperature profiles from the Atmospheric Infrared Sounder (AIRS). In this study, this issue is further examined using currently available 7-yr version 5 AIRS data (2002–09) to test its dependence on the AIRS data record lengths, AIRS retrieval versions, and MJO event selection and compositing methods employed. The results indicate a strong consistency of the large-scale vertical moist thermodynamic structure of the MJO between different AIRS data record lengths (2.5 vs 7 yr), different AIRS retrieval versions (4 vs 5), and different MJO analysis methods [the extended empirical orthogonal function (EEOF) method vs the multivariate empirical orthogonal function (MEOF) method]. The large-scale vertical moist thermodynamic structures of the MJO between the AIRS retrievals and the ECMWF Interim Re-Analysis (ERA-Interim) products are also compared. The results indicate a much better agreement of the MJO vertical structure between AIRS and ERA-Interim than with the NCEP–NCAR reanalysis, although a significant difference exists in the magnitude of moisture anomalies between ERA-Interim and AIRS. This characterization of the vertical moist thermodynamic structure of the MJO by AIRS and ERA-Interim offers a useful observation-based metric for general circulation model diagnostics

Imatinib plus Granulocyte Colony-Stimulating Factor in Chronic Myeloid Leukemia Patients Who Have Achieved Partial or Complete Cytogenetic Response while on Imatinib

Background: The BCR/ABL tyrosine kinase inhibitor imatinib is highly effective in the treatment of chronic myeloid leukemia (CML) but fails to eliminate all leukemia cells. In this study, we investigated whether the addition of granulocyte colony-stimulating factor (G-CSF) could reduce the level of residual disease in patients with Ph-positive CML who appeared to have achieved a suboptimal response to imatinib alone. Methods: Eleven patients with CML who had achieved ≧35% Ph-negativity on imatinib were enrolled. The starting dose of imatinib was 400 mg or 600 mg orally daily, and of G-CSF 5 µg/kg s.c. daily. The administration of G-CSF was postponed or interrupted in the event of leukocytosis (≧30 ×109 leukocytes/l) until the white blood cell count fell below 20 × 109/l. Efficacy was assessed by serial monitoring of blood levels of BCR-ABL transcripts. Results: Of 11 evaluable patients, 9 had an appreciable decline in BCR-ABL transcript levels; in 7 cases the reduction was greater than 1 log. Conclusions: We conclude that the addition of G-CSF should be considered for patients on imatinib who fail to obtain optimal response to imatinib alone and that this approach deserves further evaluation as frontline therapy for newly diagnosed CML.Baijun Fang and Ling Mai are equal contributors

Cross-lingual Pre-training Based Transfer for Zero-shot Neural Machine Translation

Transfer learning between different language pairs has shown its effectiveness for Neural Machine Translation (NMT) in low-resource scenario. However, existing transfer methods involving a common target language are far from success in the extreme scenario of zero-shot translation, due to the language space mismatch problem between transferor (the parent model) and transferee (the child model) on the source side. To address this challenge, we propose an effective transfer learning approach based on cross-lingual pre-training. Our key idea is to make all source languages share the same feature space and thus enable a smooth transition for zero-shot translation. To this end, we introduce one monolingual pre-training method and two bilingual pre-training methods to obtain a universal encoder for different languages. Once the universal encoder is constructed, the parent model built on such encoder is trained with large-scale annotated data and then directly applied in zero-shot translation scenario. Experiments on two public datasets show that our approach significantly outperforms strong pivot-based baseline and various multilingual NMT approaches.Comment: Accepted as a conference paper at AAAI 2020 (oral presentation