Genetic analysis of human H2N2 and early H3N2 influenza viruses, 1957-1972: evidence for genetic divergence and multiple reassortment events

Phylogenic analysis of all gene segments of human H2N2 viruses isolated from 1957 to 1968 was undertaken to better understand the evolution of this virus subtype. Human H3N2 viruses isolated from 1968 to 1972 were also examined to investigate genetic events associated with their emergence in humans and to identify the putative H2N2 ancestral virus. All gene segments of human H2N2 viruses demonstrated divergent evolution into two distinct clades (I and II) among late H2N2 isolates. All gene segments of 1968 H3N2 viruses that were retained from human H2N2 viruses were most similar to clade I H2N2 genes. However, genes of both clades were found among H3N2 isolates of 1969-1971. Unique phylogenic topologies reflected multiple reassortment events among late H2N2 or H3N2 viruses that resulted in a variety of different genome constellations. These results suggest that H2N2 viruses continued to circulate after 1968 and that establishment of H3N2 viruses in humans was associated with multiple reassortment events that contributed to their genetic diversity.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.     

Oxidation and biotinylation of beta 2 glycoprotein I glycan chains induce an increase in its affinity for anionic phospholipids similar to that obtained by the addition of anti-beta 2 glycoprotein I or anti-cardiolipin antibodies

Binding of beta 2 glycoprotein I (β₂GPI) to apoptotic cells plays a key role in the opsonization of apoptotic bodies and the formation of antiphospholipids antibodies. Here, we describe the binding of β₂GPI to apoptotic cells using β₂GPI labelled with biotin-hydrazide (β₂GPI-bh) after oxidation of its glycan chains. Flow cytometry analyses and confocal microscopy showed that β₂GPI-bh, contrary to native β₂GPI, bound to apoptotic cells, either permeable or non-permeable to propidium iodide (PI), as did annexin-V–FITC. But, in the absence of divalent ions, β₂GPI-bh, contrary to annexin V, was still able to bind to apoptotic cells. Binding equilibrium studies, performed on solid-state anionic phospholipids (AnPL), revealed that β₂GPI-bh had a greater apparent affinity for AnPL than native β₂GPI. In presence of the anti-β₂GPI mAb 8C3, the ability of native β₂GPI to bind to AnPL was increased and binding to apoptotic PI⁺ and PI⁻ CEM cells was observed whereas binding of β₂GPI-bh was barely affected by the addition of 8C3. However, the 8C3-enhanced ability of native β₂GPI to bind to AnPL was still weaker than that of β₂GPI-bh. It is not clear why the oxidation and biotinylation of glycan chains of β₂GPI increases its affinity for AnPL, but it seems that if such oxidative process occurs naturally, it could participate in enhancing antiphospholipid formation.     

Role of alpha chain-IL-2 complex in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor

Using anti-Tac (anti-alpha chain) and 2R-B (anti-beta chain) antibodies, we studied the roles of IL-2 receptor subunits (alpha and beta chains) in the formation of IL-2 and high-affinity IL-2 receptor complex, which is the initial event of IL-2 induced T cell growth. High-affinity IL-2 binding which was undetectable in the presence of 2R-B antibody at 4 degrees C became fully detectable when examined at 37 degrees C, which explained the lack of inhibition by 2R-B antibody of IL-2-induced proliferation of the cells expressing high-affinity IL-2 receptor. We further studied the mechanism of the 'reappearance' of high-affinity IL-2 binding in the presence of 2R-B antibody. The addition of IL-2 to the cells preincubated with radiolabeled or fluorescence-labeled 2R-B antibody resulted in a marked decrease in the antibody bound to the cells expressing high-affinity IL-2 receptor at 37 degrees C. This decrease was blocked by the presence of anti-Tac antibody, which inhibited IL-2 binding to alpha chain, but not by 7G7/B6 antibody, which recognized a non-IL-2 binding site of its chain. Furthermore, the decrease in cell-bound 2R-B antibody was not due to the internalization of beta chain-2R-B antibody complex, because the amount of cell-bound Mik-beta3 antibody recognizing a non-IL-2 binding epitope of beta chain remained unchanged, nor to the inhibition by simple competitive binding of IL-2 molecules to beta chain as judged from comparative studies of competitive binding inhibition. Taking these data together, the reappearance of high-affinity IL-2 binding was considered to be caused by the replacement of 2R-B antibody at the IL-2 binding site of beta chain by alpha chain-mediated IL-2, and it was strongly suggested that alpha chain-IL-2 complex has a key role in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor. alpha chain may function as a dimension converter of IL-2 to effectively deliver IL-2 molecules to a relatively small number of beta chains in the dynamics of the formation of high-affinity IL-2 binding in T cells.     

Metabolism of 2-acetylaminofluorene in the Chinese hamster ovary cell mutation assay

Chinese hamster ovary (CHO) cells were exposed to 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF), and several of their N-oxidized metabolites in order to study the mechanisms by which arylamides and arylamines produce mutations in mammalian cells. The number of mutations induced at the hypoxanthine-guanine phosphoribosyl transferase locus by each compound (mutants/10(6) CHO cells/nmol compound/ml) was estimated to be: N-acetoxy-2-AAF, 310; N-hydroxy-2-AF, 3; N-hydroxy-2-AAF (with and without hepatic S9 activation), 0.7; 2-AAF (with S9), 0.1; and 2-AF (with S9), 0.09. With each compound, DNA adducts were also identified and quantified, and in all cases the major adduct was N-(deoxyguanosin-8-yl)-2-AF. 2-AAF and N-hydroxy-2-AAF also formed minor amounts of N-(deoxyguanosin-8-yl)-2-AAF and 3-(deoxyguanosin-N2-yl)-2-AAF. The relationship between mutation induction and adduct formation for each of the derivatives was similar to that previously reported for N-hydroxy-2-AF. Inclusion of the deacetylase inhibitor, paraoxon, reduced the mutagenicity of 2-AAF, N-hydroxy-2-AAF and N-acetoxy-2-AAF, and the DNA adducts produced by N-acetoxy-2-AAF to background levels. Acetyl coenzyme A increased the mutations and CHO cytosol-mediated DNA binding of N-hydroxy-2-AAF, but did not substantially increase these responses from N-hydroxy-2-AF. N-Hydroxy-2-AAF was not detectably metabolized by CHO cells. Taken together, these data indicate that CHO cells metabolized N-acetoxy-2-AAF to a reactive derivative by N-deacetylation to N-acetoxy-2-AF, while N-hydroxy-2-AF reacted directly with DNA. The major pathway of N-hydroxy-2-AAF activation appeared to be an initial O-acetylation to N-acetoxy-2-AAF and this occurred to only a limited extent in the CHO cells. N-Hydroxy-2-AAF also seemed to form an additional unknown ester intermediate that gave rise to acetylated DNA adducts. The initial step in the activation of 2-AAF and 2-AF was an N-oxidation to N-hydroxy-2-AAF and N-hydroxy-2-AF, respectively. The limited O-acetylase activity in CHO cells appeared to contribute to the low sensitivity of these cells toward mutation induction by arylamines and arylamides.     

A paracrine role for chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) in mediating chemotactic activation of CRTH2+ CD4+ T helper type 2 lymphocytes

Activation of human CRTH2(+) CD4(+) T helper type 2 (Th2) cells with anti-CD3/anti-CD28 led to time-dependent production of prostaglandin D(2) (PGD(2)) which peaked at 8 hr. The production of PGD(2) was completely inhibited by cotreatment with the cyclo-oxygenase inhibitor diclofenac (10 microm) but was not affected by cotreatment with ramatroban, a dual antagonist of both the thromboxane-like prostanoid (TP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Supernatants from activated CRTH2(+) CD4(+) Th2 cells caused a concentration-dependent increase in the migration of naive CRTH2(+) CD4(+) Th2 cells compared to supernatants from unstimulated CRTH2(+) CD4(+) Th2 cells. The level of chemotactic activity peaked at 8 hr after activation, corresponding to the peak levels of PGD(2), but production of chemotactic activity was only partially inhibited by the cyclo-oxygenase inhibitor diclofenac. In contrast, ramatroban completely inhibited the chemotactic responses of naive Th2 cells to supernatants from activated CRTH2(+) CD4(+) Th2 cells collected up to 8 hr after activation, although supernatants collected 24 hr after activation were less sensitive to inhibition by ramatroban. The selective TP antagonist SQ29548 did not inhibit migration of Th2 cells, implicating CRTH2 in this response. These data suggest that CRTH2 plays an important paracrine role in mediating chemotactic activation of Th2 cells. Interestingly, although PGD(2) is produced from Th2 cells and contributes to this paracrine activation, it appears that additional CRTH2 agonist factors are also produced by activated Th2 cells and the production of these factors occurs independently of the cyclo-oxygenase pathway of the arachidonic acid metabolism.     

Taste responses to deuterium oxide

Substitution of deuterium oxide (D2O) as the solvent in taste stimuli elicits neural responses which differ from ordinary water (H2O). Previous reports have shown that D2O is toxic to many animals and rats avoid drinking it when offered H2O simultaneously. In the current study, summated responses were recorded from the chorda tympani nerves of rats after NaCl, KCl, sucrose and quinine were applied to the tongue in solutions of either D2O or H2O. Both solvents were used as the adapting or rinse solution in separate series. On tongues adapted to H2O, D2O elicited mean responses which were equivalent to 29% of the response to 0.1 M NaCl. The threshold concentration of D2O in H2O was between 25% and 50%. Solutes in D2O yielded responses which were greater than corresponding solutions of H2O when adapting rinse was H2O. Adaptation to D2O diminished the responses to D2O solutions of NaCl, KCl and sucrose but not quinine. This observation suggests that some portion of the augmented response to stimuli in D2O is due to the solvent itself. The taste of water has been examined by both electrophysiological methods and by behavior, but none of the mechanisms espoused for its effect seem adequate to explain the response to D2O. Water structure at the interface between molecular components of the cell membrane and the bulk phase of the surrounding medium is considered as a locus for disparity in the taste responses to D2O and H2O in the rat.     

Investigating the functional role of CD2BP2 in T cells

The adaptor protein CD2-binding protein 2 (CD2BP2) confers binding to proline-rich sequences (PRS) via its GYF domain. In addition to the cytoplasmic domain of CD2, several other proteins were identified as interaction partners of CD2BP2, but the in vivo significance of these findings is unclear. We now show that CD2BP2's nuclear localization is not changed when CD2 and CD2BP2 are co-expressed in HeLa cells, indicating that other PRS compete effectively for CD2BP2 binding in the nucleus. Since the CD2BP2-binding motifs of CD2 are known to be involved in cytokine signaling, we tested the effect of CD2BP2 knockdown in PBMCs on the expression of T-cell cytokines. No major difference in cytokine expression can be observed for primary cells transfected with CD2BP2-specific small interfering RNA. We conclude that CD2 signaling is at least partially independent of its in vitro binding partner CD2BP2.     

Cross-reactivity and clinical impact of the antibody response to hepatitis C virus second envelope glycoprotein (E2)

The genotype of hepatitis C virus (HCV) can profoundly affect the success of antiviral therapy for HCV infection. A possible contributing factor is a varied immune response elicited by infection with different HCV genotypes. In this study, full-length E2 proteins of HCV genotypes 1a, 1b, 2a, and 2b were used to determine the fraction of the humoral immune response to HCV E2 that is genotype specific. Greater than 90% of all infected individuals had serum antibodies to the four E2 proteins. Overall, individuals infected with genotype 1a or 1b were characterized by variable immune responses to HCV E2 with relatively high amounts of cross-reactivity with other E2 proteins. Individuals infected with genotype 2a or 2b exhibited a strong preferential reactivity to genotype 2a and 2b E2 proteins. Individuals with elevated titers to HCV E2 were more likely to be infected with genotype 2a and had a significantly lower median viral load. These findings indicate that the antibody response to HCV E2 is affected by the genotype of the virus and that induction of a strong humoral immune response to HCV E2 may contribute to a decreased viral load.     

The incidence of topoisomerase II-alpha genomic alterations in adenocarcinoma of the breast and their relationship to human epidermal growth factor receptor-2 gene amplification: a fluorescence in situ hybridization study

Clinical and in vitro evidence supports the concept that human epidermal growth factor receptor-2 ( HER2 ) gene amplification prediction of response to anthracycline-based chemotherapy in breast cancer is not a direct effect of HER2 overexpression, but the result of coamplification of topoisomerase II-alpha ( TOP2A ). We investigated the relationship of TOP2A to HER2 genomic alterations by fluorescence in situ hybridization (FISH) and the correlations with polysomic states for chromosome 17 (CEP17). One hundred thirty-eight cases of breast cancer HER2 gene amplified by 2-color FISH ( HER2 /CEP17) were reevaluated with a 3-color probe set ( HER2 /CEP17/ TOP2A ) to investigate the frequency of coamplification and deletion of TOP2A . TOP2A was never amplified in the absence of HER2 amplification and was coamplified with HER2 in 68 (50%) of 137 cases; HER2 gene copy number was higher than the TOP2A copy number ( P < .01). Of the 137 cases with HER2 amplification, 23 (16%) showed a monoallelic deletion of TOP2A . Of the 43 cases not amplified for HER2 , 27 (63%) were CEP17 eusomic, 13 (30%) polysomic, and 3 (7%) monosomic. Of the HER2 nonamplified cases, 2 (5%) showed monoallelic deletion of both the HER2 and TOP2A . The current study demonstrates the complex interrelationship between the HER2 and TOP2A genes in breast cancer. The clinical implications of TOP2A amplification and deletion in breast cancer need to be further defined. If TOP2A gene dosage can be confirmed to correlate with tumor responsiveness to anthracycline-based therapy in the clinical setting, FISH testing for TOP2A status may be warranted to aid in the selection of the most appropriate therapy.     

Substitutions at the Glu62 residue of human interleukin-2 differentially affect its binding to the α chain and the βγ complex of the interleukin-2 receptor

Human interleukin-2 (IL-2) α helix B is more conserved than the whole molecule, but has been less studied than other α helices of IL-2. Using site-directed mutagenesis, several IL-2 mutants in this helix were obtained. We found that the IL-2 mutant containing Leu at position 62 (Leu⁶²-IL-2) loses its ability to bind IL-2 receptor subunit α (IL-2Rα), but retains binding affinity to IL-2R subunit βγ as well as some bioactivity; nevertheless, another substitution at the same residue, Arg⁶² IL-2, loses its binding ability to both IL-2Rα and IL-2Rβγ, and can no longer stimulate IL-2-dependent cell growth, showing that Glu⁶² not only takes part in IL-2Rα binding, but can also affect IL-2 binding to IL-2Rβγ. In this regard, Glu⁶² may be a key site in the IL-2/IL-2Rα interaction, and can facilitate IL-2R ternary-complex formation, leading to IL-2Rα-mediated, IL-2-stimulated signal transduction.     

Different epitopes of the CD31 antigen identified by monoclonal antibodies: cell type-specific patterns of expression

3 mAb-5A2.G5, B2B1 and 2BD4-all of IgG1 isotype were identified as belonging to the CD31 cluster by their binding to transfected murine cell lines expressing the CD31 antigen and by sequential immunoprecipitation experiments. Competitive binding experiments were carried out using the human myelomonocytic cell line RC-2A. mAb B2B1 and 2BD4 did not cross block. mAb 5A2.G5 partly inhibited binding of 2BD4 but not B2B1. Thus the epitopes identified by 5A2.G5 and 2BD4 appear to overlap but to be quite separate from that recognized by B2B1. All 3 antibodies bound to a 130 kDa species on Western blots after nonreducing polyacrylamide gel electrophoresis of platelet lysates. Culture of RC-2A cells in the presence of tunicamycin (after removal of surface antigens by pronase) blocked re-expression of the epitopes recognised by all 3 mAb suggesting that they involve N-linked glycosylation. Furthermore, treatment of platelet lysates with Endoglycosidase F prior to electrophoresis and Western blotting abolished the binding of the mAb but not a rabbit polyclonal antiserum to CD31. Nevertheless, neuraminidase treatment of RC-2A cells failed to affect mAb binding. The antibodies displayed typical properties of CD31 antibodies in that all 3 precipitated at 130 kDa cell surface protein and bound strongly to platelets, monocytes, neutrophils and vascular endothelium. All 3 antibodies were positive on hemopoietic progenitor cells which give rise to colonies in the CFU-C assay. However, differences in binding to peripheral blood lymphocytes and to certain human leukemic cell lines were noted. In particular, mAb 5A2.G5 bound weakly to lymphocytes and to the lymphoid cell line HSB-2 compared with the other 2 antibodies.     

Influence of prostaglandin A2 and 2-methoxyestradiol on Bax and Bcl-2 expression levels in cervical carcinoma cells

Proteins of the Bcl-2 family are key regulators of apoptosis. Bax can be regarded as pro-apoptotic, whereas Bcl-2 is perceived as anti-apoptotic. It has been proposed that an increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2 can be associated with apoptosis. Since prostaglandin A2 (PGA2) and 2-methoxyestradiol (2-ME) play an active role in the induction of apoptosis, the influence of 20 microg/ml PGA2 and 1 microM 2-ME was investigated on Bax and Bcl-2 expression levels in cervical carcinoma cells. Both PGA2 and 2-ME exposure led to statistically significant increases in Bax expression levels. Cells were shown to be more susceptible to the effects of 2-ME than to the effects caused by PGA2. In contrast, no statistically significant effects were observed on Bcl-2 expression levels after exposure to PGA2 and 2-ME. The Bax/Bcl-2 ratios for PGA2- and 2-ME-exposed cells were 2.06 and 1.87 respectively, normalised against Bcl-2 levels. Further investigation of the function and regulation of the Bcl-2 family will allow researchers to consider potential pathways of apoptosis signaling mechanisms for diseases where apoptosis can potentially be controlled.     

鼠尾胶原及C-erbB-2蛋白抗体对肝癌（HepG-2）细胞粘连和增殖的影响

目的：揭示胶原及C-erbB-2蛋白抗体对肝癌细胞粘连和增殖的影响，提供抗癌治疗的线索。方法：以人肝癌细胞细胞系（HepG-2）为靶子，利用C-erbB-2抗体及鼠尾胶原体外作用于靶细胞，观察C-erbB-2抗体及鼠尾胶原对肝癌细胞粘连和增殖的影响。结果：鼠尾胶原有加强肝癌细胞粘附作用而对其增殖作用无显著影响；C-erbB-2抗体能明显抑制其细胞的增殖和粘连，也明显拮抗鼠尾胶原对其细胞粘连性的加强作用。结论：人肝癌细胞HepG-2的粘连和增殖与胶原的作用存在与否有关，这与C-erbB-2蛋白介导的信号传递作用存在着密切的关联，阻断C-erbB-2的信号传递作用对肝癌治疗是一个有前景的战略性靶点。     

Activation of caspase-3 pathway by expression of sGalphai2 protein in BHK cells

Treatment with dopamine and other dopamine D2 receptor agonists has been shown to induce cell death through activation of caspase-3 pathway. However, initial step that leads to the activation of caspase-3 in D2 receptor-mediated apoptotic pathway remains unclear. Recently, it was shown that a spliced variant of Galphai2 protein (sGalphai2) forms intracellular complex with D2 receptors by protein-protein interaction and that D2 drugs treatment causes the liberation of sGalphai2 protein from complex. Now, we show that the unbound form of sGalphai2 protein is able to activate caspase-3 pathway in baby hamster kidney (BHK) cells. Expression of sGalphai2 protein in BHK cells led to the production of active form of caspase-3 and activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated kinase 1/2 (ERK1/2). Co-expression of sGalphai2 with either D2 short (D2S) or D2 long (D2L) isoforms of dopamine D2 receptors blocked the activation of caspase-3 pathway. Thus, our results demonstrate that high level of unbound sGalphai2 protein can affect the cell survival and engagement of this protein with D2 receptors can block this process. It is suggested that this process may be a crucial step in the initiation of D2 receptor-mediated cellular apoptosis through this pathway.     

Nucleosome-binding activities within JARID2 and EZH1 regulate the function of PRC2 on chromatin

Polycomb-repressive complex 2 (PRC2) comprises specific members of the Polycomb group of epigenetic modulators. PRC2 catalyzes methylation of histone H3 at Lys 27 (H3K27me3) through its Enhancer of zeste (Ezh) constituent, of which there are two mammalian homologs: Ezh1 and Ezh2. Several ancillary factors, including Jarid2, modulate PRC2 function, with Jarid2 facilitating its recruitment to target genes. Jarid2, like Ezh2, is present in poorly differentiated and actively dividing cells, while Ezh1 associates with PRC2 in all cells, including resting cells. We found that Jarid2 exhibits nucleosome-binding activity that contributes to PRC2 stimulation. Moreover, such nucleosome-binding activity is exhibited by PRC2 comprising Ezh1 (PRC2–Ezh1), in contrast to PRC2–Ezh2. The presence of Ezh1 helps to maintain PRC2 occupancy on its target genes in myoblasts where Jarid2 is not expressed. Our findings allow us to propose a model in which PRC2–Ezh2 is important for the de novo establishment of H3K27me3 in dividing cells, whereas PRC2–Ezh1 is required for its maintenance in resting cells.     

恶性外周神经鞘膜瘤中MMP-2和TIMP-2的表达

目的 探讨恶性外周神经鞘膜瘤(malignant peripheral nerve sheath tumors,MPNST)中基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)及其相应的组织金属蛋白酶抑制剂-2(tissue inhibitor of metalloproteinase-2,TIMP-2)蛋白表达与病理分级、转移及预后的关系。方法 采用免疫组化S-P法检测MPNST中MMP-2及TIMP-2表达,并行回顾性随访。结果 58例MPNST中MMP-2阳性表达51例,阳性表达率是87．9％,TIMP-2阳性表达36例,阳性表达率是62．1％。MMP-2蛋白表达与病理学分级、远处转移率呈正相关,与术后生存率呈负相关;而TIMP-2则相反。结论 MMP-2、TIMP-2与MPNST病理学分级、远处转移及术后生存期有关,可作为判断肿瘤恶性程度及预后的有用的参考指标。     

Species-specific measurement of the second group of Dermatophagoides mite allergens, Der p 2 and Der f 2, using a monoclonal antibody-based ELISA

Background The group 2 Dermatophagoides mite allergens. Der p 2 and Der f 2, were known to he highly crossreactive, and previous assays to measure Der p 2 and Der f 2 were not species-specific. Objective The aim of this study was to develop a monoclonal antibody-based ELISA (MoAb-ELISA) to specics-spccifically measure Der p 2 and Der f 2. Methods The MoAb-ELISA lor Der p 2 and Der f 2 was performed using species-specific MoAbs for Der p 2 and Der f 2 and a biotinylated second MoAb which recognized a common epitope on both Der p 2 and Der f 2. Rcsuits The assay was highly specics-spccific, reproducible and sensitive. Thirty-two house dust samples were assayed by the MoAb-ELISA for Der p 2 and Der f 2 and by a previously reported radioimmunoassay for Der 2 with rabbit anti-Der 2 antibodies. The summed values for Der p 2 and Der f 2 by the MoAb-ELISA detnonstrated a good correlation with the Der 2 values using the radioimmunoassay (r = 0.978). Furthermore, the proportion of the Der p 2 level in the total Der 2 level (Der p 2 divided by Der p 2 plus Der f 2) correlated well with that of the D. pteronyssinus mite number to the total Dermalophagoides mite number identificd by species (r = 0.970). Conclusion The MoAb-ELISA for Der p 2 and Der f 2, as well as that for Der p 1 and Der f 1, will be useful for the standardization of mite extracts and for the assessments of mite allergen exposure.     

c‐Cbl regulates MICA‐ but not ULBP2‐induced NKG2D down‐modulation in human NK cells

The NKG2D activating receptor on human NK cells mediates “altered self” recognition, as its ligands (NKG2DLs) are upregulated on target cells in a variety of stress conditions. Evidence collected in the past years shows that, even though expression of NKG2DLs acts as a danger signal that renders tumor cells susceptible to cytotoxicity, chronic exposure to soluble or membrane‐bound NKG2DLs can lead to down‐modulation of receptor expression and impairment of NKG2D‐mediated cell functions. Here, we evaluated whether different cell‐bound NKG2DLs, namely MICA and ULBP2, are equivalently able to induce NKG2D down‐modulation on human NK cells. We found that although both ligands reduce NKG2D surface expression, MICA promotes a stronger receptor down‐modulation than ULBP2, leading to a severe impairment of NKG2D‐dependent NK‐cell cytotoxicity. We also provide evidence that the ubiquitin pathway and c‐Cbl direct MICA‐induced but not ULBP2‐induced NKG2D internalization and degradation, thus identifying a molecular mechanism to explain the differential effects of MICA and ULBP2 on NKG2D expression. A better understanding of the molecular mechanisms employed by the different NKG2DLs to control NKG2D surface expression could be useful for the development of anti‐tumor strategies to restore a normal level of NKG2D receptors on human NK cells.