Gangliosides with O-Acetylated Sialic Acids in Tumors of Neuroectodermal Origin

Gangliosides, carrying an O-acetylated sialic acid in their carbohydrate moiety, are often found in growing and developing tissues, especially of neuro-ectodermal origin. The most prominent one is 9-O-Ac-GD3, which is considered as an oncofetal marker in animal and human tumors like neuronal tumors, melanoma, basalioma or breast cancer, as well as in psoriatic lesions. Also other gangliosides like GD2 or GT3 were found to be O-acetylated in their terminal sialic acid. In this review we are summarising the occurrence of such gangliosides in normal and transformed tissues and delineate a more general theory that O-acetylated sialic acids in gangliosides are a universal marker for growing cells and tissues.     

Subpopulations of B cells in germinal centers. III. HJ6, a monoclonal antibody, binds globoside and a subpopulation of germinal center B cells.

To identify surface Ag uniquely expressed on human germinal center B cells, we produced a mouse mAb, HJ6. When tonsillar lymphocytes were examined, HJ6 did not label T cells and labeled only about half of PNA+ B cells that were HK23-. HJ6 did not label mononuclear cells from peripheral blood, splenocytes, and any of 29 cell lines including 23 B cell lines. This binding pattern of HJ6 was very similar to that of a mAb named 5B5. It was shown previously that 5B5 bound a glycolipid named CTH (CD77) and its Ag was expressed on HK23- PNA+ tonsillar lymphocytes and Burkitt's lymphoma cell lines. Despite the similarity, HJ6 differed from 5B5: HJ6 did not stain Burkitt's lymphoma cell lines and stained PNA+ tonsillar lymphocytes in the presence of a large concentration of galactose. When its binding to isolated glycolipids was studied, HJ6 was found to bind globoside and Forssman Ag and not to other glycolipids including CTH. When its binding to neutral glycolipids extracted from tonsillar lymphocytes was studied, HJ6 bound only globoside; Forssman Ag was not detected in tonsillar lymphocytes. Taken together, we conclude that globoside is a B cell Ag expressed on a subpopulation of germinal center B cells.     

Developmentally regulated O-acetylated sialoglycans in the central nervous system revealed by a new monoclonal antibody 493D4 recognizing a wide range of O-acetylated glycoconjugates

We have previously detected an alkali-labile and developmentally regulated antigen in rat embryonic cerebral cortex, which may be 9-O-acetylsialylated GT3 ganglioside (Hirabayashi Y, Hirota M, Suzuki Y, Matsumoto M, Obata K, Ando S (1989) Neurosci Lett 106:193-98). In this study we established a mouse monoclonal antibody, 493D4, that recognizes 9-O-acetyl GT3 ganglioside, but not non-O-acetyl gangliosides. This antibody also reacted with 9-O-acetyl GD3 to a much lesser extent. By using this antibody, we found that O-acetyl GT3 as well as O-acetyl GD3 were expressed strongly in fetal murine cerebral cortex and decreased to an undetectable level after birth. With the assistance of TLC-immunostaining using 493D4 together with Q-Sepharose column chromatography, O-acetyl gangliosides of bovine brain were purified and the structural analysis showed the presence of O-acetyl GD3, O-acetyl LD1, O-acetyl GD2 and O-acetyl GD1b in the adult brain as extremely minor components. Interestingly, the antibody 493D4 could detect O-acetyl sialoglycoproteins in rat brain tissues. One of the major immunoreactive proteins was shown to be synaptophysin, an integral membrane protein specifically present in synaptic vesicles. This monoclonal antibody was therefore useful for sensitive detection of both O-acetylated gangliosides and glycoproteins with O-acetylated sialic acids. This revised version was published online in November 2006 with corrections to the Cover Date.     

Participation of NK1.1+ T Cells in the Rejection of lpr αβT Cells When Bone Marrow Cells of Ipr Mice Are Transplanted into B6 Mice

When C57BL/6 (B6) mice were irradiated (9 Gy) and received bone marrow (BM) cells of B6-lpr/lpr mouse origin (i.e., lpr→B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3^? IL-2Rβ⁺ NK cells and IL-2Rβ CD3^int cells (i.e., CD3^int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1⁺ and NK1.1^?, among the IL-2Rβ⁺ CD3^int cells. However, the NK1.1⁺ subset (i.e., NK1.1⁺ T cells) was much more radioresistant, and the majority of CD3^int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6-lpr/lpr mice injected with both BM cells of B6-lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti-NK1.1 mAb or anti-asialoGM₁ antibody to eliminate NK cells alone or both NK cells and NK1.1⁺ T cells. When irradiated B6 mice were pretreated with anti-NK1.1 mAb, the mice could survive. These results suggest that intact NK1.1⁺ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double-negative CD4^?8^? cells, in B6-lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.     

Identification of 9-O-acetylated sialoglycans on peripheral blood mononuclear cells in Indian Visceral Leishmaniasis

Although the existence of O-acetylated sialic acids is well known, it is only in recent years that steady refinement of analytical techniques have enabled detailed mapping of their structural diversity [1]. Fluorimetric analysis of peripheral blood mononuclear cells (PBMC) of patients with Visceral Leishmaniasis (VL) showed six fold increase in the percentage of surface 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) as compared to normal human donors. Using Achatinin-H, a 9-O-acetyl sialic acid- binding lectin, an enhanced presence of 9-O-AcSGs in an 2 6 linkage was demonstrated by flow cytometry; abolition of its binding by pretreatment with a recombinant 9-O-acetylesterase corroborated the presence of this glycotope. Western blotting of PBMC from VL patients indicated the presence of five O-acetylated sialoglycans corresponding to 144, 65, 56, 36 and 19 kDa as compared to 144 and 36 kDa in normal individuals. Taken together our data indicates that during active disease, there is an overexpression of 9-O-AcSGs on the surface of PBMC of VL patients, thus opening up new research avenues wherein the expression of this biomarker could be exploited to monitor the clinical status of VL patients. Published in 2004..     

Isolation and properties of two sialate-<Emphasis Type="Italic">O</Emphasis>-acetylesterases from horse liver with 4- and 9-<Emphasis Type="Italic">O</Emphasis>-acetyl specificities

Sialate-O-acetylesterase was purified almost 900-fold from particle-free supernatants of horse liver by gel filtration, ion-exchange chromatography and isoelectric focussing. The native enzyme on gel filtration exhibits a molecular weight of 54,000 Da. It was separated by isoelectric focussing into two forms with pI values of 4.8 and 5.7, respectively. The esterase with a lower pI hydrolyses only 9-O-acetyl groups from sialic acids (K_M 1.1 mM), while that with the higher pI esterifies both 4- and 9-O-acetylated monosaccharides at similar rates (K_M 0.3 M and 1.3 mM, respectively). Both forms are inactive with 7-O-acetylated N-acetylneuraminic acid. Enzyme assays were carried out at the pH optimum (pH 8.4–8.6) using free O-acetylated sialic acids followed by direct analysis of the reaction products by isocratic anion-exchange HPLC. Glycosidically bound sialic acids can also be de-O-acetylated. Horse liver esterase seems to be an essential enzyme for the catabolism of 4-O-acetylated sialoglycoconjugates, since sialidase from this tissue cannot act on 4-O-acetylated sialic acids.     

Impact of Methimazole Treatment on Magnesium Concentration and Lymphocytes Activation in Adolescents with Graves’ Disease

The aim of this research was to assess plasma magnesium (Mg) concentration, the frequencies of activated T CD4+ and T CD8+ lymphocytes and B lymphocytes in adolescents with hyperthyroidism due to Graves’ disease (GD), and to assess changes in the above-mentioned parameters during methimazole (MMI) treatment. The frequencies of activated T and B cells were measured by flow cytometry method and plasma Mg concentration was determined by spectrophotometry method in 60 adolescents at the time of GD diagnosis and after receiving the normalisation of the thyroid hormones levels. The control group consisted of 20 healthy volunteers. We observed lower plasma Mg concentration, and higher frequencies of activated T and B lymphocytes in the study group before the treatment in comparison with healthy controls, and with study group in MMI-induced euthyreosis (p?<?0.01).Statistically significant negative correlations between the percentages of activated T CD3+, T CD4+, T CD8+ and B CD19+ lymphocytes, and plasma Mg concentration before the treatment were found (r?<??0.335, p?<?0.002). After the treatment no vital differences in plasma Mg concentration, and in percentages of activated cells between GD patients and controls were found, except CD8+CD25+ cells (p?=?0.03). The present study demonstrates that both activated T and B cells might play an important role in the pathogenesis of GD, and activation is related to Mg plasma level. The use of MMI in treatment of hyperthyroidism due to GD leads to decrease the frequencies of activated lymphocytes and normalisation of Mg levels.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

Relationship between CTLA-4 and CD28 molecule expression on T lymphocytes and stimulating and blocking autoantibodies to the TSH-receptor in children with Graves' disease

The present study was performed to elucidate the relationship between CTLA-4/CD28 molecules and stimulating (TSAb) and blocking (TBAb) antibodies to the TSH-receptor (TSH-R) in Graves' disease. CD28 and CD152 (CTLA-4) are glycoprotein molecules which provide a potent costimulatory signal for T-cell activation and proliferation via interactions with their ligands, B7.1/B7.2 molecules, which are present on the surface of antigen-presenting cells. The aim of the study was to estimate the expression of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152), CD28, B7.1 (CD80), and B7.2 (CD86) molecules on peripheral blood cells in patients with Graves' disease (GD) (n = 55, mean age 15.5 +/- 5.1 years) and nontoxic nodular goiter (NTNG) (n = 55, mean age 15.2 +/- 4.5 years), in comparison with sex and age-matched healthy control subjects (n = 55, mean age 15.2 +/- 3.9 years). The expression of the costimulatory molecules on mononuclear cells was analyzed by three-color flow cytometry using a Coulter EPICS XL cytometer. Detection of TSAb and TBAb to the TSH-R using JPO9 CHO cells in unfractionated serum was measured by a highly sensitive commercial radioimmunoassay. When compared with healthy control subjects and euthyroid patients with GD, untreated patients with GD showed a significant increase of CD152+ (p < 0.001, p < 0.001) and CD28+ (p < 0.01, NS) T lymphocytes, respectively. After 6-12 months of methimazole therapy, the percentage of these cells in the peripheral blood of hyperthyroid patients returned to normal values. In addition, patients with GD showed an increase in the percentage of both B7.1 (3.8%) and B7.2 (18.4%) molecules on activated monocytes, compared to patients with NTNG (0.5% p < 0.05, 2.5% p < 0.01, respectively) and healthy control subjects (0.2% p < 0.05, 0.8% p < 0.003, respectively). In patients with untreated GD there was a statistically significant positive correlation between the expression of CTLA-4 on the surface of peripheral blood T cells and the index of TSAb antibodies (R = 0.54, p < 0.001) as well as a negative correlation with TBAb antibody titer (R = -0.58, p < 0.001). However, no such correlations were noted with regard to CD28 and anti-TPO, anti-TG, and TRAb antibodies. We conclude that changes in the expression of costimulatory molecules on the surface of peripheral blood T cells and their significant relationship with the level of antithyroid antibodies indicate an involvement of these molecules in the pathogenesis of GD.     

6-O-Angeloylenolin induced cell-cycle arrest and apoptosis in human nasopharyngeal cancer cells

High incidence of lymph node spread and distant metastasis make poor prognosis in human nasopharyngeal carcinoma (NPC). Therefore, better treatments for NPC are needed. This study investigated the anticancer activity of 6-O-angeloylenolin, a plant-derived sesquiterpene lactone, on human nasopharyngeal cancer (CNE) cells. 6-O-Angeloylenolin was found to significantly inhibit the proliferation of CNE cells. The rate of inhibition was comparable to that of cisplatin, a well known chemotherapeutic agent used to treat NPC. Further mechanistic studies revealed that 6-O-angeloylenolin caused cell-cycle arrest in the S and G2/M phases and, subsequently, the induction of apoptosis. Rapid repressions of cyclin D1, cyclin D3, p27, cdc25c and p-cdc25c (Ser216) were observed after 1-h treatment, followed by decreases in the expression of CDK4, cdc2 and p-cdc2 (Tyr15) after 12 h. Down-regulations of p-Rb (Ser780), p21^Waf1/Cip1, cyclin A, and cyclin E were also detected as later events. Two early events that marked the occurrence of apoptosis were phosphatidylserine exposure and mitochondria membrane potential depletion, which occurred after 12 h of treatment, while a sub-G1 peak was also detected after 36-h treatment. Apoptosis induction was further confirmed by other apoptotic features, including nuclear fragmentation, and PARP cleavage. Moreover, 6-O-angeloylenolin caused the release of cytochrome c and AIF to the cytosol by regulating the expression of the Bcl-2 family proteins. However, pretreatment of the general caspase inhibitor failed to attenuate the apoptosis induction effect, suggesting that apoptosis induction of 6-O-angeloylenolin was independent of caspase activation. While 6-O-angeloylenolin also triggered the activation of Akt, ERK and JNK, only the JNK inhibitor significantly decreased the extent of cell death and apoptosis in CNE cells. Taken together, these results suggest the potential applicability of 6-O-angeloylenolin as a candidate for NPC treatment.     

Melanoma Cells Treated with GGTI and IFN-γ Allow Murine Vaccination and Enhance Cytotoxic Response against Human Melanoma Cells

Background

Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1].

Methodology/Principal Findings

In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-γ and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-γ and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-γ and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-γ; and iv) anti-melanoma specific cytotoxic cells.

Conclusions/Significance

These data indicate that pharmacological treatment of melanoma cell lines with IFN-γ and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.     

CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves' disease

Activation of T cells requires at least two signals transduced by the Ag-specific TCR and a costimulatory ligand such as CD28. CTLA-4, expressed on activated T cells, binds to B7 present on APCs and functions as a negative regulator of T cell activation. Our laboratory previously reported the association of Graves' disease (GD) with a specific CTLA-4 gene polymorphism. In theory, reduced expression or function of CTLA-4 might augment autoimmunity. In the present study, we categorized autoimmune thyroid disease patients and normal controls (NC) by genotyping a CTLA-4 exon 1 polymorphism and investigated the function of CTLA-4 in all subjects. PBMCs and DNA were prepared from GD (n = 45), Hashimoto's thyroiditis (HT) (n = 18), and NC (n = 43). There were more GD patients with the G/G or A/G alleles (82.2% vs 65.1% in NC), and significantly fewer patients with the A/A allele (17.8% vs 34.9% in NC). In the presence of soluble blocking anti-human CTLA-4 mAb, T cell proliferation following incubation with allogeneic EBV-transformed B cells was augmented in a dose-dependent manner. Augmentation induced by CTLA-4 mAb was similar in GD and NC (GD, HT, NC = 156%, 164%, 175%, respectively). We related CTLA-4 polymorphism to mAb augmentation of T cell proliferation in each subgroup (GD, HT, NC). Although PBMC from individuals with the G/G alleles showed 132% augmentation, those with the A/A alleles showed 193% augmentation (p = 0.019). CTLA-4 polymorphism affects the inhibitory function of CTLA-4. The G allele is associated with reduced control of T cell proliferation and thus contributes to the pathogenesis of GD and presumably of other autoimmune diseases.     

<Emphasis Type="Italic">O</Emphasis>-acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL)

Exploiting the selective affinity of Achatinin-H towards 9-O-acetylneuraminic acid(α2-6)GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac₂-GPs) on hematopoietic cells of children suffering from acute lymphoblastic leukemia (ALL), indicative of defective sialylation associated with this disease. The carbohydrate epitope of Neu5,9Ac₂-GPs_ALL was confirmed by using several synthetic sialic acid analogues. They are functionally active signaling molecules as demonstrated by their role in mediating lymphoproliferative responses and consequential increased production of IFN-γ due to specific stimulation of Neu5,9Ac₂-GPs on PBMC_ALL with Achatinin-H. Cells devoid of 9-O-acetylations (9-O-AcSA⁻) revealed decreased nitric oxide production as compared to 9-O-AcSA⁺ cells on exposure to IFN-γ. Under this condition, a decrease in viability of 9-O-AcSA⁻ cells as compared to 9-O-AcSA⁺ cells was also observed which was reflected from increased caspase 3 activity and apoptosis suggesting the protective role of this glycotope. These Neu5,9Ac₂-GPs are also capable of inducing disease-specific anti-Neu5,9Ac₂-GPs antibodies in ALL children. Additionally, we have observed that disease-specific anti-Neu5,9Ac₂-GPs have altered glycosylation profile, and they are incapable of exerting a few Fc-glycosylation-sensitive effector functions. These observations hint toward a disbalanced homeostasis, thereby enabling the cancer cells to escape host defense. Taken together, it may be hypothesized that Neu5,9Ac₂-GPs and their antibodies play a prominent role in promoting the survival of lymphoblasts in ALL.     

Changed expression of 9-O-acetyl GD3 (CDw60) in benign and atypical proliferative lesions and carcinomas of the human breast

 Expression of gangliosides is affected in various ways by malignant cell transformation. In the present study, we investigated the expression of CDw60, a constituent of O-acetylated disialogangliosides, in benign and atypical proliferative breast diseases, and preinvasive and invasive carcinomas by immunohistochemistry and thin-layer chromatography (TLC). In normal ducts, antibodies to CDw60 (mAb M-T21) reacted to membranes of the Golgi apparatus in the juxtaluminal cell compartment. A similar polarized distribution of Golgi cisterns in epithelial cells was observed in several benign lesions, i.e., fibroadenomas, intraductal papillomas, and gynecomastia. In contrast, blunt duct adenosis and duct hyperplasia exhibited an abnormal cytosolic and cell surface staining, whereas atypical duct hyperplasia showed randomly dispersed immunoreactive Golgi cisterns, indicating loss of epithelial polarity. In mammary carcinomas and in two breast carcinoma cell lines (MCF-7 and EFM-19) the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were distributed in a disorderly fashion throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Additionally, only well differentiated ductal carcinomas in situ or invasive ductal carcinomas disclosed a strong cell surface labelling, which was absent in lower differentiated carcinomas of the same types. In all carcinomas, the intensity of CDw60 immunostaining decreased with progressing loss of differentiation (grade of dedifferentiation), as demonstrated by staining intensity in paraffin sections and by evaluation of the relative amounts of extracted 9-O-acetyl GD3 by TLC. Our results indicate that abnormal CDw60 expression is already detectable in benign proliferative breast lesions with different risk rates to develop into malignant lesions. Downregulation of CDw60 expression in poorly differentiated invasive carcinomas may be the consequence of loss of cell functions usually associated with poor prognosis. Received: 19 February 1998     

Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B-cell-activating molecule (5c8 antigen) and CD40 in contact-dependent help.

In lymphoid follicles, CD4+ T lymphocytes provide contact-dependent stimuli to B cells that are critical for the generation of specific antibody responses in a process termed Th function. The CD4+ T cell-restricted surface activation protein, 5c8 Ag (T-BAM), has recently been shown to be a component of the contact-dependent helper signal to B cells. To further dissect this process, we utilized a Jurkat T cell lymphoma clone, termed D1.1, that constitutively expresses T-BAM and activates peripheral B cells to express surface CD23 in a contact-dependent mechanism that is inhibited by mAb anti-T-BAM (5c8). Similar to its effect on peripheral B cells, Jurkat D1.1 activates B cells from lymphoid organs, as well as a B cell lymphoma clone, RAMOS 266,4CN 3F10 (RAMOS 266), to up-regulate surface CD23. Interestingly, mAb to the B cell surface molecule, CD40 (mAb G28-5 and B-B20), inhibit D1.1 induced activation of RAMOS 266 and peripheral and lymphoid B cells. In contrast, mAb to CR2 or the adhesion molecules, LFA1, LFA3, or ICAM-1, have little effect. The inhibitory effect of anti-CD40 mAb on B cell activation induced by D1.1 is specific because anti-CD40 potentiates, rather than inhibits, the up-regulation of CD23 on B cells induced by rIL-4. Moreover, cross-linking CD40 molecules by anti-CD40 mAb bound to Fc gamma RII+ (CD32) L cells induces B cell CD23 expression. In vivo, T-BAM-expressing cells are CD4+ T cells that are restricted to lymphoid organs and are localized in the mantle and centrocytic zones of lymphoid follicles and the spleen periarteriolar lymphoid sheath in association with CD40+ B cells. Taken together, these data demonstrate that T-BAM on T cells and CD40 on B cells are involved in contact-dependent T-B help interactions that occur in lymphoid follicles.     

Costimulation of T-cell proliferation by a chimeric B7-antibody fusion protein

 T cells require at least two signals for activation and clonal expansion. The first signal conferring specificity is initiated by interaction of the T cell receptor with peptide-bearing MHC molecules. The second, costimulatory signal can be provided by cell-surface molecules on antigen-presenting cells such as B7-1 (CD80) and B7-2 (CD86), which interact with CD28 on T cells. To direct the costimulatory B7-2 molecule to the surface of tumor cells we have constructed a chimeric fusion protein, which consists of the extracellular domain of human B7-2 fused to a single-chain antibody domain (scFv) specific for the ErbB2 protein, a type I growth factor receptor overexpressed in a high percentage of human adenocarcinomas. This B7-2₂₂₅-scFv(FRP5) molecule, expressed in the yeast Pichia pastoris and purified from culture supernatants, binds to B7 counter-receptors and to ErbB2. B7-2_225-scFv(FRP5) localizes specifically to the surface of ErbB2-expressing target cells, thereby providing a costimulatory signal, which results in enhanced proliferation of syngeneic T cells. Accepted: 14 October 1997     

Recombinant viral sialate-O-acetylesterases

Viral O-acetylesterases were first identified in several viruses, including influenza C viruses and coronaviruses. These enzymes are capable of removing cellular receptors from the surface of target cells. Hence they are also known as receptor destroying enzymes. We have cloned and expressed several recombinant viral O-acetylesterases. These enzymes were secreted from Sf9 insect cells as chimeric proteins fused to eGFP. A purification scheme to isolate the recombinant O-acetylesterase of influenza C virus was developed. The recombinant enzymes derived from influenza C viruses specifically hydrolyze 9-O-acetylated sialic acids, while that of sialodacryoadenitis virus, a rat coronavirus related to mouse hepatitis virus, is specific for 4-O-acetylated sialic acid. The recombinant esterases were shown to specifically de-O-acetylate sialic acids on glycoconjugates. We have also expressed esterase knockout proteins of the influenza C virus hemagglutinin-esterase. The recombinant viral proteins can be used to unambiguously identify O-acetylated acids in a variety of assays. Published in 2004..     

Brucella abortus induces apoptosis of human T lymphocytes

Immune evasion is essential for Brucella abortus to survive in the face of robust adaptive CD4+ T cell response. We have previously demonstrated that B. abortus can indirectly inhibit CD4+ T cells by down-regulating MHC-II expression and antigen presentation on macrophages. However, whether B. abortus is able to directly interfere with T lymphocytes is not known. We report here that B. abortus induces apoptosis of human T lymphocytes, even though invasion of T lymphocytes was low and non-replicative. The ability of heat-killed B. abortus to reproduce the same phenomenon suggested that there was a bacterial structural component involved. We demonstrated that a prototypical B. abortus outer membrane lipoprotein (l-Omp19), but not its unlipidated form, induced T lymphocyte apoptosis. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also induced an increase in T lymphocyte cell death, indicating that the structural component implicated in the phenomenon could be any B. abortus lipoprotein. B. abortus-induced T lymphocyte apoptosis was dependent on the secretion of TNF-α since pre-incubation of T lymphocytes with anti-TNF-α mAb inhibited the apoptosis of the cells. Overall, these results represent a new mechanism whereby B. abortus by directly inhibiting T cell-mediated responses may evade adaptive immune responses.     

Characteristics of U-937 and HL-60 leukemia cells differentiated by spinach extract

Some characteristics of U-937 and HL-60 leukemia cell lines treated with a fraction of non-dialyzable extract of spinach are reported. The absorbed fraction separated by a DEAE-Tyopearl 650 column chromatography of the non-dialyzable extract induced NBT reducing activity of U-937 and HL-60 cells. This fraction also induced substrate adhesion of U-937 cells, and the non-specific esterase activity of HL-60 cells. The expression of CD11b, CD11c and CD36 antigens on the U-937 cell surface was enhanced by the treatment with the fraction, whereas CD24 antigen was not. The treatment of HL-60 cells with the fraction also induced the expression of CD11b and CD11c antigens, but CD24 and CD36 were not expressed. These results indicated that the non-dialyzable extract of spinach induced immature differentiation of U-937 and HL-60 cells into monocyte/macrophages.Abbreviations NBT nitroblue tetrazolium - TPA 12-O-tetradecanoyl-phorbol-13-acerate - PBS phosphate buffered saline - FITC fluorescein isothiocyanate