Monoclonal C219 Research Articles

Isolation and Characterization of Laminin-10/11 Secreted by Human Lung Carcinoma Cells: LAMININ-10/11 MEDIATES CELL ADHESION THROUGH INTEGRIN α3β1

A panel of human tumor cell lines was screened for selective expression of laminin alpha5 chain, a newly identified laminin subunit comprising laminin-10 (alpha5 beta1 gamma1) and -11 (alpha5 beta2 gamma1). The lung adenocarcinoma cell line A549 was found to express the alpha5 chain at relatively high levels but no detectable amounts of other alpha chains. The laminin variants containing alpha5 chain were purified from the conditioned medium of A549 cells by immunoaffinity chromatography using the anti-laminin monoclonal antibody 4C7 which was shown recently to recognize the laminin alpha5 chain (Tiger, C.-F., Champliaud, M.-F., Pedrosa-Domellof, F., Thornell, L.-E., Ekblom, P., and Gullberg, D. (1997) J. Biol. Chem. 272, 28590-28595). The purified laminin variants consisted of three chains with molecular masses of 350, 220, and 210 kDa. The 350-kDa chain was specifically recognized by another anti-alpha5 chain monoclonal antibody capable of recognizing denatured alpha5 chain on immunoblots, whereas the 210-kDa chain was recognized by an anti-gamma1 chain antibody. The purified alpha5 chain-containing laminin variants (hereafter referred to as laminin-10/11) were highly active in mediating adhesion of A549 cells to the substratum with potency as high as that of laminin-5 and significantly higher than those of laminin-1, laminin-2/4, or fibronectin. Adhesion to substrata coated with laminin-10/11 was specifically inhibited by anti-integrin antibodies directed against the integrin alpha3 or beta1 subunit but not by those against alpha2 or alpha6 subunit, indicating that laminin-10/11 is specifically recognized by integrin alpha3 beta1. Given the wide distribution of laminin-10/11 in the basement membrane of various tissue types and dominant expression of integrin alpha3 beta1 in most epithelial cells, specific interaction of laminin-10/11 with integrin alpha3 beta1 may play an important role in in vivo regulation of proliferation and differentiation of epithelial cells through the basement membrane.

Cytotoxicity of alpha-particle-emitting astatine-211-labelled antibody in tumour spheroids: no effect of hyperthermia.

The high linear energy transfer, alpha-particle-emitting radionuclide astatine-211 (211At) is of interest for certain therapeutic applications; however, because of the 55- to 70-microm path length of its alpha-particles, achieving homogeneous tracer distribution is critical. Hyperthermia may enhance the therapeutic efficacy of alpha-particle endoradiotherapy if it can improve tracer distribution. In this study, we have investigated whether hyperthermia increased the cytotoxicity of an 211At-labelled monoclonal antibody (MAb) in tumour spheroids with a radius (approximately 100 microm) greater than the range of 211At alpha-particles. Hyperthermia for 1 h at 42 degrees C was used because this treatment itself resulted in no regrowth delay. Radiolabelled chimeric MAb 81C6 reactive with the extracellular matrix antigen tenascin was added to spheroids grown from the D-247 MG human glioma cell line at activity concentrations ranging from 0.125 to 250 kBq ml(-1). A significant regrowth delay was observed at 125 and 250 kBq ml(-1) in both hyperthermia-treated and untreated spheroids. For groups receiving hyperthermia, no increase in cytotoxicity was seen compared with normothermic controls at any activity concentration. These results and those from autoradiographs indicate that hyperthermia at 42 degrees C for 1 h had no significant effect on the uptake or distribution of this antitenascin MAb in D-247 MG spheroids.

Functional Expression of P-Glycoprotein in the Hepatic Canalicular Membrane of Developing Rats

□ P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte. The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22days old) and adult rats. No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations. The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp. DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups. In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue. Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, γ) between groups. Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups. Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult. Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.

Decreased expression and activity of P-glycoprotein in rat liver during acute inflammation.

Drug disposition is often altered in inflammatory disease. Although the influence of inflammation on hepatic drug metabolism and protein binding has been well studied, its impact on drug transport has largely been overlooked. The multidrug resistance (MDR) gene product, P-glycoprotein (P-gp) is involved in the active secretion of a large variety of drugs. Our goal was to ascertain the influence of acute inflammation (AI) on the expression and functional activity of P-gp. AI was induced in rats through turpentine or lipopolysaccharide (LPS) administration. Expression of P-gp in liver was detected at the level of protein on Western blots using the monoclonal antibody C-219 and at the level of mRNA using an RNase protection assay. P-gp mediated transport activity was assessed by measuring the verapamil-inhibitable efflux of rhodamine 123 (R123) in freshly isolated hepatocytes. Turpentine-induced AI significantly decreased the hepatic protein expression of P-gp isoforms by 50-70% and caused a significant 45-65% reduction in the P-gp mediated efflux of R123. Diminished mRNA levels of all three MDR isoforms were seen. LPS-induced AI similarly resulted in significantly reduced levels and activity of P-gp in liver. Although differences in the constitutive levels of P-gp were seen between male and female rats, the influence of AI on P-gp expression and activity was not gender specific. Experimentally-induced inflammation decreases the in vivo expression and activity of P-gp in liver. This is the first evidence that expression of P-gp is modulated in response to experimentally-induced inflammation.

MDR1 in taste buds of rat vallate papilla: functional, immunohistochemical, and biochemical evidence.

Multidrug resistance P-glycoprotein (MDR1) is a membrane protein of 150-170 kDa that catalyzes the ATP-driven efflux of hydrophobic xenobiotics, including fluorescent dyes, from cells. Expressed in many epithelial tissues and in the endothelia of the blood-brain barrier, the MDR1 protein provides major routes of detoxification. We found that taste cells of the rat vallate papilla (VP; posterior tongue) had only a slow increase in fluorescence due to uptake of the hydrophobic dye calcein acetoxymethyl ester. However, the development of fluorescence was accelerated two- to threefold by substrates and/or inhibitors of MDR1, such as verapamil, tamoxifen, and cyclosporin A, and by addition of the transport-blocking antibody to MDR1, UIC2. Western blots of vallate tissue rich in taste buds with the MDR1-specific monoclonal antibodies C219 and C494 revealed an immunoreactive protein at approximately 170 kDa. In contrast, the lingual epithelium surrounding the VP showed a much weaker band with these antibodies. Furthermore, using the antibodies C494 and UIC2 with tissue sections, MDR1-like immunoreactivity was found in taste cells. These results show that MDR1 is present and functional in vallate taste cells of the rat. MDR1-related transport may achieve active elimination of xenobiotics from the sensory cells and thereby protect the peripheral taste organs from potentially harmful molecules contained in an animal's food.

Evaluation of the role of P-glycoprotein in ivermectin uptake by primary cultures of bovine brain microvessel endothelial cells.

The P-glycoprotein efflux system located on the apical membrane of brain capillary endothelial cells functions as part of the blood-brain barrier. In this study, primary cultures of bovine brain microvessel endothelial cells (BMECs) were investigated for the presence of a P-glycoprotein system and its contribution in regulating ivermectin distribution across the blood-brain barrier. Results of rhodamine 123 uptake studies with cyclosporin A and verapamil as substrates indicated that a functional efflux system was present on BMECs. Immunoblot analysis with the C219 monoclonal antibody to the product of the multidrug resistant member 1(MDR1) gene also confirmed the expression of MDR1 in the BMECs. Unbound ivermectin was shown to significantly increase the uptake of rhodamine 123 in BMECs, however, the drug only modestly enhanced the transcellular passage of rhodamine. The results of these studies affirmed that unbound ivermectin is an inhibitor of the MDR1 efflux system in BMECs.

A Single Chain Fv Fragment of P-glycoprotein-specific Monoclonal Antibody C219: DESIGN, EXPRESSION, AND CRYSTAL STRUCTURE AT 2.4 Å RESOLUTION

A construct encoding a single chain variable fragment of the anti-P-glycoprotein monoclonal antibody C219 was made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding the flexible linker (GGGGS)3, an OmpA signal sequence, a c-myc identification tag, and a five-histidine purification tag. The construct was expressed in Escherichia coli and purified from the periplasmic fraction using a nickel chelate column and ion exchange chromatography. Three-step Western blot analysis showed that the construct retains binding affinity for P-glycoprotein. Crystals of 1.0 x 0.2 x 0.2 mm were grown in 100 mM citrate, pH 4.5, 21% polyethylene glycol 6000 in the presence of low concentrations of subtilisin, resulting in proteolytic removal of the linker and purification tags. The structure was solved to a resolution of 2.4 A with an R factor of 20.6, an Rfree of 28.5, and good stereochemistry. This result could lead to a clinically useful product based on antibody C219 for the diagnosis of P-glycoprotein-mediated multidrug resistance. The molecule will also be useful in biophysical studies of functional domains of P-glycoprotein, as well as studies of the intact molecule.

Presence of Laminin α5 Chain and Lack of Laminin α1 Chain during Human Muscle Development and in Muscular Dystrophies

There is currently a great interest in identifying laminin isoforms expressed in developing and regenerating skeletal muscle. Laminin alpha1 has been reported to localize to human fetal muscle and to be induced in muscular dystrophies based on immunohistochemistry with the monoclonal antibody 4C7, suggested to recognize the human laminin alpha1 chain. Nevertheless, there seems to be no expression of laminin alpha1 protein or mRNA in developing or dystrophic mouse skeletal muscle fibers. To address the discrepancy between the results obtained in developing and dystrophic human and mouse muscle we expressed the E3 domain of human laminin alpha1 chain as a recombinant protein and made antibodies specific for human laminin alpha1 chain (anti-hLN-alpha1G4/G5). We also made antibodies to the human laminin alpha5 chain purified from placenta. In the present report we show that hLN-alpha1G4/G5 antibodies react with a 400-kDa laminin alpha1 chain and that 4C7 reacts with a 380-kDa laminin alpha5 chain. Immunohistochemistry with the hLN-alpha1G4/G5 antibody and 4C7 revealed that the two antibodies stained human kidney, developing and dystrophic muscle in distinct patterns. Our data indicate that the previously reported expression patterns in developing, adult, and dystrophic human muscle tissues with 4C7 should be re-interpreted as an expression of laminin alpha5 chain. Our data are also consistent with earlier work in mouse, indicating that laminin alpha1 is largely an epithelial laminin chain not present in developing or dystrophic muscle fibers.

Cross-reactivity of C219 anti-p170(mdr-1) antibody with p185(c-erbB2) in breast cancer cells: cautions on evaluating p170(mdr-1)

Increased expression of the multidrug resistance gene (MDR-1)-encoded P-glycoprotein (p170[mdr-1]) is a major cause of tumor cell multidrug resistance. p170(mdr-1) functions as a drug-efflux pump to reduce the cellular accumulation of specific drugs. MDA-MB-435 human breast cancer cells that have been transfected with oncogene c-erbB2 complementary DNA (435.eb cells) express high levels of the transmembrane glycoprotein p185(c-erbB2) and exhibit increased resistance to the chemotherapeutic agent paclitaxel via p170(mdr-1)-independent mechanisms. We have recently discovered that the widely used monoclonal antibody C219, which is specific for p170(mdr-1), may cross-react with p185(c-erbB2) in 435.eb cells. In this study, we have investigated the nature of this cross-reactivity. Immunoprecipitation experiments involving the use of breast cancer cells that express different levels of p185(c-erbB2) were performed, and C219 was used for western blot analysis of immunoprecipitated proteins. Immunohistochemical analyses were performed on acetone-fixed slides of human breast cancer cells. Peptide sequence comparisons and enzyme-linked immunosorbent assays were performed to determine the molecular basis of C219 cross-reactivity with p185(c-erbB2). The cross-reactivity of C219 with p185(c-erbB2) was demonstrated by both western blot and immunohistochemical analyses. Peptide sequence comparisons revealed that C219 recognizes an epitope in p170(mdr-1) (C219 epitope) that shares sequence homology with p185(c-erbB2). Enzyme-linked immunosorbent assays demonstrated that C219 recognizes synthetic peptides derived from both the C219 epitope in p170(mdr-1) and the C219 epitope-homologous region in p185(c-erbB2). The anti-p170(mdr-1) monoclonal antibody C219 cross-reacts with p185(c-erbB2) through a peptide sequence in p185(c-erbB2) that is homologous to the C219 epitope in p170(mdr-1).

Evaluation of P-glycoprotein expression in human oral oncogenesis: correlation with clinicopathological features.

To determine whether the multidrug-resistance-gene product phospho-glycoprotein (P-gp) is implicated in progression of oral tumours and/or drug resistance, the expression of P-gp was examined in different stages of oral oncogenesis using monoclonal antibody C-219. Cryosections from normal (41 cases), dysplastic lesions (32 cases), untreated primary SCCs (50 cases) and recurrent tumours (31 cases) were used for immunostaining, and the results were corroborated by immunoblotting. Chi-square test for trend analysis showed a significant increase in P-gp immunopositivity across the normal, leukoplakia, primary oral SCC and recurrent SCC groups (p < 0.01). Expression of P-gp in dysplastic lesions showed significant association with severity of dysplasia, the level of P-gp protein being higher in severe and moderate dysplasia. Among the primary tumours, significant correlation was observed between P-gp positivity as well as level of P-gp expression and tumour stage. The recurrent tumours showed significant increase in P-gp expression as compared with untreated primary oral tumours. We conclude that differential expression of P-gp may be an index of the disease prognosis in oral-cancer patients in the context of the Indian population.

Evaluation of P‐glycoprotein expression in human oral oncogenesis: Correlation with clinicopathological features

To determine whether the multidrug-resistance-gene product phospho-glycoprotein (P-gp) is implicated in progression of oral tumours and/or drug resistance, the expression of P-gp was examined in different stages of oral oncogenesis using monoclonal antibody C-219. Cryosections from normal (41 cases), dysplastic lesions (32 cases), untreated primary SCCs (50 cases) and recurrent tumours (31 cases) were used for immunostaining, and the results were corroborated by immunoblotting. Chi-square test for trend analysis showed a significant increase in P-gp immunopositivity across the normal, leukoplakia, primary oral SCC and recurrent SCC groups (p < 0.01). Expression of P-gp in dysplastic lesions showed significant association with severity of dysplasia, the level of P-gp protein being higher in severe and moderate dysplasia. Among the primary tumours, significant correlation was observed between P-gp positivity as well as level of P-gp expression and tumour stage. The recurrent tumours showed significant increase in P-gp expression as compared with untreated primary oral tumours. We conclude that differential expression of P-gp may be an index of the disease prognosis in oral-cancer patients in the context of the Indian population. Int. J. Cancer 72:728–734, 1997. © 1997 Wiley-Liss, Inc.

Expression of P-glycoprotein in inner ear capillary endothelial cells of the guinea pig with special reference to blood–inner ear barrier

Expression of P-glycoprotein (P-gp) was detected immunohistochemically in the guinea pig inner ear using mouse anti-P-gp monoclonal antibody C219. P-gp was found only in the capillary endothelial cells of the cochlea and vestibule. The pattern of P-gp immunostaining in the inner ear was similar to that of the brain. The present investigation suggested that P-gp expression in inner ear capillary endothelial cells might play an important role in the blood–inner ear barrier by acting as an extrusion pump.

Smoking enhanced the expression of MDR-1 in rat prostatic carcinoma

Smoking is associated with the development of different malignancies, especially lung carcinoma. Considerably less interest has been devoted whether the outcome of cancer treatment can be affected by ongoing smoking. In the present study, the effects of ongoing smoking was evaluated on the expression of classical multidrug resistance (MDR-1) in a tumour tissue not usually related to smoking, prostatic carcinoma in rats. The expression of mdr1 was evaluated by quantitative RNA-RNA solution hybridisation and the distribution of the glycoprotein P-170 was examined by immunohistochemistry using the monoclonal antibodies C219 and C494. A group of animals was exposed to cigarette smoke in a nose-only exposure system for 1 h/day, 5 days/week for four weeks. The results demonstrated a significant enhanced expression of mdr1 RNA in the rumours from smoke exposed animals and the immunohistochemical analysis also displayed an increase of the positively stained cells following smoking. These results indicate that alteration at least at the level of transcription is involved in the overexpression of P-170 following smoking. An effect on translation or post-translation cannot be excluded. The observations might add to the experience of drug resistance seen in the clinical situation in tumours associated with carcinogens such as smoking.

Method for radioiodination of proteins using N-succinimidyl 3-hydroxy-4-iodobenzoate.

A conjugation method has been developed for the radioiodination of proteins which should be adaptable to kit formulation. m-Hydroxybenzoic acid was converted to 3-hydroxy-4-[131I]iodobenzoic acid in 65% radiochemical yield using Chloramine-T as the oxidant. This intermediate was then converted to N-succinimidyl 3-hydroxy-4-[131I]iodobenzoate ([131I]mSHIB) in 75% yield by reaction with N-hydroxysuccinimide and dicyclohexylcarbodiimide in a reaction time of only 10 min. Monoclonal antibody (mAb) 81C6 was labeled in 40-60% yield by reaction with [131I]mSHIB. Performing purifications of radioiodinated compounds using cartridges instead of HPLC did not alter conjugation efficiency, mAb immunoreactivity, or tissue distribution. Thyroid uptake of labeled mAb was low but up to 2.4 times higher than that seen when the mAb was labeled with N-succinimidyl 3-[125I]-iodobenzoate. These results suggest that [131I]mSHIB may be a useful reagent for the radioiodination of proteins, particularly in contexts when less complicated purification methods would be advantageous.

P-glycoprotein expression in capillary endothelial cells of the 7th and 8th nerves of guinea pig in relation to blood–nerve barrier sites

Expression of P-glycoprotein (P-gp) was detected by immunohistochemical staining and Western blot analysis in the peripheral nerves (7th and 8th nerves) of the guinea pig using anti-P-gp monoclonal antibody C219. P-gp was detected in the capillary endothelial cells of these nerves. Immunoreactivity in these nerves was similar to that in the brain. Besides these nerves, positive staining was observed in the sciatic nerve, although immunoreactivity was somewhat lower than that of the 7th and 8th nerves. The present investigation suggested that P-gp in the endothelial cells of the peripheral nerves might play a very important role as a part of the blood–nerve barrier function, since P-gp acts as an extrusion pump in these cells.

Immunocytochemical detection of the multidrug resistance-associated protein and P-glycoprotein in acute myeloid leukemia: impact of antibodies, sample source and disease status.

Immunocytochemical detection of the expression of the MRP gene and the MDR1 gene in clinical specimens might be affected by several factors. Thus, we studied the impact of monoclonal antibodies, sample source (peripheral blood vs bone marrow) and disease status on the expression of multidrug resistance-associated protein (MRP) as well as P-glycoprotein (P-gp) in leukemic cells of patients with acute myeloid leukemia (AML). MRP expression was determined by means of anti-MRP antibodies (QCRL-1, QCRL-3, QCRL-1/QCRL-3 or MRPr1). In the case of P-gp, monoclonal antibodies C219 and MRK16 were used. High MRP expression ranged from 5 to 35% and high P-gp expression from 5 to 14% of the specimens. A fair correlation between results obtained with QCRL-1/QCRL-3 and those obtained with MRPr1, as well as a moderate correlation between C219 and MRK16, were seen. MRP and P-gp expression of peripheral blood blasts were similar to those of bone marrow blasts in the majority of cases. The degrees of MRP expression at the time of diagnosis were also similar to the degrees of expression at relapse, albeit an analysis of sequential MRP expression in 13 patients indicated an increase of expression at relapse in six patients as compared to the time of diagnosis.

Carbohydrate recognition on MUC1-expressing targets enhances cytotoxicity of a T cell subpopulation.

The influence of the epithelial mucin MUC1 on T cell-mediated lysis was analysed using lymph node lymphocytes (LNL) from patients with colorectal carcinoma. LNL were stimulated with allogeneic, MUC1-transfected B cells and the bulk cultures were cloned. Alloreactive cytotoxic T cell clones were obtained which preferentially lysed MUC1-expressing targets. The majority was CD4+ and MHC-class II-restricted, and a minor group was CD8+ and MHC-class I-restricted. All the clones expressed CD3 and TCR alpha beta, and were CD56-. The capacity to preferentially kill MUC1-expressing targets was stable in several clones for up to 6 months in culture. The enhancing effect of MUC1 on the lysis was investigated in more detail. It was only seen after inhibition of O-linked glycosylation in the targets. Furthermore, this effect was completely abrogated by the monoclonal antibody 3C9, directed against the Thomsen-Friedenreich antigen (T-antigen, Gal beta 1-3GalNAc bound alpha 1-3 to Ser/Thr) as well as by the soluble disaccharide Gal beta 1-3GalNAc, but not by other similar disaccharides. The authors conclude that in their system the preferential killing of MUC1-expressing targets is due to the recognition of an internal carbohydrate epitope accessible on under-glycosylated MUC1, possibly T-antigen, by an auxiliary receptor molecule on T cells.

Inverse Relationship between P-Glycoprotein Expression and Its Proliferative Activity in Hepatocellular Carcinoma

Hepatocellular carcinomas (HCCs) from 31 patients, cirrhotic livers from 7 patients and normal liver from 8 patients were immunohistochemically assessed in frozen sections using anti-P-glycoprotein (Pgp) monoclonal antibody C219. Immunohistochemical staining with anti-Ki-67 monoclonal antibody MIB-1 was performed for 22 HCCs to assess proliferative activity. Tumor tissues expressed Pgp on the biliary surface and on the luminal surface of cancer cells, which was less extensive than in normal and cirrhotic liver hepatocytes. Expression of Pgp was closely associated with the degree of histological differentiation and the histological type of HCC. Labeling indices (LI) of Pgp expression were 0.68 +/- 0.06 (mean +/- SD) in well, 0.51 +/- 0.14 in moderately and 0.04 +/- 0.06 in poorly differentiated HCC (significant differences among the three groups). LI of Pgp expression in the trabecular (0.55 +/- 0.15) and in the pseudoglandular types (0.44 +/- 0.10) were remarkably higher than in the compact type (0.04 +/- 0.06). The extent of Pgp expression in HCC was significantly inversely related to the extent of Ki-67 expression, in which Pgp expression decreased in highly proliferating tumors. In conclusion, Pgp expression in HCC was clearly related to its proliferative activity.

P-Glycoprotein Expression in De Novo Acute Myeloid Leukemia

Detection of the multidrug resistance P-glycoprotein (PGP) phenotype was performed at the time of diagnosis in 223 patients with acute myeloid leukemia (AML) by flow cytometry using C219 Monoclonal Antibody (MoAb). On the other hand, JSB1 MoAb was tested in 173 of these samples. At onset, PGP was detected in 57.4% of cases with C219 and 75.9% of cases with JSB 1. There was no correlation between PGP expression and sex, age, marrow blast percentage or extramedullary disease. On the contrary, strict correlations were noted either between C219 negativity and FAB M3 subtype or between C219 positivity and FAB M5 group (P = 0.003). Significant correlation was found between PGP phenotype and CD7, as 143 of 223 samples had similar patterns of staining with C219 (P < 0.0001). Finally, there was a close relationship between C219 and JSB1 positivity: all the C219+ cases were positive for JSB1 (P < 0.0001). Concerning the karyotype, most patients with monosomy or del (7) were MDR positive; on the other hand, most patients with t(8;21) or t(15;17) were MDR negative. Rh123 accumulation studies showed a significant decrease of mean fluorescence intensities both in C219 and in JSB1 positive cases in comparison with PGP negative ones (P < 0.001). A significant decrease of remission induction rates (CR) was highlighted both between C219+ and C219− and between JSBT+ and JSB1− cases (32.1% v 62.1% and 32.6% v 73.8%, respectively, with P < 0.0001). The overall survival and the remission duration (CCR) were significantly shorter both in C219+ and in JSB1+ patients with no relationship to age. Furthermore, a higher rate of early relapses was noted among MDR+ when compared with MDR− patients both for C219+ and JSBT cases. The combination (C219− JSB1+) identified a subset of patients with an intermediate prognosis. On multivariate analysis, C219 and JSB1 were confirmed to be independent prognostic factors for achievement of CR, overall survival and CCR. In conclusion, the assessment of MDR phenotype by flow cytometry is a crucial prognostic factor of treatment outcome in AML.

Immunogenicity of Neisseria gonorrhoeae Lipooligosaccharide Epitope 2C7, Widely Expressed In Vivo with No Immunochemical Similarity to Human Glycosphingolipids

Natural infection with Neisseria gonorrhoeae may elicit a substantial antibody response directed against gonococcal lipooligosaccharide. Monoclonal antibody (MAb) 2C7 recognized a gonococcal lipooligosaccharide epitope, identified the epitope directly in 94% of 68 consecutive culture-positive genital secretions, and recognized 95% of 101 randomly chosen fresh (second-passage) gonococcal isolates. The epitope was stably maintained after multiple in vitro passages and did not compete with any of the known cross-reactive human glycosphingolipid structures. MAb 2C7 mediated in vitro killing and phagocytosis by human polymorphonuclear leukocytes of 1 serum-sensitive (sialylated or not) and 1 stably serum-resistant gonococcal isolate that expressed the epitope. Gonococcal endometritis and disseminated infection elicited increases (6.5-fold IgM, 4.4-fold IgG; 18-fold IgM, 17-fold IgG, respectively) in anti-2C7 epitope antibody. Immunization with a gonococcal outer membrane vaccine elicited a mean 44.5-fold increase in IgG anti-2C7 epitope antibody in 20 of 28 subjects. The epitope identified by MAb 2C7 may represent an excellent target for a potentially protective gonococcal vaccine candidate.