Immunolymphoscintigraphy for the detection of lymph node metastases from breast cancer

The presence of metastases in the regional lymph nodes is the major prognostic factor in breast cancer in the absence of overt distant metastases and is also an important indicator of the need for adjuvant therapy in "early" breast cancer. Currently, the accurate assessment of axillary lymph node status requires axillary dissection which has an associated morbidity. An alternative method of identifying patients who are "node positive" has been developed by means of immunolymphoscintigraphy with s.c. administered radioiodinated monoclonal antibody. The 131I-labeled anti-breast cancer antibody (RCC-1; 400 micrograms) and cold iodine-labeled "blocking" antibody (Ly-2.1; 2 mg which is nonreactive with breast cancer) were injected s.c. into both arms and scintigraphy images were obtained 16-18 h after the injection, using the axilla contralateral to the side of the breast cancer as the control. Studies were reported as positive (and therefore indicative of lymph node metastases) if the amount of background-subtracted radioactive count in the axilla of interest exceeded the normal side by a radio equal to or greater than 1.5:1.0 as assessed by computer analysis. In 38 of 40 patients the findings on scintigraphy were correlated with operative and histopathological findings on the axillary dissection specimen or cytological findings of fine needle aspiration of axillary lymph nodes. In a prospective study of 26 patients, the method is more sensitive (86%) and specific (92%) than preoperative clinical assessment (57% sensitivity, 58% specificity) in the detection of axillary lymph node metastases; and by combining both modalities of assessment, there was an improvement in the sensitivity (100%) but a deterioration in the specificity (50%). There was no significant complication from this essentially outpatient procedure and only 1 of 40 patients developed a human anti-mouse antibody response. This novel and safe method of imaging may become a most useful adjunct in the surgical management of breast cancer.     

Development of immunofiltration assay by light addressable potentiometric sensor with genetically biotinylated recombinant antibody for rapid identification of Venezuelan equine encephalitis virus

A genetically biotinylated single chain fragment variable antibody (scFv) against Venezuelan equine encephalitis virus (VEE) was applied in a system consisting of an immunofiltration enzyme assay (IFA) with a light addressable potentiometric sensor (LAPS) for the rapid identification of VEE. The IFA involved formation of an immunocomplex sandwich consisting of VEE, biotinylated antibody, fluoresceinated antibody and streptavidin, capture of the sandwich by filtration on biotinylated membrane, and labeling of the sandwich by anti-fluorescein urease conjugate. The concentration ratio of biotinylated to fluoresceinated antibodies was investigated and optimized. By the IFA/LAPS assay, the limit of detection (LOD) of VEE was approximately 30 ng/ml, similar to that achieved when chemically biotinylated monoclonal antibody (mAb) was applied. Total assay variance of the IFA/LAPS assay for both intra- and inter-assay precision was less than 20%. Assay accuracy was measured by comparing VEE concentrations estimated by IFA/LAPS standard curve to those obtained by conventional protein assay. VEE concentrations were found to differ by no more than 10%. The IFA/LAPS assay sensitivity was approximately equal to that of a conventional enzyme-linked immunosorbent assay (ELISA) utilizing polystyrene plates and a chromogenic substrate; however, less time and effort were required for performance of the IFA/LAPS assay. More importantly, use of genetically biotinylated scFv in the IFA/LAPS assay obviates the need for chemical biotinylation of antibody with resultant possible impairment of the antigen-binding site. Furthermore, the potential for batch-to-batch variability resulting from inequality in the number of biotin molecules labeled per antibody molecule is eliminated.     

A natural musaceas plant extract inhibits proteasome activity and induces apoptosis selectively in human tumor and transformed, but not normal and non-transformed, cells

Animal studies have demonstrated that a dietary polyphenol known as tannic acid (TA) exhibits anticarcinogenic activity in chemically induced cancers. Most recently, we have reported that TA and ester-bond containing green tea polyphenols are potent proteasome inhibitors in vitro and in vivo. We hypothesize that CellQuest, a patented formula which contains high level of TA obtained from a musaceas (plantain) plant extract, will inhibit the tumor cell proteasome activity. Here, we report that a partially purified CellQuest fraction, S3, potently inhibits the proteasomal chymotrypsin-like activity of Jurkat T cell extracts in a concentration-dependent manner. Inhibition of the proteasome by S3 in leukemia Jurkat T, simian virus 40-transformed and prostate cancer LNCaP cells results in accumulation of ubiquitinated proteins and the natural proteasome substrate p27Kip1, followed by induction of apoptosis. In contrast, non-transformed, immortalized human natural killer cells and normal human fibroblasts are resistant to S3-mediated proteasome inhibition and apoptosis induction. Our present study suggests that CellQuest targets and inhibits the proteasome selectively in tumor cells, which may contribute to the claimed anticancer activity.     

Phenotypic differences within the AB blood type of the feline AB blood group system

Flow cytometry immunolabeling, tube agglutination tests, and thin-layer chromatography immunostaining with two different anti-A monoclonal antibodies (anti-A mAb1 and anti-A mAb2) and one anti-B mAb were used to demonstrate differences in expression of the A and B antigens among erythrocytes from type A and four different type AB cats. Although the flow cytometric patterns of reactivity and agglutination scores for erythrocytes from types A and B cats detected with the anti-A and anti-B mAbs were consistent, reactivity among erythrocytes of different type AB cats was variable. By flow cytometric analysis, 99.9% of type A erythrocytes, no type B erythrocytes, 2.5–4.0% of erythrocytes from type AB cats 1, 3, and 4, and 60.7% of erythrocytes from type AB cat 2 had detectable A antigen when anti-A mAb1 was used. In contrast, 86.4% of type A erythrocytes, no type B erythrocytes, 20.2–38.0% of erythrocytes from type AB cats 1, 3, and 4, and 68.5% of erythrocytes from type AB cat 2 had detectable A antigen when anti-A mAb2 was used. In addition, 86.9% of type B erythrocytes, no type A erythrocytes, 83.1–96.8% of erythrocytes from type AB cats 1, 3, and 4, and 73.0% of erythrocytes from type AB cat 2 had detectable B antigen when the anti-B mAb was used. Agglutination scores of type AB cats were comparable to the percent binding on flow cytometry. Thin-layer chromatography immunostains confirmed differences in the amount of A antigen between erythrocyte glycolipids of type A and AB cats and those of type AB cats 1 and 2. These results suggest that at least two different phenotypes exist within the feline AB blood type, which differ in the amount of A antigen expressed on the erythrocyte surface.     

NADPH oxidase activity of neutrophil specific granules: requirements for cytosolic components and evidence of assembly during cell activation

Neutrophils and other phagocytic cells support host defense by ingesting microbes and destroying them with reactive oxygen species or oxygen independent mechanisms. Production of ROS is initiated by the phagocyte NADPH oxidase (phox), an enzyme system composed of several constituents. During activation of the cell cytosolic phox proteins (p47phox, p67phox, p40phox, and Rac2) translocate to the plasma membrane and specific granules fuse with the plasma membrane increasing the amount of flavocytochrome b(558). The resultant assembly of phox components results in formation of a complete complex and expression of activity. In this study, we evaluated the oxidase activity of specific granules. In the SDS cell-free system, specific granules expressed oxidase activity in the presence of cytosol in a manner similar to plasma membrane. In contrast to plasma membrane, activity of specific granules was latent, diminishing rapidly over time. In addition, this subcellular fraction contained an inhibitor, possibly related to contamination with azurophilic granules explaining previously published discrepant results. Experiments with recombinant p47phox, p67phox, and dilute cytosol or fractionated cytosol as a source of Rac demonstrated that specific granules have requirements identical to specific granules for oxidase activity. Finally, analysis of neutrophils stimulated with PMA demonstrated translocation of p47phox and to p67phox to specific granules as well as plasma membrane. Both plasma membrane and specific granules from PMA stimulated cells expressed oxidase activity with addition of NADPH demonstrating an assembled oxidase complex. These studies establish a critical role for specific granules as a site for assembly and activation of the oxidase enzyme system and an important constituent for the microbicidal activity of the neutrophil.     

Regulation of human basophil adhesion to endothelium under flow conditions: Different very late antigen 4 regulation on umbilical cord blood-derived and peripheral blood basophils

BACKGROUND: Although soluble mediators released by basophils in tissue sites contribute to the chronic injury that occurs in hypersensitivity diseases, only limited information is available about how circulating basophils are recruited to tissues. In particular, the interaction of basophils with endothelium under conditions that mimic physiologic flow has not been explored. OBJECTIVE: We sought to identify adhesion molecules regulating the attachment of human basophils to IL-4-activated human umbilical vein endothelial cells (HUVECs) under flow conditions. METHODS: A parallel-plate flow chamber and blocking mAbs were used to define the adhesion molecules involved in the interactions of peripheral blood basophils (PBBs) and cord blood-derived basophils (CBDBs) with IL-4-activated HUVECs and with Chinese hamster ovary (CHO) cell transfectants expressing specific adhesion molecules. A fluorescent ligand specific for very late antigen 4 (VLA-4) was used to directly examine the VLA-4 affinity state of basophils. RESULTS: Flowing PBBs and CBDBs attached to activated HUVECs and to CHO cells expressing P- or E-selectin. However, only CBDBs attached to vascular cell adhesion molecule 1 (VCAM-1)-transfected CHO cells under flow conditions. The attachment of CBDBs to CHO cells was blocked by mAbs directed against E-selectin, P-selectin, and VCAM-1, whereas attachment of PBBs was blocked by E-selectin and P-selectin mAbs. Activating VLA-4 with Mn(2+) on PBBs resulted in adhesion to the VCAM-1-transfected CHO cells, indicating that VLA-4 activity on PBBs can be regulated, at least in part, through affinity changes. The Mn(2+)-induced upregulation of basophil VLA-4 affinity was demonstrated directly by using a fluorescent ligand for VLA-4 and flow cytometry. CONCLUSIONS: The interaction of human CBDBs and PBBs with endothelium under flow conditions is mediated in part by both P- and E-selectin. VLA-4 additionally contributes to the adhesion of flowing CBDBs. However, the affinity of VLA-4 is too low to support the adhesion under flow conditions of unstimulated PBBs.     

Detection of cytomegalovirus infection during a vaccine clinical trial in healthy young women: seroconversion and viral shedding.

BACKGROUND: An antibody method based on absorption of serum with cytomegalovirus (CMV) glycoprotein B (gB) was developed for detection of infection during clinical trials of CMV gB vaccine. Previous study showed that this method detected the antibody response to infection and was negative with vaccine induced immunity. OBJECTIVES: In an ongoing efficacy trial of CMV gB vaccine the ability of the gB-absorbed CMV IgG assay to detect CMV infection was assessed and compared with viral culture results. STUDY DESIGN: Two hundred and ninety two healthy, seronegative young women in a phase II, double-blind, placebo-controlled, clinical trial of recombinant CMV gB vaccine (sanofi pasteur) with MF59 adjuvant (Chiron) were randomized to receive CMV gB vaccine or placebo (1:1) on a 0, 1 and 6 month schedule. Participants were screened every 3 months for CMV infection using the gB-absorbed CMV IgG assay, and a subgroup was also screened for infection with viral cultures. Viral culture (urine, vaginal swab and saliva) was used to confirm CMV infection in all subjects with a positive gB-absorbed CMV IgG result. RESULTS: Evidence of CMV infection (gB-absorbed CMV IgG levels>or=5.0 AU/ml) was found in 23/292 (7.88%) study participants. The gB-absorbed CMV IgG levels of their first positive serum ranged from 15.7 to 251.0 AU/ml with a mean of 77.0 AU/ml and a median of 44.9 AU/ml. Cytomegalovirus was isolated from all 23 of them from culture specimens collected after their first positive gB-absorbed CMV IgG. The time to first CMV positive culture from first positive gB-absorbed CMV IgG ranged from 0 to 12 weeks with a median of 2 weeks. CONCLUSIONS: The gB-absorbed CMV IgG assay detects CMV infection in CMV gB vaccine clinical trials earlier and more rapidly than virus culture and does not reveal whether subjects received CMV gB vaccine or placebo.