Characterization of human IgG1 monoclonal antibody against gangliosides expressed on tumor cells

In this study, our intention was to describe the decision making of nurses practicing in intensive care, and the differences of nurses' decision making in Canada, Finland, Northern Ireland, Switzerland, and the United States. The instrument used in the study was a 56-item Likert-type questionnaire that has been used in previous studies and has proved to be a reliable tool. The target group comprised a nonrandom sample of nurses (N = 314) from five countries. The samples are not representative; therefore, the results in these cases cannot be generalized. The results showed that the decision making of nurses practicing in intensive care was broadly based, and that there were some country differences in data collection, problem definition, and planning. In contrast, decision making related to the implementation and evaluation of nursing is quite similar in the different countries. Canada and the United States on the one hand, and Finland, Northern Ireland, and Switzerland on the other, showed more similarities with each other in data collection, problem definition, and nursing planning related to decision making. Neither experience nor nurse's knowledge structure was associated with different decision-making approaches.     

Characterization of a monoclonal antibody recognizing mast cells

Immunohistochemical screening for monoclonal antibodies prepared by immunization of mice with a rat osteoblastic cell population led to identification of one antibody that reacted against a small population of cells present in the soft connective tissue compartment of 21 days fetal rat calvaria. The morphology of the cells and the immunohistochemical staining characteristics (a distinct intracellular granular pattern) suggested that the antibody might be reacting specifically against mast cells. We used combined histochemistry and immunohistochemistry to further characterize this antibody, designated RCJ102. Cryosections containing calvaria bone, soft connective tissues and skin were prepared from the top of the head of 21 days fetal rats, and from adult rats cryosections of lung, muscle, adipose tissue and small intestine were prepared. Some sections were labelled by indirect immunofluorescence with RCJ102; corresponding sections were labelled histochemically with toluidine blue. There was a direct correspondence between mast cells identified histochemically and cells labelling with RCJ102 in all tissues except intestine, in which the mast cell detectable by histochemistry were not labelled by RCJ102. These results suggest that the RCJ102 antibody will be a valuable new reagent for further elucidation of the heterogeneity described between connective tissue and intestinal mucosal mast cells.     

Characterization of Reed-Sternberg cells with granulocyte specific monoclonal antibody

Monoclonal antibody Leu M1 represents a highly specific marker to locate granulocyte antigen in Reed-Sternberg cells in Hodgkin's disease. Except the L&H variants of reed-sternberg cells in lymphocyte predominance variety in which antigens are probably sialylated. All the cases of non-Hodgkin's lymphoma were negative with this marker, because of absence of antigen. Therefore this specific marker characterizes granulocyte origin of reed-sternberg cells.     

CNA.42, a new monoclonal antibody directed against a fixative-resistant antigen of follicular dendritic reticulum cells

A new monoclonal antibody (MAb), CNA.42, was generated using the CEM T-cell line. It recognizes a 120-kd formalin-resistant glycosylated antigen that is mainly expressed by follicular dendritic reticulum cells (FDRCs). This antigen is also expressed by a few mononuclear cells in the paracortical area of reactive lymph nodes and by some cortical thymocytes. Two hundred and eighty-nine cases of hematopoietic tumors of various types were tested with this antibody. They showed either intact FDRC networks or FDRC networks dispersed among malignant cells. In follicular lymphomas, the follicular pattern was highlighted by CNA.42 MAb. Expanded FDRC networks were found in angioimmunoblastic T-cell lymphomas. Neoplastic cells were positive in 43.6% (24/55) of T-cell and 4.6% (6/129) of B-cell lymphomas. The highest percentage of cases with positive neoplastic cells was found in anaplastic large-cell lymphomas (62.5%; 15/24). In Hodgkin's disease, FDRC networks, sometimes encasing Hodgkin and Reed-Sternberg (HRS) cells, were found. HRS cells were also stained by this antibody in 23 (21.9%) of the 105 cases examined. A variety of normal nonlymphoid tissues and nonhematopoietic tumors, such as some neurogenic tumors, carcinoma, and occasional sarcomas, were found to be positive. Analysis of the reactivity of CNA.42 antibody with FDRCs of lymphoid tissue from different animal species showed similar reactivity to that observed in humans, suggesting widespread evolutionary conservation of the antigen recognized by this antibody. In daily diagnostic practice, CNA.42 MAb seems to be a suitable FDRC marker and possibly has an auxiliary role in recognizing T-cell lymphomas.     

Immunotherapy of multiple myeloma with a monoclonal antibody directed against a plasma cell-specific antigen, HM1.24

Multiple myeloma remains an incurable malignancy because of marked resistance of tumor cells to conventional chemotherapeutic agents. Alternative strategies are needed to solve these problems. To develop a new strategy, we have generated a monoclonal antibody (MoAb), which detects a human plasma cell-specific antigen, HM1.24. In this report, we evaluated the in vivo antitumor effect of unconjugated anti-HM1.24 MoAb on human myeloma xenografts implanted into severe combined immunodeficiency (SCID) mice. Two models of disseminated or localized tumors were established in SCID mice by either intravenous or subcutaneous injection of human myeloma cell lines, ARH-77 and RPMI 8226. When mice were treated with a single intraperitoneal injection of anti-HM1.24 MoAb 1 day after tumor inoculation, the development of disseminated myeloma was completely inhibited. In mice bearing advanced tumors, multiple injections of anti-HM1.24 MoAb reduced the tumor size and significantly prolonged survival, including tumor cure, in a dose-dependent manner. The proliferation of cultured human myeloma cells was inhibited in vitro by anti-HM1.24 IgG-mediated complement-dependent cytotoxicity, but not by the antibody alone. Moreover, spleen cells from SCID mice mediated antibody-dependent cell cytotoxicity against RPMI 8226 cells. These results indicate that anti-HM1.24 MoAb can be used for immunotherapy of multiple myeloma and related plasma cell dyscrasias.     

Detection of mouse tyrosinase with a monoclonal antibody MAT-1 against human tyrosinase

Monoclonal Antibody (MoAb) HNK, or anti-leu-7, is reactive with several neuroendocrine and nonneuroendocrine tumors. The aim of this study is to examine anti-leu-7 reactivity in thyroid neoplasms and its relationship to cellular proliferation as determined by anti-PCNA reactivity. The expression of anti-leu-7 in 56 thyroid neoplasms (24 papillary carcinomas, 14 follicular carcinomas, two medullary carcinomas and 16 follicular adenomas) was examined immunohistochemically. Papillary and follicular thyroid carcinomas reacted with anti-leu-7 in a membranous and cytoplasmic pattern in 88% and 93% of cases, respectively. The adjacent benign tissues were nonreactive. Only eight cases diagnosed as follicular adenomas were reactive with anti-leu-7. Furthermore, the mean proliferative index (PI), as measured by the percentage of nuclei immunoreactive with anti-PCNA, was greater than 30% in all thyroid neoplasms reactive with anti-leu-7. The PI was 58% for papillary carcinomas and 68% and 48% for follicular carcinomas, and follicular adenomas, respectively. Lesions originally classified as follicular adenomas that were nonreactive with anti-leu-7 had a PI of 24% and were reclassified as hyperplastic nodules. These data suggest that anti-leu-7 may be useful for characterizing thyroid neoplasia.     

Kinetic analysis of T cells and antibody production in chickens infected with Marek's disease virus

In chickens inoculated with a Marek's disease (MD) vaccine and subsequently with virulent MD virus (MDV), CD4+ T cell population was drastically decreased following a transient increase at 21 days after hatching (16 days after MDV infection). To elucidate the immune response after the decrease of CD4+ T cell population, the antibody production against sheep red blood cells (SRBC) was examined in these chickens. Chickens challenged with a virulent MDV after MD vaccination produced lower titers, of anti-SRBC antibody than untreated control chickens. Antibody production against SRBC was also lowered in vaccinated chickens or chickens challenged with a virulent MDV.     

Characterization of fertilization-blocking monoclonal antibody 1G12 with human sperm-immobilizing activity

A mouse hybridoma (1G12) producing sperm-immobilizing MoAb to human sperm was established and characterized in order to study the antigens relevant to sperm immobilization by antibodies. MoAb 1G12 had strong sperm-immobilizing and agglutinating activities and also showed a fertilization-blocking activity on in vitro fertilization tests. The antibody absorption experiments showed that MoAb 1G12 reacted not only to ejaculated sperm but also human seminal plasma, suggesting that the corresponding antigen might be a sperm coating antigen. The MoAb also reacted with peripheral blood lymphocytes. In histochemical studies, the epithelia of corpus epididymis were most strongly stained. Ejaculated sperm were stained with a granular pattern for their entire surface by immunofluorescence. MoAb 1G12 recognized polymorphic glycoproteins of 15-25 kD in the ejaculated sperm extract in Western blot analysis. After deglycosilation of the sperm extract, only a single staining band of under 15 kD was detected by MoAb 1G12. This suggests that the antigen epitope recognized by MoAb 1G12 might be a peptide of the core portion of the glycoprotein. MoAb 1G12 might be a useful tool for studying the mechanism of egg-sperm interaction, and also be applied to identifying the corresponding antigen by using gene technology.     

Labeling of cycling corneal endothelial cells during healing with a monoclonal antibody to the Ki67 antigen (MIB-1)

PURPOSE: To assess the efficacy of labeling actively cycling corneal endothelial cells by using a monoclonal antibody to the Ki67 antigen (MIB-1) and to determine what changes in f-actin and ZO-1 organization are associated with entry into the cell cycle during wound healing under different culture conditions. METHODS: Three corneal buttons (6 mm diameter) were punched from each cornea of 15 cats. After a mechanical scrape injury (2 mm diameter) was made, buttons were cultured for 24, 48, or 72 h in serum-free media (SFM), SFM plus 10% fetal calf serum, or SFM plus basic fibroblast growth factor (bFGF). Buttons were single and double labeled by using phalloidin, anti-ZO-1, and MIB-1. Counts of Ki67-positive cells were used to determine the number of actively cycling endothelial cells. RESULTS: After culture in SFM, wounds healed by cell spreading with maintenance of normal apical f-actin and ZO-1 organization; Ki67-positive cells were detected near the leading edge in some areas. A significant increase in the number of cycling cells was measured after 48 h of culture in bFGF as compared with SFM (p<0.05); serum increased the number of cycling cells more than both SFM and bFGF (p<0.05). In all cases, positive MIB-1 staining was not observed until 48 h after injury, was limited to cells actively spreading over the wound area, and was diminished after wound closure (72 h). Double labeling demonstrated that endothelial cells exhibited a fibroblastic phenotype in some central areas of cell proliferation after culture in serum or bFGF, but, in general, apical cell border-associated f-actin and ZO-1 organization was partially maintained in most Ki67-positive cells. CONCLUSION: The data suggest that spreading corneal endothelial cells are capable of proliferating and can respond to growth factors, but that dedifferentiation or fibroblastic transformation is not required before entry into the cell cycle. Overall, the MIB-1 antibody appears to be ideally suited to the study of corneal endothelial proliferation during wound healing.     

Proliferative potentials of glioma cells and vascular components determined with monoclonal antibody MIB-1

We present four cases of esophageal rupture (three iatrogenic, one Boerhaave syndrome) to demonstrate the difficulty in diagnosis and therapy. The current literature is discussed and conclusions are drawn as regards the modus operandi. All our patients were operated on. The site of esophageal rupture was always closed with a primary suture and substantial irrigation and drainage were performed. In two cases the suture line was in addition covered with fibrin glue or by an omentum flap, respectively. All patients survived and recovered was unremarkable. Our own results and a subsequent analysis of literature allow the following conclusions. At an early stage of esophageal rupture surgical intervention is indicated. The method of choice is primary closure of the rupture site by suture, possibly combined with a muscle or omentum flap. In cases of delayed diagnosis with advanced mediastinitis, suture of the rupture site should also be striven for. Additional coverage is advisable in these cases. Resection procedures with or without reconstruction should be done only in exceptional cases before of the high surgical risk.     

Circulating immunoglobulin G1 antibody in patients with ulcerative colitis against the colonic epithelial protein detected by a novel monoclonal antibody

Autoimmunity has been implicated in the pathogenesis of ulcerative colitis (UC). Several studies have shown amplified immunoglobulin G1 (IgG1) antibody response in UC; however the immunoreactive antigen(s) is unknown. To study this antigen(s), mucosal colonic extract was prepared by sonication, ultracentrifugation followed by ion exchange chromatography in fast protein liquid chromatography. The fraction (enriched colonic peptide), that was most reactive to a novel monoclonal antibody, 7E12H12 (IgM isotype), was isolated and used to examine the immunoreactivity against the patients' serum samples. Two hundred and thirteen coded samples from 111 patients with UC (symptomatic and untreated (63), symptomatic and treated (26), remission (22)); 47 with Crohn's disease (CD) (40 were symptomatic and untreated, and 30 had colonic disease); 29 with acute diarrhoea caused by specific pathogen(s); 10 with systemic lupus erythematosus, and 16 normal subjects were examined against the enriched colonic peptide by IgG subtype specific enzyme linked immunosorbent assays (ELISAs). Total IgG antibody reactivity was significantly (p < 0.01) higher only in symptomatic and untreated UC patients compared with each of the non-UC group, but the sensitivity was only 50%. IgG2 and IgG3 reactivities were not different among various groups. The IgG1 antibody reactivity against the enriched colonic peptide, however, differentiated UC patients from CD and each of the other non-UC groups. Seventy nine per cent of the patients with UC, treated or untreated, symptomatic or in remission, had significantly (p < 0.0001) higher IgG1 antibody against the enriched colonic peptide when compared with each of the other non-UC groups. Only 12% of CD serum samples and none of the other control serum samples reacted. Using purified serum IgG1 and 7E12H12-IgM, by 7E12H12 reactive peptide indeed reacts with UC-IgG1 antibody but not with control IgG1.     

Protection of chickens with or without maternal antibodies against both Marek's and Newcastle diseases by one-time vaccination with recombinant vaccine of Marek's disease virus type 1

We constructed a stable recombinant Marek's disease virus type 1 (rMDV1) expressing the fusion protein (F) of Newcastle disease virus (NDV) by inserting the coding sequence within the US10 gene of MDV1 by homologous recombination and designated this as rMDV1-US10L(F). The NDV-F protein was significantly expressed under control of the SV40 late promoter in cultured cells infected with the rMDV1. To examine the protective efficacy of the rMDV1, specific pathogen-free (SPF) chickens were vaccinated with rMDV1 at one-day-old. Almost all birds (> 95%) were protected from NDV challenge via intramuscular, ocular, intranasal and intratrachial routes at 4 weeks after vaccination. The rMDV1 showed 100% protection against virulent MDV1 challenge in SPF chickens. Antibody responses against NDV-F and MDV1 antigens were observed at least up to 11 weeks after immunization. When the sera from chickens vaccinated with the rMDV1 were examined for the presence of anti-NDV-F antibody on the day of NDV challenge, the vaccinated bird group which did not survive from NDV challenge were found to show lower antibody titers than the surviving group. The rMDV1 also provided sufficient protection against NDV and MDV1 challenges in commercial chickens with maternal antibodies against NDV-F and MDV1 antigens.     

Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity

The transit of immature to mature sIgM+ B cells, the life span, maturation kinetics and response to polyclonal activators have been analyzed with the help of a new mAb (493), that distinguishes immature, 493+ from mature, 493 B cells in a variety of mouse strains tested. Analysis of the turnover of immature 493+ B cells by bromodeoxyuridine (BrdU) labeling kinetics indicate that only 10-20 % of the cells reach the spleen as immature 493+ cells. The life span of 493+ B cells in bone marrow and spleen is around 4 days. BrdU chase experiments show that most of the immature cells in spleen enter the pool of mature, 493+ B cells where they gain a longer life span of 15-20 weeks. Immature and mature B cells respond equally well to LPS stimulation; anti-CD40, however, stimulates mature B cells better than immature B cells. IgM cross-linking of mature B cells results in proliferation, while it induces apoptosis in immature B cells. This apoptosis of immature cells can be inhibited by costimulation with anti-CD40 or by overexpression of bcl-2. We speculate that Ig receptor ligand-mediated apoptosis (negative selection) plays a major role in the transit of immature B cells from bone marrow to spleen, but only a minor role in the transit from immature B cells to mature B cells in the spleen.     

Immunochemical study of polysaccharide antigen in Streptococcus sobrinus and Streptococcus downei with a cross-reactive monoclonal antibody

A monoclonal antibody (mAb h-448) was prepared after cell fusion of mouse myeloma cells (SP2/0-Ag-14) to the spleen cells of mice immunised with serotype h strain (MF25) of Streptococcus downei. The antibody (IgM class) reacted in enzyme immunoassay only with whole cells as well as purified polysaccharide (PS) antigen of Streptococcus sobrinus (types d and g) and Streptococcus downei (serotype h), but not with cells or purified PS antigen from any other serotypes of the mutans group of streptococci. mAb h-448 also quantitatively precipitated in solution with the purified antigens. Competitive hapten inhibition tests demonstrated that beta-methylgalactopyranoside inhibited the reaction most strongly. Although rhamnose also showed a substantial inhibitory effect, the results of this study indicate that the antigenic determinant of the PS antigen has a structure similar to the beta-methylgalactopyranoside molecule.     

Immuno-capillary electrophoresis for the characterization of a monoclonal antibody against DNA

A monoclonal antibody against DNA established from a mouse strain that spontaneously develops systemic lupus erythematosus was characterized by migration shift immuno-capillary electrophoresis. The minimal size for DNA binding antibody was > 16 bases and the interaction with a double-stranded 32-mer oligonucleotide was almost one order of magnitude stronger than the interaction with a single-stranded oligonucleotide. The binding was highly dependent on the ionic strength conditions with an increase in binding with a decrease in ionic strength. The estimate of the dissociation constant for the antibody binding of a single stranded 32-mer oligonucleotide was 0.62 microM at pH 7.90. This value was in good agreement with the value of 0.44 microM measured by an independent method using biosensor (surface plasmon resonance) technology.     

Characterization of complex formation by humanized anti-IgE monoclonal antibody and monoclonal human IgE

The interaction of human IgE with high-affinity IgE Fc receptors on cells of the immune system plays an essential role in the type I hypersensitivity reaction. A proposed therapy is to use an anti-IgE monoclonal antibody to block the binding of IgE to its high-affinity receptor on mast cells and basophils, thus preventing subsequent release of the inflammatory agents after exposure to allergen. We report here the solution characteristics of immune complexes formed by a humanized anti-IgE monoclonal antibody (rhuMAb E25) and IgE using sedimentation analysis and size exclusion chromatography. We demonstrate that the rhuMAb E25 is able to form a variety of complexes with IgE at different molar ratios. The largest complex was identified by sedimentation equilibrium analysis as a heterohexamer with very high stability. The intermediate complex formed when one of the interacting components is in large molar excess appears to have a trimeric structure. The high-affinity interaction of rhuMAb E25 and IgE has also been confirmed. Furthermore, by using hydrodynamic modeling, we show that the largest complex may be represented by a cyclic structure.     

Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue

The presence of > or = 25% blasts in a marrow aspirate obtained on day 7 of induction followed by a remission at day 28 has been associated with a poor prognosis in children with acute lymphoblastic leukemia (ALL). We evaluated whether a day 7 marrow biopsy may be used to more accurately assess therapeutic reduction of leukemia tumor burden. Studied were 76 children with ALL enrolled on CCG protocols at B.C's Children's Hospital who received both a day 7 aspirate and biopsy and were in remission by day 28. Evaluation for the correlation of the percentage aspirate blasts on day 7 with the biopsy demonstrated a moderate correlation with the percentage biopsy blasts (R = 61), but not correlation with the biopsy cellularity. We saw a similar prediction of outcome by the percentage blasts on day 7 marrow aspirate in the study as reported previously although it was not significant. Outcome analysis was done using leukemia burden as measured by the day 7 absolute blast index-aspirate (ABI-aspirate) calculated as the product of the biopsy cellularity with the percentage blasts on the aspirate. The ABI-aspirate significantly predicted patient outcome with 83% survival in those with an ABI-aspirate of < .06 compared to 51% in those > or = .06 (P = .01) and was highly significant when analyzed as a continuous predictor (P = .004). This is the first study to demonstrate that information gained from the day 7 marrow biopsy can improve prediction of outcome in children with ALL. Based on this preliminary study, we recommend that large population ALL therapy trials evaluate the role of the day 7 marrow biopsy for outcome prediction in children with ALL.     

Characterization of a unique factor-independent variant derived from human factor-dependent TF-1 cells: a transformed event

A factor-independent variant (TF-1a) has been isolated from the factor-dependent TF-1 cell line. The subline has been grown continuously in culture for > 1.5 years without added cytokines. The cells retain the ability to respond to multicytokines, with a different response pattern from its parental cell line. The TF-1 cells appeared singly in liquid culture. In contrast. TF-1a cells formed aggregates which increased markedly in size and in number upon TGFbeta1 treatment and showed a diminished TGFbeta-mediated growth inhibition. TF-1a, but not TF-1 cells, formed colonies in soft agar culture in the absence of any added growth factors, and developed the capacity to generate an invasive tumor(s) in nude mice. There was a constitutive activation of MAPK and MEK in TF-1a but not in TF-1 cells, which may be one of the mechanisms leading to factor-independent growth of TF-1a cells. Phenotypically, TF-1 cells were CD34+ /CD38+, whereas TF-1a cells were CD34+ /CD38-. This suggests that TF-1a may represent a less mature hematopoietic cell than TF-1. In conclusion, TF-1a is different from TF-1 in many important aspects which are associated with neoplastic transformation. The variant appears to be an excellent model for studying the process of progressive malignant transformation of myeloid cells and for studying signal pathways involved in the spontaneous and factor-induced growth of the cells.     

In vitro and in vivo properties of murine monoclonal antibody for a novel immature thymocyte-differentiated antigen, JL1

Bactericidal activities of 8 antibiotics (ticarcillin, piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, imipenem, amikacin, ciprofloxacin) and of 7 combinations have been studied by time-killing curve technique for 16 Pseudomonas aeruginosa selected for their beta-lactams resistance phenotypes (3 wild strains, 5 penicillinases, 2 extended-spectrum beta-lactamases, 3 high level cephalosporinases and 3 non enzymatic resistance). Bactericidal activities of beta-lactams used alone were as follows: imipenem > cefepime > ceftazidime, piperacillin, piperacillin-tazobactam > ticarcillin. All the antibiotics combinations were synergistic or additive. However, only the combination of imipenem with amikacin was bactericidal for all strains. For the strains with penicillinases, piperacillin-tazobactam was not more effective than piperacillin. For the extended-spectrum beta-lactamases, we have observed a synergy with piperacillin-tazobactam and ciprofloxacin or amikacin.