Monoclonal Cocktail Research Articles

Significance of immunohistochemically demonstrated micrometastases to lymph nodes in esophageal cancer with histologically negative nodes

Background: We examined the prevalence, patterns, and clinical significance of nodal micrometastases in patients with esophageal cancer. Methods: Cervical, mediastinal, and abdominal lymph nodes systematically removed from 37 patients without conventional histologic evidence of lymph node metastasis from esophageal squamous cell carcinoma were immunohistochemically examined to detect cells that were stained for cytokeratins by the monoclonal antibody cocktail AE1/AE3. Postoperative care and survival were compared in cases with and without such micrometastases. Results: Nodal micrometastases were found in 14 of 37 patients (38%). Among these patients, 9, 7, and 4 had micrometastases to abdominal, mediastinal, and cervical lymph nodes, respectively. Postoperative tumor recurrence was significantly more frequent in patients with micrometastases (50%) than in those without (9%, P =.008). Overall and relapse-free survival in the former group was significantly worse than in the latter group (P =.042 and P =.002, respectively). Nodal micrometastases had an independent prognostic importance for relapse-free survival as determined by multivariate analysis. Conclusions: Metastatic tumor cells are frequently present in lymph nodes, even in patients without histologic evidence of nodal metastasis from esophageal cancer. Nodal micrometastases indicates a poorer prognosis after a curative esophagectomy procedure in histologically node-negative cases. (Surgery 2000;127:40-6.)

Autologous bone marrow transplantation for high risk acute lymphoblastic leukemia: clinical relevance of ex vivo bone marrow purging with monoclonal antibodies and complement.

Herein we describe our experience with 75 consecutive autologous BM transplants for patients with high-risk ALL, with special attention to the clinical impact of BM purging. Fifty-two patients received purged BM using monoclonal antibody (MoAb) cocktails and complement, and 23 patients received untreated BM. The distribution of prognostic factors was similar in both groups. Hemopoietic reconstitution was adequate and did not differ in the two groups. Transplant-related mortality was 9.6% and 13% in 'purged' and 'unpurged' groups. Median follow up was 11 months (2-71) and overall actuarial probability of disease-free survival (DFS) at 5 years was 40% (53% relapse probability). We found a beneficial effect of purging in patients over 15 years of age and in patients needing more than 1 month to reach CR1. Patients in CR1 receiving purged marrow had a longer DFS and a lower relapse probability (52% vs 12%, P = 0.02 and 35% vs 86%, P = 0.005, respectively) which were related to the efficacy of the purging procedure (more or less than one log of depletion). In further CR, no advantage of purging has been found. Our data strongly suggest the clinical relevance of BM purging in autologous BMT in high-risk ALL patients and support the need for prospective randomized studies.

Multiplex Western Blotting System for the Analysis of Muscular Dystrophy Proteins

A multiplex system of Western blotting is presented in which most of the current muscular dystrophy proteins can be analyzed simultaneously on one pair of blots. This represents a significant improvement in efficiency and cost for this type of analysis. The final diagnosis is more quickly achieved in patients where several possible diagnoses are indicated after clinical appraisal, and those with unusual presentations may be quickly resolved. The method uses a biphasic polyacrylamide gel system, which enables the corresponding blot to be probed simultaneously with a cocktail of monoclonal antibodies. The gel is optimized so that large proteins of more than 200 kd (eg, dystrophin, dysferlin, and myosin heavy chain) can be analyzed in the top part, while smaller proteins under 150 kd (eg, calpain 3, the 80-kd fragment of laminin alpha2 chain, all of the sarcoglycans, and caveolin 3) are separated in the lower phase. This basic system could be used for different combinations of antibodies as new muscular dystrophy proteins are identified and require examination. In addition, analysis of the laminin alpha2 chain of merosin showed that this protein was expressed as a doublet or triplet set of bands in many patients with active muscle pathology. This may indicate the existence of an embryonic isoform, which is re-expressed in regenerating fibers.

Co-expression of tumor antigens and their modulation by pleiotrophic modifiers enhance targeting of human monoclonal antibodies to pancreatic carcinoma

Cocktails of human monoclonal antibodies (HuMAbs) have been used to increase the likelihood of identifying heterogeneous cancer cells. We utilized 3 HuMAbs termed SK-1, GM4, and GMA1, which recognized a 42-46 kDa two chain structure, a 57 kDa antigen, and the ganglioside GD3, respectively. An estimated two dozen cell lines were tested for the coexpression of these antigens and this was found to be present only on pancreatic carcinoma cell line, PANC-1; A 24 hr treatment of PANC-1 cells with interferon gamma (IFN gamma; 100 units), interferon beta (IFN beta; 1000 units), as well as interferon alpha (IFN alpha; 1000 units) resulted in roughly a four fold increase in the co-expression of the 42-46 kDa/GD3 antigens as well as the 42-46 kDa/57 kDa antigens. After a 4 day incubation the co-expression of these antigens progressed and IFN alpha treatment had the most pronounced effect, which was 8 fold higher than background for the 42-46 kDa/57 kDa antigens, whereas IFN beta resulted in a five fold antigen upregulation. The pronounced effect of vinblastine on the co-expression of the 42-46 kDa/GD3 antigens (4 fold on day 1 and, 10 fold on day 4) and 42-46 kDa/57 kDa antigens on PANC-1 (5 fold on day 1 and 7 fold over background on day 4) cells can be seen at concentrations as low as 10(-7)M. Colchicine and vincristine dramatically enhanced co-expression of these tumor antigens on day 4 but not on day 1 PANC-1 cells. The expression of these antigens was also found to be cell cycle dependent.

Partial restoration of hair growth in the DEBR model for Alopecia areata after in vivo depletion of CD4+ T cells.

Alopecia areata (AA) is widely believed to be an autoimmune disease. Hair loss is associated with a peri- and intrafollicular inflammatory infiltrate of anagen hair follicles primarily composed of CD4 + and CD8 + cells. A previous investigation involved in vivo depletion of CD8 + cells in the DEBR rat model to examine the cells' potential pathogenic activity in AA. The rat model is used here in a comparable study of CD4 + cell pathogenic activity. Eight AA affected DEBR rats were given intraperitoneal injections of a CD4 + cell depleting OX-35/OX-38 monoclonal antibody (MoAb) cocktail over a 15-day therapy course. A further eight AA-affected rats comprised a control group and were injected with equivalent volumes of an irrelevant MoAb, OX-21. Changes in both CD4 + and CD8 + peripheral blood cell populations were analysed by flow cytometry, and macrophotography was used to record any changes in hair growth. Of the eight CD4 + cell-depleted rats six responded with hair growth. The rats revealed significant hair growth within 23 days of treatment initiation. With rapid replacement of the CD4 + cell population the newly generated pelage hair was eventually lost. Two control rats also showed limited hair growth within the 112-day study period. In vivo depletion of CD4 + cells partially restores hair growth in AA affected rats. The response suggests that CD4 + cells may be actively involved in the pathogenesis of AA. Further research may elucidate whether CD4 + cells have a direct effect on hair follicles or exert their influence through their classic T helper cell supporting role for CD8 + cells.

Enhanced radioimmunotherapeutic efficacy of a monoclonal antibody cocktail against SMMC-7721 human hepatocellular carcinoma.

The improved tumoricidal effect of the radioantibody mixture ("cocktail") has been reported recently for the treatment of colon tumor. In the present study, we demonstrated the enhanced radioimmunotherapeutic efficacy of a monoclonal antibody (MAb) cocktail against human hepatocellular carcinoma. Therapeutic efficacy was determined by measuring the change in tumor size over a period, determining the percentage of growth inhibition of each treatment at various times after radioantibody therapy. Radioimmunotherapy of SMMC-7721 human hepatoma xenografts in athymic nude mice with combination of 131I-labeled Hepama-1 and 131I-labeled 9403 mouse MAbs was more effective than using either Hepeam-1 or 9403 MAb alone The MAb cocktail could target a greater number of hepatoma cells and increase the magnitude of hepatoma cell uptake of radioantibodies. The in vitro results explain the enhanced effect of the MAb cocktail in in vivo model system.

Micrometastasis and tumor cell microinvolvement of lymph nodes from esophageal squamous cell carcinoma

BACKGROUND The purpose of this study was to investigate micrometastasis (MM) and tumor cell microinvolvement (TCM) in the regional lymph nodes of patients with esophageal squamous cell carcinoma (SCC). METHODS MM was defined as individual tumor cells or tumor cell clusters <0.5 mm in greatest dimension with a surrounding stromal reaction. TCM was defined as individual tumor cells or tumor cell clusters without a surrounding stromal reaction. One thousand nine hundred and fifty-four lymph nodes were dissected from 69 complete (R0) resection specimens of TNM classified pT1-3, pN0 or pN1, and M0 esophageal SCC. These lymph nodes were examined immunohistochemically using the monoclonal antibody cocktail AE1/AE3 for cytokeratins. The primary tumors were immunostained with an anti-E-cadherin monoclonal antibody. RESULTS MM ± TCM was found in 13 cases (31.7%) and TCM alone in 2 cases (4.9%) of the 41 pN0 cases. The pN0 patients with MM (but not TCM) had the same shorter survival as the original pN1 cases (P < 0.05). Of the 69 primary tumors, 49 (71.0%) had reduced or negative E-cadherin expression that showed a correlation with the occurrence of lymph node metastases (original pN1), MM, and TCM, but not prognosis. CONCLUSIONS The results of the current study show that, in SCC of the esophagus, MM, but not TCM, in the regional lymph nodes is prognostically equivalent to metastasis and should be examined by immunohistochemistry to classify these cases correctly as pN1. Cancer 1998;83:858-866. © 1998 American Cancer Society.

Micrometastasis and tumor cell microinvolvement of lymph nodes from esophageal squamous cell carcinoma

The purpose of this study was to investigate micrometastasis (MM) and tumor cell microinvolvement (TCM) in the regional lymph nodes of patients with esophageal squamous cell carcinoma (SCC). MM was defined as individual tumor cells or tumor cell clusters <0.5 mm in greatest dimension with a surrounding stromal reaction. TCM was defined as individual tumor cells or tumor cell clusters without a surrounding stromal reaction. One thousand nine hundred and fifty-four lymph nodes were dissected from 69 complete (R0) resection specimens of TNM classified pT1-3, pN0 or pN1, and M0 esophageal SCC. These lymph nodes were examined immunohistochemically using the monoclonal antibody cocktail AE1/AE3 for cytokeratins. The primary tumors were immunostained with an anti-E-cadherin monoclonal antibody. MM +/- TCM was found in 13 cases (31.7%) and TCM alone in 2 cases (4.9%) of the 41 pN0 cases. The pN0 patients with MM (but not TCM) had the same shorter survival as the original pN1 cases (P < 0.05). Of the 69 primary tumors, 49 (71.0%) had reduced or negative E-cadherin expression that showed a correlation with the occurrence of lymph node metastases (original pN1), MM, and TCM, but not prognosis. The results of the current study show that, in SCC of the esophagus, MM, but not TCM, in the regional lymph nodes is prognostically equivalent to metastasis and should be examined by immunohistochemistry to classify these cases correctly as pN1.

Cell-surface antigen expression in early and term gestation fetal hematopoietic progenitor cells.

The objective of this study was to compare the expression of primitive cell-surface antigens on CD34+ cells from early in gestation to those from term gestations. Fetal blood samples were obtained from 10 early gestation (21.0+/-0.8 [SE] weeks) and 12 term gestation (39.3+/-0.4 weeks) fetuses. The mononuclear cell population was separated by red cell lysis. Two-color flow cytometry was used to assess cell surface antigen coexpression of CD34 with CD33, CD38, and HLA-DR as well as staining by a cocktail of monoclonal antibodies for lineage-associated (Lin) antigens (CD2, CD10, CD11b, CD19, CD20, CD33, CD36, 7B9, and Glycophorin-A). The frequency of CD34+ cells (5.5+/-0.9 versus 1.5+/-0.2, p < 0.001) was significantly higher in the early gestational age group. Within the CD34+ population, the frequency of CD34+/CD38- cells (81.8+/-9.9 versus 51.3+/-7.7, p = 0.02) and CD34+/DR- cells (15.3+/-7.4 versus 8.2+/-2.7, p = 0.05) was also higher in the early gestational age group. In contrast, CD34+/CD33- (51.8+/-10.1 versus 83.0+/-6.1, p = 0.02) and CD34+/Lin- cells (15.9+/-7.0 versus 51.8 +/-6.9, p < 0.01) were higher in the term gestation group. The high percentage of CD34+, CD34+/CD38-, and CD34+/DR- cells supports our hypothesis that early gestational age fetal blood has a higher frequency of primitive hematopoietic progenitor/stem cells than does umbilical cord blood at term. This suggests that hematopoietic progenitor/stem cells in early fetal blood may be a desirable target for in utero gene therapy. However, further studies to characterize the functional properties of CD34+ cell subsets at different stages of fetal development will be necessary to determine the appropriateness of targeting fetal hematopoietic cells for in utero gene therapy. The higher frequency of CD34+/CD33- and CD34+/Lin- cells from term gestational age fetuses was unexpected, and the significance of this finding is unclear at this time.

Production and Characterization of a Set of Mouse-Human Chimeric Immunoglobulin G (IgG) Subclass and IgA Monoclonal Antibodies with Identical Variable Regions Specific forPseudomonas aeruginosaSerogroup O6 Lipopolysaccharide

The heavy- and light-chain variable regions from a murine monoclonal antibody that recognize Pseudomonas aeruginosa serogroup O6 lipopolysaccharide (LPS) were used to generate a series of chimeric mouse-human monoclonal antibodies with identical variable regions. The murine variable-region gene segments were cloned into an immunoglobulin (Ig) cDNA expression vector that contained the human kappa light-chain and IgG1 constant regions. The IgG1 heavy-chain constant region was then replaced with the human IgG2, IgG3, IgG4, or IgA1 heavy-chain constant region. The five different expression vectors were transfected into Chinese hamster ovary cells for antibody production. The chimeric antibodies exhibited immunoreactivity and affinity similar to that of the parental murine IgG antibody toward whole cells of a serogroup O6 strain. In vitro complement deposition assays demonstrated that the chimeric IgG4 and IgA antibodies did not mediate the deposition of complement component C3 onto the surface of either purified LPS or whole bacteria. The chimeric IgG1 and IgG3 antibodies were similar in their ability to deposit C3 onto the surface of both bacteria and LPS, while IgG2 antibody was more effective at depositing C3 onto the surface of bacteria than onto purified LPS. The pattern of opsonophagocytic activity of the chimeric monoclonal antibodies was similar to that of complement deposition onto bacterial cells in that the chimeric IgG1 and IgG3 had the highest opsonic activity. Although IgG2 deposited more C3 onto the bacterial surface than did IgG4 or IgA, all three of these isotypes had low opsonic activity against the serogroup O6 target strain. This series of related antibodies will help reveal functional differences in efficacy among protective antibodies to P. aeruginosa and will be critical for defining the optimal formulation of either a vaccine for active immunization or a polyclonal intravenous IgG or monoclonal antibody cocktail for passive immunotherapy.

RACIAL DIFFERENCES IN CLINICALLY LOCALIZED PROSTATE CANCERS OF BLACK AND WHITE MEN

Purpose Tumor grade, deoxyribonucleic acid (DNA) ploidy, proliferation, p53 and bcl-2 expression were examined in clinically localized prostate cancers of black and white American men to learn whether these features showed racial differences. Materials and Methods A total of 117 prostate cancers (43 black and 74 white patients) obtained at radical prostatectomy for clinically localized disease were assigned Gleason scores by a single pathologist. Enzymatically dissociated nuclei from archival prostate cancers were examined by DNA flow cytometry using propidium iodide staining and the multicycle program to remove debris and sliced nuclei and to perform cell cycle analysis. For immunostaining after microwave antigen retrieval we used a DO-1/DO-7 monoclonal antibody cocktail for p53 and the clone 124 antibody for bcl-2. Results Significantly more black than white men had Gleason score 7 tumors. The DNA ploidy distribution of Gleason 6 or less tumors was similar for both races. As anticipated, the ploidy distribution of higher grade prostate cancer in white men was more abnormal but, unexpectedly, this was not found for higher grade prostate cancer in black men. No significant racial differences were found in S phase fractions, p53 or bcl-2 immunopositivity. However, for prostate cancer in black men there was a significant association between bcl-2 immunopositivity and higher S-phase fractions. Conclusions The aggressive prostate cancers of black men may be characterized by the 2 features of high proliferation and a block to programmed cell death.

Expression of CD26/dipeptidyl-peptidase IV in benign and malignant pigment-cell lesions of the skin.

The T-cell activation antigen CD26 or dipeptidyl-peptidase IV (DPP-IV) belongs to a group of membrane-bound proteases that are variably expressed by melanoma cell lines. In vitro studies have suggested that loss of CD26 is associated with tumour progression. To correlate its expression with the histological stage of tumour progression of malignant melanoma (MM), we studied the distribution of CD26/DPP-IV in paraffin sections of a series of 110 benign and malignant pigment-cell lesions of the skin using a cocktail of anti-CD26 monoclonal antibodies and the three-step ABC method. Only two of 44 benign lesions focally expressed CD26 in their junctional compartment. In MM, expression of CD26 was not related to any of the known histological prognostic factors, but was associated with the stage of tumour progression; thus, CD26 was expressed in the situ or invasive radial growth phase in 34% of MM, whereas only 12% of MM expressed CD26 in the vertical growth phase. No CD26 expression occurred in metastatic melanomas. These data suggest that this proteinase plays a part in the early invasion of MM. Thus, CD26 may serve in the binding to, and enzymatic degradation, components of the extracellular matrix of the papillary dermis. Loss of CD26 in the vertical growth phase may contribute to the insufficient inactivation of regulatory peptides and unlimited action of growth factors.

Binding of a human monoclonal antithyroglobulin antibody to cultured human thyroid cancer cells.

To develop a new method of radioimmunodetection for thyroid cancer, we tested the binding ability of a human antithyroglobulin monoclonal antibody, VB5, to primary culture of human thyroid cancer cells. VB5 was able to immunostain cytoplasmic thyroglobulin (Tg) in the acetone-fixed cancer cells when used in a labeled streptavidin-biotin method but not in a conventional indirect immunoperoxidase technique. The antibody was readily labeled with I-125 in the standard chloramin-T method, and showed specific binding to the antigen on cultured malignant thyrocytes displaceable with non-labeled VB5 or with excess Tg antigen. Although these initial results in vitro are encouraging, the observed low specific binding (about 1% at room temperature) to intact cells with a single monoclonal antibody seems insufficient to conduct any in vivo immunolocalization experiments in animals. To obtain more binding, we would need a cocktail of several monoclonal antibodies to different epitopes, and also fragmentation of antibody molecules to penetrate into cytoplasm.

Combination of monoclonal antibodies for radioimmunoguided surgery.

The use of a cocktail of monoclonal antibodies in intraoperative radioimmunodetection of colorectal tumour has been evaluated in 14 patients. Eight patients had primary and six recurrent colorectal cancer. Three different monoclonal antibodies were used included B72.3, recognizing TAG72 antigen, FO23C3 and FO23C5 directed against two epitopes of CEA. The antibodies were labeled with 125I. During surgery the radioactive emission of tumour and normal tissue was evaluated by a gamma detecting probe. Ten patients showed positive intraoperative results and in particular 6 (75%) of the 8 patients with primary tumour and 4 (67%) of the 6 patients with recurrence, with a mean tumour to normal tissue ratio of 2.2. Intraoperative radioimmunodetection was instrumental in modifying the therapeutic approach in two patients with recurrent cancer but in none of the patients with a primary presentation. Immunohistochemical analysis, to evaluate TAG72 and CEA antigen expression, was performed in 13 cases. CEA antigen was expressed in 8/13 cases and TAG72 in 5/13. Thus the sensitivity was 38.5% and 61.5% with B72.3 and anti-CEA monoclonal antibodies respectively, while in combination the sensitivity was 71.4%. The use of a cocktail of different antibodies improved the sensitivity of intraoperative radioimmunodetection of colorectal tumours, when compared with a single antibody injection. This approach might improve the clinical use of radioimmunodetection.

Double-label Suspension Immunohistochemistry for the Detection of Gene Dysfunction in Sputum

A method was developed to identify molecular markers and gene alterations in exfoliated cells present in preparations of sputum. A double-label immunocytochemistry procedure was performed in suspension, with antibodies to cytokeratin intermediate filaments and to the p53 tumor suppressor gene product. The procedure was carried out in sputum samples obtained from individuals with lung cancer, and p53 expression in exfoliated cells was correlated with p53 expression in corresponding lung tumors. An alkaline phosphatase substrate, which is readily discerned by light microscopy and enhanced by fluorescence at the FITC excitation wavelengths, was used to detect immunolabeling by the monoclonal antibody DO-7 to the nuclear protein p53. The epithelial cells in the sputum were confirmed by fluorescence immunocytochemistry using a cocktail of monoclonal antibodies to a broad range of cytokeratins. Coexpression of cytokeratin and the p53 protein was detected in exfoliated cells with morphologies consistent with neoplasia and/or premalignant disease. These results demonstrate the feasibility of identifying gene alterations by immunocytochemistry in sputum from individuals with lung cancer. The detection of other gene alterations in individuals at risk for lung cancer using this method may enhance the benefit of traditional sputum cytology as a screening tool. (The J Histotechnol 20:13, 1997)

Double-label Suspension Immunohistochemistry for the Detection of Gene Dysfunction in Sputum

A method was developed to identify molecular markers and gene alterations in exfoliated cells present in preparations of sputum. A double-label immunocytochemistry procedure was performed in suspension, with antibodies to cytokeratin intermediate filaments and to the p53 tumor suppressor gene product. The procedure was carried out in sputum samples obtained from individuals with lung cancer, and p53 expression in exfoliated cells was correlated with p53 expression in corresponding lung tumors. An alkaline phosphatase substrate, which is readily discerned by light microscopy and enhanced by fluorescence at the FITC excitation wavelengths, was used to detect immunolabeling by the monoclonal antibody DO-7 to the nuclear protein p53. The epithelial cells in the sputum were confirmed by fluorescence immunocytochemistry using a cocktail of monoclonal antibodies to a broad range of cytokeratins. Coexpression of cytokeratin and the p53 protein was detected in exfoliated cells with morphologies consistent with neoplasia and/or premalignant disease. These results demonstrate the feasibility of identifying gene alterations by immunocytochemistry in sputum from individuals with lung cancer. The detection of other gene alterations in individuals at risk for lung cancer using this method may enhance the benefit of traditional sputum cytology as a screening tool. (The J Histotechnol 20:13, 1997)

A MONOCLONAL ANTIBODY TO HUMAN PLACENTAL LACTOGEN HORMONE FACILITATES ISOLATION OF FETAL CELLS FROM MATERNAL BLOOD IN A MODEL SYSTEM

A cocktail of trophoblast-reactive monoclonal antibodies (MAbs) is required for efficient isolation of trophoblasts from maternal blood. A modified antibody screening procedure was used to identify a clone, in a COS cell placental cDNA expression library, that expressed the gene product recognized by MAb FDO202N. The antigen recognised by MAb FDO202N was identified as human placental lactogen (hPL) hormone. hPL hormone is secreted into the maternal blood by trophoblasts at high levels during pregnancy. Immunohistochemical localization of hPL hormone was consistent with expression in the syncytiotrophoblast and extravillous cytotrophoblast. A model system was used where known numbers of syncytiotrophoblast sprouts were seeded into saline or maternal blood, bound by trophoblast-specific MAbs, recovered magnetically, and then counted. MAb FDO202N was shown to facilitate the efficient recovery of trophoblast sprouts from saline and maternal blood.

Presence of the intermediate filaments cytokeratins and vimentin in the rat corpus luteum during luteal life-span.

The presence of the intermediate filament (IF) proteins cytokeratins and vimentin in corpus luteum (CL) and other parts of the ovary from adult pseudopregnant rats was investigated using immunohistochemistry and immunoblot analysis. To induce pseudopregnancy, female rats were mated with sterile male rats. The mating procedure induces a prolonged luteal life-span of 13 +/- 1 days. Positive staining for cytokeratin could be seen in CL, in the theca layer of follicles, and the ovarian surface epithelium with the broad-spectrum monoclonal antibody cocktail AE1/AE3. Weak staining was also seen in CL with antibodies against cytokeratins 8 and 18. A similar distribution was also seen for vimentin, which furthermore was detected in blood vessels. No changes in staining intensity was seen in CL of different luteal age. The strong staining for vimentin in CL was confirmed by immunoblot analysis, where one main band of 57 kDa was observed from day 1 to day 19 of pseudopregnancy. Expression of the IF proteins investigated seems to start in the newly formed CL and the continuous expression indicates that they are not directly regulated by luteal steroids.

Autologous bone marrow transplantation for leukemia: Monoclonal antibody-mediated purging of multidrug-resistant leukemia

Nine drug-resistant variants of human leukemic/lymphoma cell lines were evaluated for the expression of antigens which are commonly targeted in antibody-dependent purging protocols used for autologous bone marrow transplantation. Flow cytometry revealed that most drug-resistant variants differ in antigen expression from parental lines. Both epipodophyllotoxin-selected T-ALL variants tested expressed lower levels of CD5 than parental counterparts. In contrast, other antigens (CD7, CD3, CD4, CD8, CD38, CD9, CD10, CD24, CD19, CD20) were not consistently altered in drug-resistant variants. Drug-resistant cells were lysed as effectively as drug-sensitive cells by murine antibody and baby rabbit complement, but only when particular monoclonal antibodies or cocktails of monoclonal antibodies were utilized. Two of the nine drug-resistant variants proved to be more resistant than parental cells to antibody-dependent cell-mediated cytolysis (ADCC).

Further studies on the feasibility of one-day Salmonella detection by enzyme-linked immunosorbent assay.

A model system previously developed for the rapid detection of Salmonella typhimurium in foods was improved and extended to many other Salmonella serotypes. The original protocol, which consisted of an overnight nonselective culture followed by a specific enzyme-linked immunosorbent assay (ELISA), was modified and improved. A sandwich ELISA which used polyclonal antibodies for the capture stage and a cocktail of monoclonal antibodies for the detector stage was developed. The assay recognized a wide range of Salmonella serotypes; S. enteritidis, the most important serotype in the United Kingdom had a detection limit in the ELISA of about 4 x 10(2) cells ml-1. The cultural stage prior to the ELISA was either a single nonselective broth (incubated for 28 h) or a preenrichment broth (incubated for 7 h) plus a selective broth (incubated for 21 h). Antibodies which bind to cells grown in the unfavorable conditions of a selective medium were selected. It was concluded that, in the future, the shortened protocols for the detection of Salmonella spp. in foods described here will be of considerable value.