Improved detection of Beet necrotic yellow vein virus in a DAS ELISA by means of antibody single chain fragments (scFv) which were selected to protease-stable epitopes from phage display libraries.

The detection of Beet necrotic yellow vein virus (BNYVV) in stored sugar beets by means of monoclonal antibodies or antibody single chain fragments (scFv) often poses problems, because the immunodominant C-terminal epitope of the viral coat protein is readily lost due to proteolysis. Clones which produce scFv specific for protease-stable BNYVV epitopes were selected from two naive phage display libraries. Fusion proteins of the scFv with a human IgG kappa chain (expressed from the newly designed vector pCL) or with alkaline phosphatase,respectively, allow the ELISA detection of BNYVV even in stored sugar beets with a sensitivity which was comparable or often higher than that achieved with polyclonal antibodies.

Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay.

A recombinant enzyme-linked immunosorbent assay (ELISA) system, entirely based on antibody fragments, is described here as an attractive alternative to assays using polyclonal antisera or monoclonal antibodies. Two expression vectors have been developed for cloning and production of single-chain Fv (scFv) fusion proteins suitable as coating and detecting reagents, respectively, in a highly sensitive double antibody sandwich ELISA. The coating reagent is produced from the vector pZIP1, as a bivalent miniantibody with a leucine zipper for dimerization. The detection reagent is a fusion protein, in which a modified Escherichia coli alkaline phosphatase with increased specific activity is attached to the carboxy-terminus of the scFv. This conjugate is produced using pDAP2/S as cloning and expression vector. Optimized bacteria expression and simple one-step purification by hexahistidine tag-mediated metal chelate affinity chromatography yielded milligrams of ELISA reagent per liter of bacterial culture in both cases. A double antibody sandwich ELISA for the detection of beet necrotic yellow vein virus, the causal agent of sugarbeet rhizomania, was developed using fusion proteins obtained by means of pZIP1 and pDAP2/S. The plant pathogen was detected with a sensitivity higher than that reached in a conventional ELISA employing polyclonal antisera. Both plasmid vectors are compatible to phage display vectors such as pHEN1, pCOCK, and the pCANTAB series, allowing simple subcloning after isolation of scFv from phage display libraries. It is therefore easy to develop and produce an ELISA entirely by using bacterial recombination and expression techniques.

pDAP2: a vector for construction of alkaline phosphatase fusion-proteins.

BACKGROUND: Expression of enzymatically active protein fusions in Escherichia coli could facilitate the analysis of proteins and even replace some reagents frequently used in immunology such as chemically produced antibody-enzyme conjugates. For this purpose there is up to now no system of general utility available. OBJECTIVES: The vector pDAP2 has been designed for simplified fusion of single-chain Fv fragments to the N-terminus of E. coli alkaline phosphatase. The resulting immunoconjugates can be produced by expression in E. coli and purified in a single step via metal affinity chromatography. STUDY DESIGN: Several different single-chain Fv genes as well as peptide coding oligonucleotides have been cloned into pDAP2 and tested for expression levels, purification properties and usefulness in ELISA and immunowestern blotting. RESULTS: The fusion proteins from pDAP2 can be prepared at levels of several milligrams per liter culture from the periplasma of the cells. The proteins work well in different immunoassay formats such as ELISA or western blotting. CONCLUSION: pDAP2 is compatible to phage display vectors such as pHEN1, pCOCK and the pCANTAB-series (Pharmacia, Sweden). Genes of single-chain antibody fragments can be simply swapped from these vectors into pDAP2. Furthermore, we demonstrate that it is possible to produce alkaline phosphatase-active peptides with this vector by inserting synthetic coding oligonucleotides. This allows simple analysis of the binding properties of peptides and may have some advantages over other systems such as fusion to glutathione-S-transferase.

Enhancing adoption of an alcohol abuse prevention program: An application of diffusion theory.

The purpose of this study was to apply diffusion theory (Rogers, 1983) to an alcohol abuse prevention program in order to enhance program adoption. Three of the four stages of diffusion theory (dissemination, adoption, and implementation) are discussed relative to the development and implementation of a program designed to increase awareness of the destructive effects upon children who live with an addicted parent of living with that parent. The program also focused upon resiliency building strategies to use with children in order to mitigate the harmful effects of addiction in the home. Empirical data are presented concerning the dissemination, adoption and implementation phases of the program.

Use of the theory of reasoned action to predict drug and alcohol use.

The present study examines the role of self-esteem (SE) in the prediction of drug and alcohol use. Consistent with research on the theory of reasoned action, we suggest that alcohol and drug attitudes and subjective norms are more useful in the prediction of self-reported drug and alcohol consumption than SE. In the present study, measures of SE, drug attitudes, subjective norms, and drug use behaviors were collected from 2,074 high school and college students. Results indicate that drug attitudes and subjective norms do predict drug and alcohol use, but that SE does not add significantly to the prediction of the drug and alcohol behaviors.

Pulmonary fibrosis associated with low-dose adjuvant methyl-CCNU.

Pulmonary fibrosis is a serious side effect of nitrosourea therapy, occurring most frequently in patients treated with BCNU. Pulmonary fibrosis developed in a 63 year-old male patient while being treated with adjuvant methyl-CCNU for rectal carcinoma. This toxicity is rare with methyl-CCNU, having only been reported once previously. The case of methyl-CCNU-induced pulmonary fibrosis reported here occurred at a much lower total dose than the first reported case (604 mg/m2 vs. 2,733 mg/m2). Details of the case history, including radiographic and pathologic findings, are presented.

Pathologic analysis of advanced adult soft tissue sarcomas, bone sarcomas, and mesotheliomas. The Eastern Cooperative Oncology Group (ECOG) experience.

A total of 488 tumors entered in the Eastern Cooperative Oncology Group (ECOG) Study EST 3377 were evaluated histologically by a panel of pathologists from member institutions for quality control purposes. The overall agreement rate between the eligible submitting diagnosis and the pathology review panel's diagnosis was 74% (312/424). In 10% (44/424), the case was excluded because it was deemed to be nonsarcoma. In the other 16%, the disagreement concerned the type of sarcoma. The histologic type with the lowest agreement rate was rhabdomyosarcoma (17%), followed by sarcoma not otherwise specified (NOS) (27%), angiosarcoma (33%), and fibrosarcoma (48%). These figures reflect the significant degree of difficulty in the diagnosis of these tumor types. The treatment response rate of soft tissue sarcomas in the randomized study of Adriamycin (Adria Laboratories, Columbus, OH) regimens was slightly higher for those with lower grade sarcomas, i.e., 25% (four of 16) response rate for Grade 1 lesions; 22% (17/77) for Grade 2, and 21% (35/170) for Grade 3. When adjusted for type of sarcoma, there was no noticeable difference between Grade 1-2 versus 3 in response rate. A statistically significant difference in the percentage of complete responders was noted between Group A tumors (synovial sarcoma, hemangiopericytoma, sarcoma NOS, and Ewing's; 12.2%) versus Group B tumors (all other types--mostly spindle cell sarcomas; 3.5%) (P = 0.02).

Immunostaining for carcinoembryonic antigen does not discriminate for early recurrence in breast cancer. The ECOG experience.

Carcinoembryonic antigen (CEA) immunohistochemistry was evaluated by 11 surgical pathologists with sections from 147 postmenopausal women with node-positive breast cancer. Carcinoembryonic antigen staining in breast cancer tissues has been correlated with a worse prognosis. This association was studied with a clinically characterized population of Eastern Cooperative Oncology Group (ECOG) patients using precisely the peroxidase-antiperoxidase methodology which had been employed in another published study. In 50% of the cases, the study pathologists were uncertain whether CEA was or was not present in the cancers. Various groupings of the pathologists' interpretation were compared with the observed disease-free intervals in the patients. These analyses suggested no association of perceived CEA staining with the biological course of the cancers. Two reference pathologists who examined the sections in a similar way also gave non-prognostic interpretations. There is no convincing evidence that pathologists can reliably interpret the CEA content in the same breast cancer tissue sections. There is no observed correlation between immunohistochemical evidence of CEA in a breast cancer tissue section and the biological behavior of that cancer.

Interobserver reproducibility of histopathological features in stage II breast cancer. An ECOG study.

Eleven surgical pathologists studied microscopic sections from 45 mastectomy specimens of node positive breast cancer patients who had been entered into ECOG clinical trials. Inter-observer reproducibility for histoprognostic features was examined as a prerequisite before a subsequent evaluation of their possible clinical applicability could be undertaken. Histological type, nuclear grade, tubular formation, and lymphoid reactions were studied in the cancerous tissues. Lymph nodal responses (follicular and pulp prominence, sinus histiocytosis) were also examined in a manner that stimulated slide review in routine surgical pathology practice. Numerous two-way comparisons of the pathologists' findings resulted in low levels of agreement (usually much less than 90%). The degree of inter-observer reliability is clinically unacceptable using customary slide review analysis. New ways of examining breast cancer tissues need to be explored in the search for prognostic features which can be applied to the clinical management of breast cancer patients.

Interobserver variation in the identification of breast carcinoma in intramammary lymphatics.

Nine surgical pathologists participated in a microscopic review of 35 cases of pT1-2 N0 M0 breast carcinoma. The pathologists outlined strict criteria for the identification of intramammary lymphatics and blood vessels and for the identification of cancerous emboli in these vascular channels. Each mastectomy case was studied by three different pathologists. All three concurred on the presence or absence of intralymphatic cancer in 12 of the 35 cases. Observers agreed on the absence of blood vessel invasion in 30 of the 35 cases. There was no consistent bias on the part of a single reviewer, either alone or with another pathologist, in identifying the emboli. We conclude that the identification of intralymphatic cancerous emboli in mastectomy specimens is not a reliably reproducible prognostic finding on which recommendation of systemic chemotherapy in stage I breast carcinoma patients can be based.

Ultrastructural characteristics of sinus histiocytic reaction in lymph nodes draining various stages of breast cancer.

An ultrastructural study of sinus histiocytosis (SH) in axillary lymph nodes was undertaken in 20 patients with primary cancer of the breast. The lymph node responses were first classified by means of light microscopy, according to previously established criteria. The ultrastructural findings established three major patterns of SH response in cancer-draining lymph nodes: developing SH, positive SH, and degenerative SH. These patterns followed closely the classification obtained by light microscopy. An important finding of electron microscopy was the presence of early activation of histiocytes (developing SH) in microscopy (negative SH). The striking ultrastructural resemblance of histiocytes in positive SH to epithelioid cells seen in delayed hypersensitivity reactions (TB) again points to the fact that the positive SH pattern represents a tumor-host reaction of the delayed hypersensitivity type. Finally, although the presence of virus has been shown to play a role in spontaneous BALB/c murine breast tumor, no virions could be found in human cancer-draining lymph nodes.

Characteristics of yellow-pigmented nonfermentative bacilli (groups VE-1 and VE-2) encountered in clinical bacteriology.

The morphological and physiological characteristics of 20 strains of motile, gram-negative, yellow-pigmented oxidative bacilli (groups VE-1 and VE-2) isolated in clinical bacteriology are described. Electron micrographs demonstrate the polar multitrichous flagella of group VE-1 and polar monotrichous flagella of group VE-2. Data obtained from guanine plus cytosine ratio studies of 56.8% for VE-1 and 68.9% for VE-2 distinguish the two groups of bacteria.