Radioautographic studies of plaque-forming cells. I. Antigen-stimulated proliferation of plaque-forming cells.

A method has been described for obtaining radioautographs of plaque-forming cells. The method permits radioautographic analyses of small numbers of plaque-forming cells amidst large populations of non-plaque-forming cells. Spleen cells that were pulse-labeled with tritiated thymidine could be categorized readily as labeled or not labeled. Using this method it was found that (a) at least 55% of plaque-forming cells which appear 3 days after a maximal stimulus of 4 x 10(8) sheep red cells are still capable of DNA synthesis, and must have arisen by cell proliferation; (b) the rate of proliferation of plaque-forming cells is proportional to the log of the dose of antigen; (c) the S period of plaque-forming cells is at least 2 hr, appears to be constant, and is not influenced by antigen dose. The results suggest that antigen stimulates proliferation of plaque-forming cells by hastening their transit through the G(1) phase of the generative cycle.

Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography.

A high-performance liquid chromatographic method (HPLC) with combined diode array and fluorescence detection of amino acids and amines in various cheese samples is described. The proposal is based on acidic deproteinization, derivatization and gradient optimization studies, resulting in the identification and quantification of 21 amino acids and 9 amines from a single solution, by one injection. The optimized, simple protocol consists of deproteinization (1M perchloric acid), centrifugation, filtration and the subsequent derivatization with the o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate (OPA-ET-FMOC) reagent. The method can be characterized with a linearity of wide concentration range (6.25-1000 pM/injection), a good chromatographic reproducibility (average: 2.69% RSD) and an excellent recovery (average: 100.2%; average 3.84% RSD). The developed method was successfully applied in the determination of the amino acid and amine contents of port salut cheese, blue cheese and smoked cheese samples.

Analysis of amino acids and biogenic amines in biological tissues as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate derivatives by high-performance liquid chromatography. A deproteinization study.

The extraction of ornithine, lysine, putrescine, cadaverine, 1,7-diaminoheptane, spermidine and spermine from biological tissues was optimized for HPLC quantitation as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate (OPA/ET/FMOC) derivatives. In applying perchloric acid deproteinization two approaches have been followed: (i) deproteinization with subsequent neutralization by potassium hydroxide and lyophilization, and (ii) deproteinization without neutralization and lyophilization. Neutralization and lyophilization resulted in the loss of free biogenic amines. HPLC analysis of ornithine (Orn), lysine (Lys), putrescine (Put), cadaverine (Cad), 1,7-diaminoheptane (Dah), spermidine (Spd) and spermine (Spm) content of biological tissues as their OPA/ET/FMOC derivatives was performed in the supernatant of perchloric acid-deproteinized samples (model solutions and tissues) with an average reproducibility of < or =2.6% relative standard deviation (RSD), including recovery of sample treatment and chromatography.

Stability and utility of the unique human small cell carcinoma line SHP-77.

The human small cell (oat cell) carcinoma line, SHP-77, established by Fisher and Paulson in 1977 and originally described as a "large cell variant of oat cell cancer" has been evaluated by several different parameters and shown even after more than 200 passages to retain properties described for the original cell line. Karyotypic, histological, and biochemical features are retained, as well as tumorigenicity in nude mice. The original authors' suggestion that this is a propitious cell line for both in vitro and in vivo studies is supported by this report. Modulation of growth characteristics in vivo (in xenografts) emphasizes the plasticity of this unique line which serves as a valuable model for basic as well as therapeutic studies. SHP-77 can serve as an in vitro target in 51Cr and 111In release cytotoxicity assays as well as in in vivo nude mouse assays for evaluating immune reactivity of cells and serum from lung cancer patients. The potential histological variability of SHP-77, despite its biochemical stability, calls attention to the inadequacy of histological criteria for lung tumor classification.

Neural cell adhesion molecule expression and messenger RNA splicing patterns in lung cancer cell lines are correlated with neuroendocrine phenotype and growth morphology.

Diverse histological types of lung cancer express neuroendocrine (NE) markers. We studied the expression and alternative splicing forms of the neural cell adhesion molecule (NCAM) NKH-1, a member of the immunoglobulin superfamily, in 56 lung cancer cell lines representing all histological types. We found a strong correlation between expression of NCAM with both NE phenotype and lack of substrate adhesion in culture. Several cell lines expressed high levels of the leukocyte antigen Leu-7 (HNK-1) but were negative for NCAM antigen and mRNA, indicating that the Leu-7 antigen is distinct from NCAM. All of the NCAM-positive cell lines demonstrate a single 6.2-kilobase mRNA, and analysis of the known 3' alternative splices shows predominant expression of only the membrane form with the small intracytoplasmic domain. We conclude that (a) expression of NCAM is associated with NE phenotype regardless of the histological type of lung cancer; (b) these cell lines share a single form of NCAM; (c) with few exceptions, NCAM expression is associated with cell to cell adhesion and lack of substrate adhesion (growing as floating clusters); and (d) Leu-7 antigen is distinct from NCAM. This form of NCAM may play a functional role in NE differentiation or may be a part of the NE program expressed by these cells.

Behavior and characteristics of biogenic amines, ornithine and lysine derivatized with the o-phthalaldehyde-ethanethiol-fluorenylmethyl chloroformate reagent.

The stability and characteristics of the ornithine (Orn), lysine (Lys), putrescine (Put), cadaverine (Cad), 1,7-diaminoheptane (Diah), spermidine (Spd) and spermine (Spm) derivatives obtained with the o-phthalaldehyde (OPA)-ethanethiol (ET)-fluorenylmethyl chloroformate (FMOC) reagent has been investigated. The stoichiometry of the introduced, two-step derivatization process has been followed by photodiode array (DAD) and fluorescence (FL) detections, simultaneously, while the composition of derivatives was confirmed by on-line HPLC-electrospray ionization (ESI) MS measurements. Depending on the composition of the OPA reagents, in addition to the secondary amino group-containing Spd and Spm, under common aqueous conditions also Orn and Lys do react with FMOC resulting in derivatives of various compositions. Applying the OPA-ET reagent of increasing methanol (Met) content (38-80%, v/v) the formation of the FMOC group containing Orn and Lys derivatives could be considerably decreased. Optimum elution condition (18 min, including equilibration) was developed for the simultaneous quantitation of Orn, Lys, Put, Cad, Diah, Spd and Spm, in the presence of the rest of protein amino acids. The practical utility of the method was demonstrated by the analysis of mouse tissues. Average reproducibility of quantitations, characterized with the relative standard deviation percentages of fluorescence intensities and UV responses, in order of listing, proved to be 2.1% and 2.1%, respectively.

JLP5B9: new monoclonal antibody against polysialylated neural cell adhesion molecule is of value in phenotyping lung cancer.

Non-small-cell lung cancer (NSCLC) is currently one of the most prevalent malignant tumors. It displays a wide variety of phenotypes which includes neuroendocrine markers commonly found on small-cell lung cancers (SCLC) such as the neural cell adhesion molecule (NCAM) and in particular its highly polysialylated isoform, embryonic NCAM (eNCAM). NSCLC with neuroendocrine differentiation may represent a subset of tumors whose cells have a more aggressive biological behavior. A tumor marker that distinguishes this latter sub-type could be of clinical relevance. Accordingly, we have raised a monoclonal antibody of the IgM type (JLP5B9) directed against capsular polysaccharides of N. meningitidis B which bears polysialic acid groups. We have demonstrated that JLP5B9 recognizes eNCAM with high affinity and that it is specifically directed against the polysialic acid moieties of NCAM. JLP5B9 was also found to react with human SCLC, NSCLC and neuroblastoma cell lines. We then used JLP5B9 as a specific probe for the detection of tissue eNCAM and found that it was expressed on up to 20% of tumor cells obtained from 5 out of 13 patients with NSCLC. This mAb deserves further investigation to evaluate its potential as a tool for serodiagnosis of lung cancer.

Gasoline sniffing and lead toxicity in Navajo adolescents.

During a 6-year period, 23 Navajo adolescents were hospitalized 47 times for presumed lead intoxication secondary to gasoline sniffing. Most patients were male (87%) and sniffed gasoline as a social activity, more frequently in spring and summer. Sixty-five percent of the patients first presented with toxic encephalopathy. Of total episodes, 31% involved asymptomatic lead overload; 31% involved tremor, ataxia, and other neurologic signs; and 38% involved encephalopathy with disorientation and hallucinations. Free erythrocyte protoporphyrin levels were not consistently high, although blood lead levels were all elevated. One death occurred. Approximately 11% of 537 Navajo adolescents said they inhaled gasoline for enjoyment at least occasionally. Among 147 junior high school students, blood lead levels averaged 18 +/- 6 micrograms/dL with no values greater than 40 micrograms/dL. Three of these students had elevated zinc protoporphyrin levels and all three were anemic. No correlation was found between levels of blood lead or zinc protoporphyrin and whether or not the youth reported sniffing gasoline. However, sniffing gasoline was associated with poor school performance and delinquent behavior. Although apparently many Navajo adolescents experiment with gasoline inhalation, only a few engage in this activity frequently enough to develop either asymptomatic or symptomatic lead overload.

Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth.

Human small cell lung cancer (SCLC) produces and secretes BN/GRP (bombesin/gastrin releasing peptide). Because BN stimulates the growth of SCLC cells and these cells have receptors for BN-like peptides, it is important to define agents which disrupt this self-promoting autocrine growth cycle. Here, substance P analogues were evaluated as BN receptor antagonists using SCLC cell lines. (D-Arg1, D-Pro2, D-Trp7.9, Leu11) substance P [(APTTL)SP] was one of the more potent analogues tested in inhibiting BN-like peptide receptor binding with an IC50 value of 1 microM. Micromolar concentrations of (APTTL)SP antagonized BN receptor mediated elevation of cytosolic Ca2+ levels and decreased the colony formation in soft agarose. These data suggest that SP analogues function as SCLC BN receptor antagonists and may be useful in disrupting the autocrine growth function of BN-like peptides.

Chrysamine-G, a lipophilic analogue of Congo red, inhibits A beta-induced toxicity in PC12 cells.

Increasing evidence suggests that deposition of amyloid-beta (A beta) peptide leads to neurodegeneration in Alzheimer's disease. Congo red, a histologic dye that binds to amyloid has previously been shown to diminish the toxic effects of A beta in cell culture. Since Congo red is too highly charged to enter the brain in significant quantities, a lipophilic derivative, Chrysamine-G, was tested for the ability to attenuate A beta[25-35]-induced toxicity in PC12 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Chrysamine-G showed a concentration-dependent inhibition of A beta[25-35]-induced toxicity. This protective effect became significant at 0.2 microM, a concentration very close to the Ki for Chrysamine-G binding to synthetic A beta (0.37 microM). A decarboxy derivative of Chrysamine-G, which does not bind to A beta, also did not protect against A beta-induced toxicity. The protective effects of Chrysamine-G may relate to its ability to bind directly to A beta and may involve other post-binding effects as well.

186Rhenium-HNK1 monoclonal antibody targets human small cell lung cancer cells in athymic nude mice: rapid screening model for therapy.

A model is described by which in vivo tumor-MOAB interactions may be investigated. The method is rapid and may aid in the selection of appropriate MOABS from a panel of MOABS for individualized patient treatment. Groups of athymic nude mice were injected intravenously with small cell lung cancer line SHP-77 cells which are trapped primarily in the lungs. Twenty-four hours post tumor cell inoculation, 186Rhenium tagged HNK1 MOAB (CD57) was injected intravenously. Controls which received no tumor cells were injected with unbound 186Re or radioactive MOAB. Biodistribution studies at 24 hours following MOAB injection showed significantly more radioactivity in lungs of mice inoculated with both SHP-77 cells and 186Re MOAB than did lungs of controls.

Antilung cancer activities of selenium.

Selenium is considered an essential trace element in most animal and plant species, although still reported in many texts as a highly toxic material. Epidemiological investigations have reported an inverse relationship between selenium and lung cancer. Explanations of reported observations have resulted in numerous mechanistic theories. Only recently have selenium metabolites involved in excretion been considered potential agents for antilung cancer activity. Anticancer properties have been shown in occupationally exposed copper smelter workers, dietary investigations and experimental studies. Supplementation with selenium of public water supplies, as is currently done with fluoride, is a potential method for increasing the blood concentration. This may permit development of a population prevention strategy against lung cancer and other diseases.

Anti-autologous erythrocyte plaque-forming cells in pregnancy. A parameter of immunoregulatory change.

Peripheral blood leukocytes (PBL) from 16 persons (6 Rh-sensitized pregnant women, 1 pregnant non-Rh-sensitized woman, 3 nonpregnant non-Rh-sensitized women, and 6 non-Rh-sensitized males) were assayed for plaque-forming cells (PFC) against several erythrocyte targets. 6 of 7 pregnant women had PFC, whereas only 1 of 6 males had PFC to autologous red cells. Antiautologous erythrocyte PFC in all of the pregnant Rh-sensitized women as well as the 1 nonpregnant multiparous woman may be the result of alloimmunization of mothers by their fetuses during gestation. Further studies in this area would be valuable in determining not only the immune status of a mother in relation to her fetus, but also would be of value in determining the consequences of that immune status on both mother and fetus. Such information would also provide a further clue to the etiology of autoimmune disease.

Phenotypic differences among morphologically similar small-cell carcinomas detected with a panel of monoclonal antibodies and indirect immunofluorescence and flow cytometry.

Indirect immunofluorescence and flow cytometry were used to determine reactivity of a panel of 75 monoclonal antibodies (MAbs) and controls (provided by the Third International IASLC Workshop on Lung Tumor and Differentiation Antigens) with 3 morphologically similar prototype continuous-culture small-cell-carcinoma cell lines (SCC) (NCI-H69, NCI-H146, and NCI-H510). All cell lines had some reactivity with some of the MAbs. There is, however, differential expression of antigens amongst the prototype cell lines, which may provide a useful method for phenotyping and sub-classifying SCC. The reactivity of the 3 cell lines was greatest with MAbs in Clusters I, 1c, 2, 4, 6, and 9, and least with MAbs in clusters W7, 8, 13, 14, and W15, with few exceptions. Although morphologically similar, each of the SCC cell lines has a unique pattern of reactivity with the workshop MAbs. For example, although a control MAb, CD56 (NKHI), which identifies an epitope on NCAM (neural cell adhesion molecule) common among many SCC lines, stained more than 90% of cells in each of the prototype cell lines, one MAb of the current panel, SEN7, which also identifies a CD56 epitope on 15 SCC lines did not react as strongly with H-146 and H-510 as with H-69. If appropriately reactive MAbs can be identified for individual patients' tumors, they can be coupled to suitable radioisotopes or toxins for individualized patient treatment.

Fetal human hemolytic plaque-forming cells. Evidence of reactivity to maternal and other erythrocytes.

Lymphoid tissues from 24 human fetuses were assayed for hemolytic plaque-forming cells (PFC) against a variety of erythrocyte targets. PFC against maternal and other erythrocyte antigens were commonly detected in human fetal liver, lymph nodes, spleen, or thymus as early as 16 weeks gestation and were usually more abundant in liver than in spleen after 16 weeks gestation. These data corroborate studies from other laboratories which indicate that human fetuses develop some forms of immunocompetence very early during gestation.