Adverse reaction to food: assessment by double-blind placebo-controlled food challenge and clinical, psychosomatic and immunologic analysis.

Double-blind placebo-controlled food challenge (DBPCFC) with food items applied in capsules was performed in a prospective study of 17 selected patients and 34 age- and sex-matched healthy controls in the interdisciplinary clinical setting. Protein immunoblotting showed no differences in antigenicity between foods in the capsules and the corresponding fresh foods. All patients reacted to one or more food substances during DBPCFC, with a doubtful reaction to placebo in 2 patients. Agreement between diet history and provocation was seen in 53 of 85 individual food challenges, 36 being positive with both examinations. In 22 (38%) of the 58 positive provocations, the reactions were not expected from the patients' histories. No reaction to food or placebo occurred in the control group. Related to diet history, sensitivity and specificity of provocation were 62 and 63%, respectively, with a positive predictive value of 78%. Allergy, previous gastroenterologic and infectious diseases among first-degree relatives, immunologic abnormalities and elevation of total IgE were significantly more common for the patients than controls. A positive skin prick test correlated well with diet history, but both prick test and food antibodies correlated poorly with DBPCFC. Assessment by the General Health Questionnaire showed a significant difference towards the controls. After 3-4 months of follow-up on an individually based diet, 11 of 15 patients reported general improvement of their condition. DBPCFC may be a valuable diagnostic test in addition to dietary history as a basis for elimination diet on food-intolerant patients. The effect of the elimination diet on the symptoms may also suggest a therapeutic effect or provocation.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous assessment of migration and proliferation of murine fibrosarcoma cells, as affected by hydroxyurea, vinblastine, cytochalasin B, Razoxane and interferon.

Using porous cell culture chambers, we have simultaneously assessed growth and locomotion of cancer cells to investigate whether certain agents affect cell motility in addition to cell division. First, cells from a murine fibrosarcoma cell line, 1.0/L1, were grown in ordinary flask cultures to determine appropriate cell inocula. Doses of agents were selected to reduce the final 4 day culture cellularity to about 50%, when present during the last two days of culturing. Secondly, the effects of these agents on cell numbers in the porous chambers and on cell migration out of the chambers ('emigration fraction') were recorded. We also examined, using a similar type of porous chamber, whether the agents could affect leucocyte chemotaxis. Hydroxyurea (an inhibitor of DNA synthesis) reduced cancer cell emigration as well as cell growth, without interfering with leucocyte chemotaxis. Cytochalasin B (a microfilament disrupting agent) inhibited cancer cell motility and growth, as well as leucocyte chemotaxis. Vinblastine (a microtubule disrupting agent), at the very low dose chosen, reduced cancer cell growth, but did not consistently affect the migration of either cell type. The experimental anti-metastasis agent Razoxane reduced growth, but had no detectable effects on motility. High doses of natural murine interferon-alpha/beta weakly inhibited both cancer cell growth and locomotion. This motivates for further studies of these and other cytokines, as treatment with agents inhibiting cancer cell locomotion might possibly prevent peri-operative spread of cancer in patients.

99mTc-hexamethylpropyleneamine oxime leukocyte scintigraphy and C-reactive protein levels in the differential diagnosis of brain abscesses.

The demonstration and accurate localization of intracerebral mass lesions are commonly performed with computerized tomography (CT), which often cannot determine the nature of the lesion. As an aid in the differential diagnosis between brain abscess and neoplasm, the authors have evaluated both 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO) leukocyte scintigraphy and the serum C-reactive protein level. Of 23 patients with intracranial mass lesions, 22 individuals showed ring-like contrast enhancement on CT scans; the one exception was a patient treated for a meningioma who had a negative CT scan despite clinical suspicion of intra- or extracranial abscess. The final diagnosis was invariably established by microscopic examination of tissue specimens. In 10 patients the final diagnosis was brain abscess; the other 13 patients harbored a brain neoplasm (glioma in nine, astrocytoma in one, and metastasis in three). The 99mTc-HMPAO leukocyte scintigraphy detected all cases of abscess. There were no false-positive results. An elevated C-reactive protein level (> 13 mg/liter) was found in all but one patient with abscess and in three patients with neoplasm; two of these three patients had dental root infections which could account for the elevation of C-reactive protein. It is concluded that 99mTc-HMPAO leukocyte scintigraphy should be performed when there is a possibility that a brain abscess may exist. Any steroid treatment should be discontinued for 48 hours prior to leukocyte scintigraphy. Also, C-reactive protein determination should be performed and is useful even when steroids are given.

Growth patterns of pulmonary metastases and primary tumours from five murine fibrosarcoma cell clones.

The growth patterns, including the size, shape and regional preferences, of lung metastases from five murine fibrosarcoma cell clones were studied. Spontaneous metastases developed from tumours formed by subcutaneous inoculation of the cell clones. Lung colonies (experimental metastases) were established by i.v. injection of cells. The numbers of both spontaneously and experimentally formed subpleural lung metastases were counted through a stereomicroscope. The fraction of colonies that was located subpleurally was determined in histological sections of lungs. The growth kinetics of clonally derived primary tumours, and the number of spontaneous and experimental lung metastases, differed greatly between certain cell clones. The number of spontaneous lung metastases was correlated with the maximum size of primary tumours. No close correlation was observed between the size of the primary tumours and the size of experimental metastases. There were differences between the cell clones in the shape and regional preferences of their lung deposits. The subpleural colonies were generally larger than the intrapulmonary ones. Thus, both the regional distribution and the growth pattern of lung deposits differed between the clones.

Tumor-cell motility and invasion within tumors determined by applying computer simulation to histologic patterns.

Proliferation and motility are crucial prerequisites for tumor-cell invasion in vivo. While proliferation can be assessed in situ by a variety of methods, the measurement of motility is largely restricted to in vitro models. In previous studies, computer simulations of tumor growth strongly indicated that a close relationship exists between tumor-cell motility on the one hand and the resulting morphological pattern on the other. Moreover, estimates of motility parameters can be based on image analysis of the particular pattern. The objective of the present study was to examine whether tumor-cell populations differing in their in vitro motility produce particular patterns in vivo similar to those predicted by the computer simulations, and whether the motility estimates derived from these patterns are consistent with the in vitro motility results. This was done using murine fibrosarcomas grown in syngeneic animals from various cell lines which had been selected for and confirmed to show greatly increased speeds of motility in vitro relative to the unselected parent cell population. Computer simulations coupled with image analysis of the various variant tumors showed that the calculated motility of tumor cells within the different tumors agreed well with the relative levels of tumor-cell motility observed in vitro. Our results thus show that the computer simulation method, along with histological analysis, produced reliable estimates of tumor-cell motility within tumors.

Growth and metastasis of hypermotile, hyperinvasive cancer cells selected in vitro by rapid locomotion under various conditions.

Cancer cells selected from a cultured murine fibrosarcoma by rapid migration through micropore membranes moved considerably faster through such membranes and invaded biological tissues much more efficiently than did the unselected parent cells. The present data show that populations of cells selected by unstimulated migration or by haptotaxis to laminin moved not only faster, but also in larger numbers than the parent cells. However, the selected cells were far less efficient than the parent cells in forming spontaneous lung metastases in syngeneic mice, although all cell lines were 100 per cent tumorigenic. Analysis of paired data within each group showed no relationship between the primary tumor size at any observation time and the number of lung metastases finally formed. Therefore, although the parent cell line produced primary tumors growing slightly more rapidly than did the various lines of hypermotile cells, this was probably not the main cause of the difference in spontaneous metastasis formation between the groups. Lung colonization experiments performed by intravenous injection of cells could not explain the spontaneous metastasis results. In vitro, the cells selected by rapid haptotaxis to laminin grew considerably better than the other cells in 0.1 per cent fetal bovine serum, but there were no, or only minor, differences in higher serum concentrations. Combined, these results indicate that small subpopulations of cells selected by extreme efficiency in one step of the metastasis process may be so specialized that they perform poorly in other steps. Therefore, the results do not disprove the concept that tumor cell migration plays an important part in metastasis.

Marker polypeptide differences between spontaneous strongly and weakly metastatic cancer cells identified by 2-dimensional gel electrophoresis.

A recent 2-dimensional gel electrophoresis study revealed several polypeptide differences between a strongly metastatic and a weakly metastatic clone from a single chemically induced murine fibrosarcoma (Jellum et al., 1984). To exclude the possibility that this was merely coincidental, the study is extended here to 2 other fibrosarcomas recently and similarly induced in mice of the same inbred strain. Metastatic potential was defined by the number of lung metastases spontaneously formed from a transplanted primary footpad tumor. One strongly (or moderately) metastatic cell line and I weakly metastatic line from each of the 3 fibrosarcomas were examined in the same experiments. In confirmation of our previous results, the same polypeptides consistently occurred in considerably greater amounts in the weakly metastatic than in the strongly metastatic cells. One of these marker polypeptides was absent from the strongly metastatic cell lines. In comparison with 2 of the 3 most metastatic lines, the third line was only moderately metastatic, and differed least strongly from the 3 weakly metastatic cell lines with regard to expression of th marker polypeptides. Marker polypeptide expression showed no consistent correlation with tumorigenicity. No other consistent polypeptide differences between strongly metastatic and weakly metastatic cells could be identified among the approximately 2,000 cellular polypeptides separated on the gels.

Thromboplastin release, but not content, correlates with spontaneous metastasis of cancer cells.

No previous studies on the possible contribution of cancer-cell procoagulants to metastasis have fulfilled all the criteria for attaining biologically relevant and readily interpretable data (Grimstad et al., 1986), viz: (1) Spontaneous metastasis from primary tumors should be assessed in syngeneic animals; (2) cloned cell lines should be used to correlate cell properties, because heterogeneity within the cell lines employed is a source of serious error; (3) enough clones, derived from the same original tumor, should be used to identify only nonrandom correlations. Observing these criteria, we examined the procoagulant activities of 19 murine fibrosarcoma cell clones and 4 uncloned cell lines with high to moderate or low potential for lung metastases formation. The procoagulant activity found was exclusively of the thromboplastin (tissue factor, factor III) type. It occurred in all cell homogenates, but the quantities did not correlate with metastatic potential. In contrast, all highly to moderately metastatic cell clones and lines from 2 different fibrosarcomas shed thromboplastin activity into the culture medium, whereas no weakly metastatic cells did. Histological examination further supported these indications that release of thromboplastin from cancer cells can promote metastasis by initiating blood clotting and thereby facilitating arrest of the cancer cells in target organ vessels. Examination of a third fibrosarcoma showed that release of thromboplastin activity is not necessary for metastasis in all tumors.

Marker polypeptides distinguishing between cancer cell clones with high and low potential for spontaneous metastasis in murine fibrosarcoma cells.

Consistent differences in expression of specific proteins were observed between numerous cancer cell clones with high and low potential for spontaneous metastasis. Seventeen clones from two unrelated murine fibrosarcomas were examined concomitantly for spontaneous formation of lung metastases and for occurrence of individual polypeptide differences by high resolution two-dimensional gel electrophoresis. One of the identified marker polypeptides, designated Hi:2, was very strongly expressed by all 10 strongly metastatic clones, but was absent from 6 and only weakly expressed by one of the 7 weakly metastatic clones. Another marker polypeptide, designated Lo:6, was consistently most strongly expressed by the weakly metastatic clones. Among the approximately 2000 individually separated cellular polypeptides, only these two marker polypeptides consistently distinguished between the strongly and weakly metastatic clones from both fibrosarcomas. Five other polypeptides also distinguished between highly and weakly metastatic clones, but not as stringently.

Direct evidence that cancer cell locomotion contributes importantly to invasion.

This study was undertaken to clarify whether active locomotion of cancer cells is important for their ability to invade. The most rapidly moving cells were isolated from a cultured murine parent fibrosarcoma by successive cycles of migration through a micropore membrane. Cells were isolated by unstimulated locomotion and by haptotaxis to laminin, and the selected cells did indeed constitute rapidly locomoting subpopulations. These cells invaded biological tissues more efficiently than did the unselected parent cells. The cells selected by haptotaxis to laminin invaded most rapidly through amnion with basement membranes (containing laminin). Cancer cell haptotaxis to laminin in basement membranes thus promotes penetration of these tissue barriers. These results show in a direct manner that cancer cell locomotion is in fact important in invasion of biological tissues.

Cell-surface laminin-like molecules and alpha-D-galactopyranosyl end-groups of cloned strongly and weakly metastatic murine fibrosarcoma cells.

Indications from previous work that cancer cell-surface laminin-like molecules and alpha-D-galactopyranosyl end-groups may contribute to spontaneous metastasis were further investigated. Both moieties are known to mediate cell attachment to various foreign surfaces. Five strongly metastatic and 5 weakly metastatic cell clones from a murine fibrosarcoma were examined for the occurrence of both cell-surface moieties by immunofluorescence flow cytometry and microscopy. None of these clones was rich in laminin-like molecules, which were least strongly expressed by the highly metastatic clones. The alpha-D-galactopyranosyl end-groups were strongly expressed by all strongly metastatic clones and by 2 weakly metastatic clones, but were only weakly expressed by the other weakly metastatic clones. These results indicate that the laminin-like cell-surface molecules are not necessary for spontaneous metastasis formation. However, the alpha-D-galactopyranosyl end-groups may be necessary, but are not sufficient for the cancer cells to form metastases. These carbohydrates are known to occur on the laminin-like molecules. The present results show that they must also exist on other cell-surface molecules.

Attachment, spreading and growth in vitro of highly malignant and low malignant murine fibrosarcoma cells.

Highly malignant cell lines and low-malignant cell lines isolated from three different methylcholanthrene-induced murine fibrosarcomas were examined for their ability to attach to plastic dishes and collagen-coated dishes under serum-free conditions and in the presence of serum. Most of the cells from the three highly malignant lines attached and spread under all conditions. By 72 h, there was a significant increase in the number of cells indicating that at least some of the cells had undergone division (even in the absence of serum). In contrast, fewer of the cells from the three low-malignant lines attached and spread on the plastic or collagen substrates in the absence of serum or in the presence of 0.1 per cent serum. However, when 15 micrograms laminin per dish was added along with the low-malignant cells, they then attached and spread on the plastic and collagen-coated dishes. Previous studies have indicated that the highly malignant lines express cell surface antigens that cross-react with laminin while the low-malignant cell lines do not. We speculate that the differences between the high- and low-malignant cells in the expression of cell surface laminin-like antigens contribute to the dissimilarities in attachment and spreading capacity. These differences may also contribute to the dissimilarity between these cells in malignant potential.

Contribution of alpha-D-galactopyranosyl end groups to attachment of highly and low metastatic murine fibrosarcoma cells to various substrates.

There are much greater numbers of cell surface terminal, non-reducing alpha-D-galactorpyranosyl groups in highly malignant (metastatic) cells than are found in low malignant cells derived from the same murine fibrosarcoma. We have examined the contribution of these residues to attachment of the cells to various collagens and to plastic. Removal of these carbohydrate groups with alpha-galactosidase or blocking them with lectins from Griffonia simplicifolia seeds or with anti-blood group B antiserum all dramatically inhibited the attachment of both the highly malignant and the low malignant cells. Following removal with the enzyme, the alpha-D-galactopyranosyl end groups were rapidly resynthesized. This resynthesis was inhibited by tunicamycin, an inhibitor of de novo glycoprotein synthesis. This antibiotic also impaired cell attachment and, when used in addition to treatment with alpha-galactosidase, it inhibited cell attachment more than did treatment with the enzyme alone. The effects of all treatments on cell attachment were greater for the highly malignant than for the low malignant cells. With the latter cells, inhibition by lectin was seen only in the absence of serum, whereas the adhesion of highly malignant cells was affected in both the presence and the absence of serum. On their surface membrane the highly malignant cells express much more than do the low malignant cells of a glycoprotein that cross-reacts immunologically with laminin. The basement membrane glycoprotein laminin promotes cell attachment to collagen, and both glycoproteins contain terminal, non-reducing alpha-D-galactopyranosyl groups. Attachment of cells is a requirement for the formation of a metastasis, and thus the laminin-like molecule and the alpha-D-galactopyranosyl end groups (whether on the laminin-related moiety or on other cell surface molecules) may both be important for expression of the most malignant phenotype.

A new assay for leukocyte chemotaxis using cell retrieval, electronic particle counting and flow cytometry.

A new micropore membrane assay for leukocyte migration has been devised. It permits the complete retrieval in monodisperse suspension of functionally intact cells that have traversed the membrane, thus allowing the application of precise, automated techniques, including flow cytometry and electronic particle counting. Hemocytometers may also be used. Direct comparison with 2 different conventional membrane methods showed that the new method performed superiorly. It was also much more economical with regard to time and labor. This technique permitted detection of functional differences between leukocytes isolated from blood in different ways. Data on the duration of concentration gradients in chemotaxis chambers are also presented.