Reversible inhibition of proliferation of epithelial cell lines by Agaricus bisporus (edible mushroom) lectin.

Galactosyl beta-1,3-N-acetyl galactosamine (Gal beta-1,3-GalNAc) (Thomsen Friedenreich antigen), the Class I core sequence in O-linked oligosaccharide chains, behaves as an oncofetal antigen showing increased expression in many epithelial malignancies. Previous work has shown that peanut agglutinin (PNA), a lectin that binds Gal beta-1,3-GalNAc, stimulates proliferation in HT-29 (human colon cancer) cells and normal human colonic epithelium and this implies that cell surface glycoproteins which express Gal beta-1,3-GalNAc may play an important role in the regulation of epithelial cell proliferation. We have now studied the effect on epithelial cells of another dietary Gal beta-1,3-GalNAc-binding lectin, the edible mushroom Agaricus bisporus lectin (ABL). This differs from PNA in its ability to bind also to sialylated Gal beta-1,3-GalNAc. In contrast to PNA, ABL (25 micrograms/ml) inhibited incorporation of [3H]-thymidine into DNA of HT29 colon cancer cells by 87% (95% confidence limit, 85-89%), Caco-2 colon cancer cells by 16% (95% confidence limit, 12-20%), MCF-7 breast cancer cells by 50% (95% confidence limit, 47-52%), and Rama-27 rat mammary fibroblasts by 55% (95% confidence limit, 51-60%) when these cells were grown for 24 h in serum-free medium. When assessed by cell count, similar inhibition of proliferation of HT29 cells by ABL was found. In the presence of 2% fetal calf serum (which contains the ABL-binding glycoprotein fetuin), the inhibitory effect of ABL on cell proliferation was still demonstrable but at increased ABL concentration (60 micrograms/ml for 49% inhibition). Ten micrograms/ml ABL completely abolished the stimulatory effect on [3H]thymidine incorporation of epidermal growth factor (100 pg/ml) and PNA (25 micrograms/ml) and markedly inhibited the stimulatory effect of insulin (50 ng/ml). ABL (0.2 mg/ml) caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells as measured by trypan blue exclusion, and inhibition of proliferation in HT29 cells caused by 50 micrograms/ml ABL was reversible after removal of the lectin. Binding studies with 125I-labeled ABL suggested a single class of binding site with an apparent Kd value of (4.12 +/- 0.29) x 10(-7) M with (3.6 +/- 0.3) x 10(7) binding sites/cell. A. bisporus lectin is a reversible noncytotoxic inhibitor of epithelial cell proliferation which deserves study as a potential agent for cancer therapy.

The specificity of immunosuppression by alloantibody in the mouse.

The ability of alloantibody to suppress the humoral immune response to transplantation antigens in the mouse has been studied in relation to its specificity. It was found that antibody was able to suppress the cytotoxic antibody response to determinants other than those against which it was directed when there was some common antigenicity between the different H-2 types used for immunising. It was also found that suppression of the response to both parental haplotypes of an F1-hybrid cell could be achieved by antibody to only one parental haplotype, the antibody being made in the other parental strain to avoid any possibility of cross-reactivity. Such nonspecific immunosuppression was not achieved when cells of the two parental strains were administered mixed together.

Interaction of socioeconomic status and provider practices as predictors of immunization coverage in Virginia children.

OBJECTIVE: To describe the pattern of immunization in the cohort of children who entered public schools in Virginia in 1992. DESIGN: This was a historic cohort study using stratified cluster sampling. Three strata were created based on the socioeconomic status (SES) of the children in the catchment area of each public school in Virginia. SETTING: The random sample included public elementary schools throughout Virginia. PARTICIPANTS: Immunization records were obtained for a randomly selected cohort of 2519 first-grade children in Virginia. OUTCOME MEASURES: Age at completion of recommended childhood vaccines was determined from birth to school entry by SES, race, and population density. Provider practices were assessed by ascertaining missed opportunities for simultaneous administration of vaccinations according to recommended schedules. RESULTS: Although immunization completion rates were high at school entry, low levels of immunization coverage were found in all areas of Virginia at 24 months of age regardless of SES (as measured by per capita income), population density, or race. However, under-immunization was more severe for poor children in urban areas (42.3% of children in low-SES urban areas were age-appropriately immunized at 24 months of age versus 64.0% in children in high-SES rural areas). By multivariate logistic regression, race and gender were not predictors of which children were appropriately immunized at 2 years of age after adjusting for the following: SES, population density, receiving the first DTP (diphtheria, tetanus, and pertussis) or OPV (oral polio) vaccination after 3 months of age, and failure to have the first DTP administered simultaneously with the first OPV or the second DTP administered simultaneously with the second OPV. Receiving the first DTP or OPV vaccination after 3 months of age and failure to have the first and second DTP and OPV administered simultaneously were the strongest predictors of not being age-appropriately immunized at 2 years of age. The effect of failure to vaccinate simultaneously on predicting vaccination coverage at 2 years of age was strongly modified by SES. Children who attended schools located in census tracts with per capita incomes less than $10,600 and who did not have the first and second doses of DTP and OPV administered simultaneously were 33.19 times more likely not to be age-appropriately immunized at 2 years of age compared with children who attended schools located in census tracts with per capita incomes greater than $18,800 and who received the first and second doses of DTP and OPV simultaneously (95% confidence interval: 18.29 to 60.22). CONCLUSIONS: Although beginning the immunization schedule at the recommended age was crucial to appropriate vaccination later in life, provider practices were important predictors of under-immunization. Failure to administer vaccinations simultaneously strongly influenced poorer children in Virginia. Serious delays in vaccine administration were observed not only for poor children in urban areas, but also in all areas of Virginia before school entry.

The alloimmunogenicity of heated blood and lymphoid cells.

Murine lymph node cells heated to 45 degrees C for 1 h or 56 degrees C for 15 min lost their ability to provoke a primary cytotoxic alloantibody response, though they were able to provoke a secondary response in animals primed with normal lymph node cells two months previously. The primary immunogenicity of whole blood and spleen cell preparations was destroyed by 56 degrees C but not by 45 degrees C treatment. Treatment of spleen cells with 45 degrees C heat, and ammonium chloride to remove red cells, destroyed their immunogenicity, whereas ammonium chloride treatment alone did not, suggesting that the red cells were the immunogenic component of heated spleen cells and, by implication, of blood. Further evidence for a difference in the immunogenicity of 45 degrees C heated blood and normal blood was provided by the finding that heated blood did not prime for a response to 45 degrees C lymph node cells given two months later. Preliminary investigations of the tolerogenicity of heated cells were unsuccessful, indicating, in view of the published data, that the precise protocol for tolerance induction is very critical.

Distribution of O-acetylated sialomucin in the normal and diseased gastrointestinal tract shown by a new monoclonal antibody.

AIMS: To produce and characterise a monoclonal antibody specific for O-acetylated sialomucin and to assess its use in immunohistochemistry on a panel of normal and diseased intestinal tissue samples. METHODS: Mouse monoclonal antibodies were developed following immunisation with highly purified human colonic mucin. One of these (MMM-17) showed strong binding to mucin throughout the normal colon with relative lack of binding to colon cancer tissue. The binding epitope of MMM-17 was then characterised by screening for agglutination activity against a panel of human and animal erythrocytes and by assessment of its binding to a range of normal and chemically treated slot blotted mucins. Further immunohistochemical studies were then performed on formalin fixed, normal, and diseased human intestinal samples. RESULTS: Binding of MMM-17 to slot blotted human colonic mucin was reduced by 38 (SD 14%) (n = 4) by alkali treatment of the mucin, sequential alkali and sialidase treatment completely abolished binding. Sialidase treatment alone, however, caused only an 11 (11%) reduction in binding. MMM-17 failed to agglutinate any human, rabbit, rat or mouse erythrocytes. These findings were compatible with specificity of MMM-17 for sialomucins O-acetylated at the C-7 or C-8 positions on the sialic acid. Strong staining by MMM-17 was found in all goblet cells throughout all 40 normal colonic and rectal samples studied, but staining was absent in seven of 13 colorectal carcinomas. Normal duodenum (n = 16) and normal ileum (n = 3) all showed occasional positive goblet cells. The normal gastric antral mucosa was generally negative B MMM-17, but in all of 15 cases of gastritis with intestinal metaplasia the metaplastic glands were strongly positive for MMM-17. CONCLUSION: Monoclonal antibody MMM-17 has specificity for O-acetylated sialomucins and its binding depends both on the position of O-acetylation and on the adjacent oligosaccharide structure. Preliminary studies using the antibody on archival tissue samples support the previous reports of reduced O-acetylation in colon cancer demonstrated by indirect histochemistry and show the neo-formation of O-acetylated sialomucin in intestinal metaplasia in the stomach.

Quantification of intestinal mucins.

Intestinal mucins are glycoproteins containing a very high proportion of carbohydrate, up to 90%, and they can occur either in a membrane-bound or a soluble form (1). The general structure of these molecules includes discrete C-terminal and N-terminal regions, which are relatively poorly glycosylated, at either end of a long repeating structure, up to 50 repeats, which is heavily O-glycosylated on serine and threonine residues the first sugar in each oligosaccharide always being N-acetyl-galactosamine (2). These oligosaccharide chains can vary between 1 and about 15 saccharides. The C- and N-terminal regions are likely to be of importance in determining the membranous or secreted nature of the molecule (2).

The effect of glutaraldehyde fixation on the immunogenicity of allogeneic lymphoid and tumour cells.

When CBA mice were injected with allogeneic (DBA/2) lymph node cells treated with glutaraldehyde at concentrations of 0.13% and 0.013% they failed to produce a primary cytotoxic antibody response; cells fixed with 0.0013% glutaraldehyde only provoked the slightest of antibody responses. No significant secondary response was provoked by cells fixed with 0.013% glutaraldehyde in mice primed 8 weeks earlier with normal lymphoid cells. As it is well established that such cells can stimulate a secondary mixed lymphocyte reaction and have ben reported to induce a secondary haemagglutinin response their assumed antigenicity in these experiments was checked. It was found that fixed cells did not have measurable antigenicity as assessed by ability to absorb anti-H2 antibody. The organ localization of chromium-labelled glutaraldehyde-fixed lymph node cells showed a lack of localization in lymph nodes at all levels of fixation, though localization in the spleen of cells fixed with 0.0013% glutaraldehyde was very variable, consistent with the borderline immunogenicity of such cells. Mitomycin treatment only modestly reduced the immunogenicity of lymph node cells and did not affect their organ localization. When CBA mice were injected with allogeneic (DBA/2) tumour cells, P 815, fixed with 0.13% or 0.013% glutaraldehyde, no cytotoxic antibody was produced and cells fixed with 0.0013% glutaraldehyde stimulated an erratic low response again suggesting a borderline level of activity. However P 815 cells fixed with 0.13% glutaraldehyde retained their antigenicity as assessed by absorption. Mitomycin treatment of P 815 cells had only a modest deleterious effect of their immunogenicity. These differences in immunogenicity are discussed in relation to the viability of cells required to stimulate an allo-cytotoxic antibody response.

The localization of allogeneic lymphoid cells in relation to antibody-mediated immunosuppression.

Carbon blockade of mice did not affect cytotoxic antibody production after immunization with allogeneic cells, nor did it affect the immunosuppressive activity of alloantibody. Though it inhibited the alloantibody-mediated increase in hepatic localization of allogeneic lymphoid cells it had no effect on the normal hepatic localization of allogeneic lymphoid cells. Alloantibody administered before allogeneic cells or 6 h after was equally immunosuppressive, but anti-lymphocyte antibody only had potent immunosuppressive activity when given just before the antigenic cells. These results suggest than an afferent mechanism of suppression of the humoral antibody response can be exerted by anti-lymphocyte serum and that alloantibody may also act by this method when given before the antigen. As alloantibody is also highly potent when given after the antigen when an afferent mechanism is unlikely to occur, studies on the specificity of immunosuppression, particularly the likely requirement for antibody to I region determinants, would best be conducted by giving the antibody after the antigen, thereby avoiding any possible afferent inhibitory activity.

The effect of ultraviolet irradiation on the localization of syngeneic and allogeneic lymphoid cells and on alloimmunogenicity.

Ultraviolet (u.v.) irradiation was found to have only a modest inhibitory effect on the alloimmunogenecity of murine lymphoid cells in vivo when the response was assessed by the primary cytotoxic antibody response. Ultraviolet irradiation had no effect on the initial (1 hr) organ localization of chromium-labelled lymphoid cells in syngeneic or allogeneic recipients using either CBA or DBA/2 cells. However the 24 hr localization in peripheral lymph nodes was considerably and similarly depressed in syngeneic and allogeneic recipients. This effect was shown, by alloantibody treatment of recipients of allogeneic cells, to be due mainly to lack of entry into lymph nodes after 1 hr. In agreement with much of the published data, control data for these experiments indicated little difference in lymph node localization of lymph node cells in allogeneic and syngeneic recipients using both CBA and DBA/2 cells in both CBA and DBA/2 recipients. The localization of spleen cells in lymph nodes was found to be rather more sensitive to the allogeneic environment though not as much as has been previously described for spleen cells in contrast to the long established data on lymph node cells. A marked difference in the degree of lymph node localization of CBA and CBA/2 cells was found regardless of the recipient strain.

Multicenter postmarketing surveillance of ondansetron therapy in pediatric patients.

OBJECTIVE: To identify prescribing patterns of ondansetron, to provide a general overview of the therapeutic responses and possible adverse effects to ondansetron in selected children's hospitals, and to evaluate this methodology of surveillance and assess its effectiveness as a means to collect postmarketing experience with a drug in pediatric patients. DESIGN: This survey examined the use of ondansetron in 210 children. Complete drug and medical histories, indications, doses, possible ondansetron-associated adverse reactions, and daily responses to ondansetron therapy were recorded by a study pharmacist for each patient. Patients were followed until discharged from the clinic or hospital and/or until ondansetron therapy was discontinued. SETTING: The survey was conducted in seven free-standing children's hospitals across the US. Hospitals ranged in size from 100 to 331 beds (average 234). One hospital was located on the West coast, one on the East coast, one in the Rocky Mountain region, one in the Southwest region, and three in the Midwest. PARTICIPANTS: The selection of study participants was limited to member free-standing children's hospitals of the Pediatric Pharmacy Administrative Group. Selection was based on geographic location and availability of a pharmacist to coordinate the study. One pharmacist at each study site served as surveillance coordinator. Each pharmacist monitored without intervention the use of ondansetron in 30 children. Patients were enrolled consecutively from physicians' orders for ondansetron. Enrollment was open to clinic and hospital patients. Patients were excluded if more than 48 hours of retrospective review was required. MAIN OUTCOME MEASURES: The survey queried patient demographics, type of antineoplastic therapy administered, indications and dosing regimen(s) for ondansetron, additional antiemetic agents administered, and clinical response. Adverse drug reactions and prescriptions for ondansetron on discharge were recorded. An evaluation of response rates in hospital patients based on exposure to antineoplastic regimens causing acute (within 24 h) or delayed emesis (after 24 h) was formulated after data collection. Off-label use was summarized. RESULTS: Surveys from 197 of the 210 patients enrolled were complete for evaluation. Ondansetron was used to treat chemotherapy-induced emesis in 88 percent of the patients and 12 percent received it for various other indications. Ondansetron dosing was off-label in 15 percent and 73 percent prior to and after an emetogenic exposure, respectively. Twenty-six percent of the patients were younger than four years. Dosages ranged from 0.15 to 0.45 mg/kg, given in various schedules. The injectable form was given both intravenously and orally. There was a significant difference in the mean number of doses in hospital (9 +/- 7.3) versus clinic (2 +/- 1.5) patients (p < 0.0001). Eighty-seven percent of all patients had a complete or major overall response. Possible ondansetron-associated adverse reactions were similar to those of previous reports for all patients, although some recorded reactions are not currently included in package labeling. CONCLUSIONS: This study documents off-label use of ondansetron in children. Further study of ondansetron use in children less than four years of age, and for indications other than chemotherapy-induced emesis, is needed. Additional evaluation into the most cost-effective dosing of ondansetron would also be valuable.

Immune responsiveness in chronic fatigue syndrome.

We have endeavoured to find immunological indications of chronic virus infection in patients with chronic fatigue syndrome (myalgic encephalomyelitis) and to investigate immune responsiveness to viruses in such patients in comparison with normal subjects and patients with muscular dystrophy. Levels of circulating IgM immune complexes were elevated (above the 95% normal control range) in 10 (17%) of 58 patients with chronic fatigue syndrome, which was not significantly different from the normal controls or from dystrophy controls (by Mann Whitney U test). Levels of IgG complexes were only increased in 10% of patients. Lymphocyte proliferation in response to concanavalin A (Con A), assessed by increase in 3H-thymidine incorporation, did not differ between 14 patients and 18 normal subjects. The proliferative response to Coxsackie B virus antigen did not differ between chronic fatigue patients and normal subjects when expressed either as an increase in counts or as a stimulation index. Adjustment of the counts in relation to the proliferation response to Con A, as an indication of the overall proliferative response of the cell preparation, did not reveal any hidden difference. IgM antibodies to Coxsackie B viruses were not found in any of 20 patients and in 1 of 20 dystrophy controls. Significant levels of neutralizing antibodies to Coxsackie B viruses 1-5 were found in 6 out of 19 (32%) patients compared with 4 out of 17 (24%) dystrophy controls, which does not differ from currently expected normal incidence. Antibody titres to other respiratory viruses were also not notably different between the patient and control groups. In conclusion we can find no evidence for a definable viral aetiology for the chronic fatigue syndrome, neither in terms of a persistent infection nor an altered ability to respond to virus.

Ankylosing spondylitis, HLA-B27, and Klebsiella: a study of lymphocyte reactivity of anti-Klebsiella sera.

Twenty three anti-Klebsiella antisera were tested for their cytotoxic activity and four for their binding capacity for peripheral blood lymphocytes (PBL) from patients with HLA-B27 positive ankylosing spondylitis (AS+B27+) and from B27 positive (AS-B27+) and B27 negative (AS-B27-) healthy individuals. None of the antisera showed specific activity against PBL from any particular group. The antisera tested included two anti-Klebsiella K43 sera provided by an Australian group, who have reported them to be specifically cytotoxic for AS+B27+ PBL, four antisera raised against a Klebsiella K43 strain provided by this group, and an antiserum from another group, who have reported it as having increased binding capacity for AS+B27+ and AS-B27+ PBL compared with AS-B27- PBL. The results of other workers who have attempted to reproduce the results of either group are reviewed and the possible reasons for the repeated failure to confirm the reported findings are discussed.

Use of a biosensor to determine the binding kinetics of five lectins for Galactosyl-N-acetylgalactosamine.

The dietary lectins, edible mushroom (ABL) and Jacalin (JAC) inhibit the proliferation of colonic cancer cells, whereas Amaranth (ACL) and peanut (PNA) stimulate their proliferation. All these lectins share as their preferred ligand the Thomsen-Friedenreich (TF) antigen galactosyl beta1,3 N-Acetylgalactosamine (Galbeta1,3GalNAc), but differ in their finer specificities for modifications of this determinant and in their specificities for cancerous epithelia. We have investigated, using a resonant mirror biosensor, the kinetics of binding of these lectins, and Maclura pomifera lectin (MPL), which is similar to JAC, to two different Gal-GalNac bearing glycoproteins, antarctic fish antifreeze glycoprotein (AFG) and asialofetuin. JAC had the highest affinity for AFG [K(d) 0.027 microM] due to a fast association rate constant [k(ass) 610,000 (Ms)(-1)]. The other lectins had considerably lower affinities, with K(d) ranging from 0.16 microM (ABL) to 5.7 microM (PNA), largely due to slower k(ass) [ABL 74,000 (Ms)(-1) to PNA 2700 (Ms)(-1)]. Similarly, JAC had a much higher affinity for asialofetuin [K(d) 0.083 microM] than the other lectins [K(d) 1.0 microM-4.5 microM]. Affinities were also calculated from the extent of binding at equlibrium and were generally similar to those calculated from the kinetic parameters indicating the true nature of these values.

Pancreatic tumour marker anti-mucin antibody CAM 17.1 reacts with a sialyl blood group antigen, probably I, which is expressed throughout the human gastrointestinal tract.

CAM 17.1 is an antimucin monoclonal antibody which has recently been proven valuable as a reagent for serological diagnosis of pancreatic cancer. A series of studies have been performed to characterise its epitope. First it was screened immunohistochemically against a wide range of formalin-fixed normal and neoplastic human tissues and showed widespread binding to mucin throughout the gastro-intestinal tract, in both normal and malignant tissues. In pancreas, strong intracellular staining of acinar and ductal cells was found in normal tissue and in carcinoma cells in tumours. Normal stomach showed only weak staining (n = 6), but gastritis with metaplasia showed strong staining (n = 4). Staining of colonic mucosa from patients of known Lewis phenotype showed Le(a+b-) (7/8) and Le(a-b+) (4/6) samples to be positive, but not Le(a-b-) (0/3) samples. CAM 17. 1 agglutinated all donor erythrocytes tested at 4 degreesC regardless of blood group, whereas cord blood red cells were not agglutinated. Since I antigen is the only antigen known to be present on all adult red blood cells but absent from cord blood, this suggests probable involvement of this antigen in the binding site. The agglutination was abolished by sialidase treatment of the red cells and immunoblotting with slot-blotted mucin showed that binding was both acid and sialidase sensitive indicating the involvement of sialic acid in the binding site. These studies show that CAM 17.1 binds to a sialic-acid-containing determinant of mucin, probably sialyl-I, which epitope shows wide distribution throughout the gastro-intestinal tract.

Opposite effects on human colon cancer cell proliferation of two dietary Thomsen-Friedenreich antigen-binding lectins.

Increased cell surface expression of the Thomsen-Friedenreich antigen (TF antigen, Galbeta1-3GalNAcalpha-) is a common feature in malignant and pre-malignant epithelia. Our previous studies have shown that dietary TF-binding lectins from peanut (Arachis hypogea) and edible mushroom (Agaricus bisporus) produce marked but different effects on human intestinal epithelial cell proliferation. This study investigates the proliferative effects of the other two known dietary TF-binding lectins: jacalin (Artocarpus integrifolia, JAC) and amaranth lectin (Amaranthus caudatus, ACA). JAC produced dose-dependent and non-cytotoxic inhibition of proliferation in HT29 human colon cancer cells with maximal effects of 46 +/- 4% at 20 microg/ml, whereas ACA produced dose-dependent stimulation of proliferation with maximal effects of 22 +/- 3% at 20 microg/ml when assessed both by incorporation of [3H]thymidine into DNA and by cell counting. The lectin-mediated effects were inhibitable by the presence of appropriate Galbeta1-3GalNAc-expressing glycoproteins but differences existed between JAC and ACA in their patterns of inhibition by such substances. Ligand binding equilibrium studies using iodinated lectins revealed different Kd of the two lectins for HT29 cell surface glycoproteins. Lectin blots of cell membrane extracts showed different binding patterns in all the four TF-binding lectins. These results provide further evidence that dietary TF-binding lectins can have marked effects on the proliferation of human malignant gastro-intestinal epithelial cells and hence may play a role in intestinal cancer development, and also show that the biological effects of dietary lectins cannot be predicted solely from their carbohydrate binding properties.