Co-migration and internalization of transferrin and its receptor on K562 cells.

The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC-transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fab-antitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4 degrees C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20 degrees C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20 degrees C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell.

Neutral lipids in the cells and cell envelope fractions of aerobic baker's yeast and anaerobic brewer's yeast.

The neutral lipids from whole cells and cell envelopes of aerobic Saccharomyces cerevisiae and anaerobic Sacch. carlsbergensis and the cell walls isolated from the cell envelopes were analysed. The effect of anaerobiosis was particularly clear on the neutral lipid composition of the plasma membrane. Compared to the anaerobic membrane, the aerobic membrane contained more C16:1, C18:1 and other unsaturated fatty acids, more total sterol, more than ten times as much ergosterol and less than one tenth as much squalene, reflecting differences between the aerobic and anaerobic whole cellmthe main sterol in the aerobic membrane ergosterol, was mainly in the free form, whereas zymosterol, 24(28)-dehydroergosterol, epi- or fecosterol and lanosterol were predominantly esterified. In contrast, the anerobic membrane contained small amounts of biosynthetic sterol precursors of ergosterol (mainly esterified), and was clearly richer in saturated fatty acids having a greater variation in chain length and in 18:2 acid. Both plasma membranes contained a considerable amount of triacyglycerols, while the amount of lower acylglycerols was clearly higher in the anaerobic plasma membrane. The lipid composition of both cell walls were relatively similar, consisting mainly of triacylglycerols and lower acylglycerols.

Peripheral blood lymphocyte subpopulations in schoolchildren born very preterm.

AIM: To investigate whether lymphocytes or serum inflammatory markers are associated with obstructive lung disease and bronchial lability in schoolchildren born very preterm. METHOD: Lymphocyte subsets were studied in the peripheral venous blood of 29 such children (median age 8.8 years). Serum eosinophil cationic protein (ECP) and myeloperoxidase (MPO) concentrations and the association between them, lymphocyte subsets, and lung function were studied. Fourteen healthy children born at term, median age 9.1 years, served as controls. T lymphocytes (CD3), T lymphocyte subpopulations (CD4 and CD8), B lymphocytes (CD19), natural killer cells (CD16+56) and activation markers of T and B lymphocytes (CD23 and CD25) were determined using flow cytometry. Lung function was measured in all children both in the clinic and at home (Vitalograph Data Storage Spirometer). RESULTS: Compared with the controls, schoolchildren born very preterm had significantly lower CD4(+) T cell percentages and CD4:CD8 ratios (p < 0.05 for both), whereas natural killer cell percentages and serum ECP values were significantly higher (p < 0. 05). The very preterm schoolchildren had significantly lower spirometric values than the control group (p < 0.05)-except forced vital capacity. When all the subjects were considered together, a weak, but significant, negative association was observed between the bronchial responsiveness in peak expiratory flow, after a beta(2) agonist during home monitoring, and the CD4(+) T cell percentage (r = -0.45; p = 0.008) and the CD4:CD8 ratio (r = -0.50; p = 0.003), indicating a relation between bronchial lability and imbalance of T cell subpopulations. CONCLUSIONS: These results suggest that there is an inflammatory basis for lung function abnormalities in schoolchildren born very preterm.

Sensitisation through breast milk?

The benefit of breast feeding to prevent or delay the development of food allergy in infants is somewhat controversial. Although prolonged breast feeding has widely been recommended for infants at high risk of allergy, studies concerning the composition of human milk in atopic mothers are scarce. Human breast milk is not a stabile concept. We have previously found that the cellular composition of human milk is significantly different in mothers with a food allergic infant than in those with a healthy child. The number of monocytes, lymphocytes or eosinophils, was greatly elevated whereas the number of activated macrophages was decreased in the breast milk of mothers with a severely allergic infant. The high numbers of eosinophils could easily increase the permeability of the gut thereby increasing the absorption of dietary antigens, and thus enhance the development of food allergies. Breast feeding provide many advantages to the suckling infant. However, infants developing food allergic symptoms on exclusive breast feeding may benefit from changing to formula feeding to protect adequate growth and to prevent the development of a more severe allergic symptomatology.

The monoclonal antibodies Trop-4 and 4F2 detect the same membrane antigen that is expressed at an early stage of lymphocyte activation and is retained on secondary lymphocytes.

The monoclonal antibodies Trop-4 and 4F2 recognize cell surface antigens present on peripheral blood monocytes, activated lymphocytes, and on continuous cell lines, but not on resting lymphocytes in blood. The membrane antigens detected by antibodies Trop-4 and 4F2 were compared by serial immunoprecipitations from membrane lysates of surface labeled T lymphoid cells and by parallel polyacrylamide gel electrophoretic analysis. It is shown that both to these antibodies recognize the heavy subunit of the heterodimeric membrane complex of an 85,000 m.w. glycoprotein disulphide linked to a light subunit of 41,000 m.w. The kinetics of the expression of the antigen was studied by indirect immunofluorescence on peripheral blood T lymphocytes during blast transformation induced by concanavalin A in vitro and during reversion of the lymphocytes back to small "secondary" lymphocytes. Upon activation of T lymphocytes with concanavalin A, the first blast cells staining with the antibodies appear within 6 hr after the initiation of the culture. After 18 hr, all blast cells displayed strong expression of the antigen. Inhibition of DNA synthesis by hydroxyurea treatment did not affect the early blast transformation and the expression of the antigen. When the mitogen-induced blast cells reverted back to small secondary lymphocytes during prolonged culturing for up to 18 days, these cells retained the expression of the antigen detected by antibodies Trop-4 and 4F2, whereas another membrane marker of activation, the transferrin receptor, rapidly disappeared. These findings demonstrate a phenotypical difference between primed, secondary T lymphocytes and resting, unstimulated cells.

New concepts of allergy to cow's milk.

CMA is a diagnostic and therapeutic problem in the field of pediatrics. The response to oral cow's milk challenge was evaluated in patients with suspective CMA. To investigate immune mechanisms in different subtypes of CMA, suppressor activity, in vitro IFN-gamma and IL-4 generation, eosinophil activation and humoral immune response were measured. Also, changes in the function of the immune system were evaluated when patients had clinically recovered from CMA. The results of different measurements were correlated with the patients' clinical response to cow's milk challenge. The prechallenge suppressor activity was found to be low in patients with challenge-proven CMA when compared with those clinically negative to oral cow's milk challenge but had normalized after a 4-18 month milk-elimination period in patients who had recovered from CMA. IFN-gamma generation was low in patients with active CMA but had clearly enhanced in patients recovering from CMA. The serum concentration of ECP increased significantly during oral cow's milk challenge in patients with cutaneously manifested CMA. Measured by the ELISPOT method, the patients with active CMA mounted a high, non-antigen-specific immune response but were unable to direct the antigen-specific response, especially in the IgA class. After a follow-up of a mean 13.5 months on cow's milk elimination diet, the immune response to cow's milk antigens had developed in patients who had recovered from CMA. The development in the capacity to mount an adequate immune response to specific protein antigens, especially in the IgA class, can be interpreted as deriving from the development in T-lymphocyte regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

The lipolytic activities of the isolated cell envelope fracttions of Baker's yeast.

1. The existence of phospholipase and lipase activities in the isolated cell envelopes of baker's yeast was demonstrated. 2. The content of phospholipase was found to be markedly higher than that of lipase. 3. After partial enzymic digestion of the isolated cell envelopes, the bulk of the lipolytic activities was recovered in the sedimentable preparations, which consisted of the fragments of the plasma membrane. 4. During repeated washings, the lipase was completely released from the cell envelopes, as were also the bulk of the lipid components and most of the Mg(2+)-dependent adenosine triphosphatase, an enzyme connected with the plasma membrane. The phospholipase was more firmly bound to the preparation but not so firmly as the external saccharase. 5. These results indicate that the lipolytic enzymes found in the cell envelopes are mostly located in the plasma membrane.

Occurrence of long-chain fatty acids and glycolipids in the cell envelope fractions of baker's yeast.

1. The total yield of fatty acids from the whole envelopes was markedly higher than that obtained from the ordinary cell walls. In both samples the major fatty acids were C(16) and C(18) acids. 2. The whole envelopes contained C(18) acids and long-chain (C(19)-C(26)) fatty acids, in a higher proportion than did the ordinary cell walls. Fifteen fatty acids with more than 18 carbon atoms were identified, among which 2-hydroxy-C(26:0) and C(26:0) acids predominated. 3. A complex sphingolipid containing inositol, phosphorus and mannose was isolated from the whole cell envelopes. The main fatty acids of this lipid were 2-hydroxy-C(26:0) and C(26:0) acids. It was concluded that this sphingolipid is present both in the ordinary cell wall and in the plasma membrane of baker's yeast. 4. The neutral lipids amounted to over 50% and the glycerophosphatides to about 30% of the total fatty acid content of the whole envelope. The major fatty acids in these lipids were C(16:1), C(18:1) and C(16:0) acids. The proportion of fatty acids with more than 18 carbon atoms was lowest in the neutral lipids, whereas the neutral glycolipids contained the highest percentage of these fatty acids. Acidic glycolipids amounted to 14% of the total fatty acid content of the whole envelope. The presence of a cerebroside sulphate in this lipid fraction was demonstrated, whereas the high content of 2-hydroxy-C(26:0) acid found is caused by the complex inositol- and mannose-containing sphingolipid.

Distribution of membranes, especially of plasma-membrane fragments, during zonal centrifugations of homogenates from glucose-repressed Saccharomyces Cerevisiae.

1. The distributions of several enzymes and other marker components were examined after zonal centrifugations of whole homogenates from glucose-repressed Saccharomyces cerevisiae on sucrose and iso-osmotic Ficoll, and the composition and morphology of the fractions were investigated. 2. After high-speed zonal centrifugation most of the protein, acid and alkaline phosphatases, alkaline pyrophosphatase, adenosine monophosphatase, beta-fructofuranosidase, alpha-mannosidase, NADPH-cytochrome c oxidoreductase and an appreciable amount of phospholipid and sterol were non-sedimentable, i.e. were at densities below 1.09 (g/cm3). Most of the RNA was at p=1.06-1.08 in Ficoll and at p=1.09-1.11 in sucrose. 3. The bulk of the Mg2+-dependent adenosine triphosphatase (Mg-ATPase) was coincident with the main peak of phospholipid and sterol, at median density 1.10, which was also rich in smooth-membrane vesicles. In Ficoll, a minor peak of phospholipid and sterol at p-1.12-1.15 contained a smaller part of the oligomycin-insensitive Mg-ATPase and heavy membrane fragments. In sucrose, several minor peaks of Mg-ATPase were in the mitochondrial density range, and a peak of oligomycin-insensitive Mg-ATPase coincident with a minor peak of phospholipid and sterol at around p-1.25 contained heavy membrane fragments of high carbohydrate content, especially mannose. 4. Further purification of the oligomycin-insensitive Mg-ATPase containing membrane preparations was performed on Urografin gradients. 5. It is argued that the oligomycin-insensitive Mg-ATPase containing membranes are fragments of the plasma membrane, but have different densities because they contain different amounts of glycoprotein particles.

Immunologic disturbances in cow's milk allergy, 1: Delayed maturation of suppressor activity.

To assist in identifying pathogenetic mechanisms in different subtypes of cow's milk allergy (CMA), the function of immunoregulatory T-lymphocytes was studied. The study population consisted of 23 patients, mean [95% confidence interval] age of 25.6 [19.5, 33.6] months, who had challenge-proven cow's milk allergy manifested with either skin (n = 9) or gastrointestinal (n = 14) symptoms; in addition, 13 age-matched disease controls were studied. Patients with challenge-proven CMA were rechallenged to establish whether they had acquired clinical tolerance to cow's milk. The suppressor activity of isolated lymphocytes was measured in vitro by a cell coculture at rechallenge and in 10/23 patients at diagnosis. At diagnosis, patients with CMA (n = 10) showed a decreased mean [95% CI] suppressor activity, induced by either Concanavalin A, 7[-2,15]%, or cow's milk, 3[-8,14]% as compared with disease controls (n = 13), 19[15,24]% and 24[17,31]%; F = 7.1, p = 0.004 and F = 6.7, p = 0.005, respectively. At rechallenge the suppressor activity, induced both by Concanavalin A and cow's milk, reached the level of disease controls only in patients who had acquired clinical tolerance to cow's milk (n = 13/23), but not in those retaining CMA (n = 10/23). Our results indicate that the maturation of suppressor function is delayed in CMA, which might be of primary importance in the etiopathogenesis of CMA.

The cell-envelope glycolipids of baker's yeast.

Sphingolipids were found to dominate in the glycolipids from the cell envelope of baker's yeast. A relatively large quantity of ceramides was detected. Among the several complex phosphosphingolipids described, ceramide-(P-inositol)(2)-mannose was the main component. About 55% of long-chain bases in sphingolipids consisted of C(18)-phytosphingosine (4D-hydroxysphinganine). Other bases, found in decreasing concentrations, were C(20)-phytosphingosine, C(20)-dehydrophytosphingosine, C(18)-dihydrosphingosine (sphinganine) and C(19)-dihydrosphingosine. The presence of sterol glycosides, sulpholipids, cerebrosides and acylglucoses was demonstrated.

Calcitonin gene-related peptide immunoreactivity in prurigo nodularis: a comparative study with neurodermatitis circumscripta.

Eight patients with intensely pruritic lesions of chronic idiopathic prurigo nodularis and three patients with neurodermatitis circumscripta were investigated using the indirect immunofluorescence method. Results showed similarities in epidermal hyperplasia but not in nerve proliferation and neuropeptide immunoreactivity. Increased numbers of calcitonin gene-related peptide (CGRP) and substance P immunoreactive nerve fibre bundles were detected in specimens taken from prurigo nodularis lesions, but no increased immunoreactivity could be seen in specimens taken from patients having neurodermatitis circumscripta compared to normal skin. The neuropeptides, CGRP and substance P, may be responsible for the intense itching of prurigo nodularis lesions.

Lymphocyte response to cow's milk proteins in patients with cow's milk allergy: relationship to antigen exposure.

The cellular immune response to cow's milk was measured in patients with challenge-proven cow's milk allergy (CMA), manifested with either gastrointestinal or skin symptoms. After 2-4 weeks on milk elimination, 44 children, mean (SD) age 15.7 (9.4) months, were challenged, and cow's milk-induced lymphocyte transformation was measured before the clinical challenge (Day 1) and/or one week later (Day 8). During the clinical challenge period, 17 (39%) patients showed gastrointestinal reactions, 9 (20%) had urticarial or eczematous skin eruptions, and 18 (41%) were negative to challenge. On Day 1, the mean [95% confidence interval] stimulation index for lymphocytes in patients manifesting CMA with gastrointestinal symptoms, 2.60 [1.60, 4.10], was significantly higher than that in patients with skin symptoms, 1.15 [0.60, 2.30], or patients with negative clinical challenge, 0.83 [0.64, 1.08], F = 9.0, p = 0.001. After the clinical challenge (Day 8), this cow's milk-induced lymphocyte proliferation response was abrogated. At the same time, CMA patients evidenced a significantly higher spontaneous lymphocyte proliferation response in RPMI medium-containing control cultures than those with negative clinical challenge. We conclude that in patients with CMA, the number of circulating cow's milk-sensitized lymphocytes is depleted or their function is impaired after clinical exposure to cow's milk antigens.

Evidence for eosinophil activation in cow's milk allergy.

To assist in identifying pathogenetic mechanisms in different clinical manifestations of cow's milk allergy (CMA), the involvement of eosinophil cells in immunoinflammatory reactions was evaluated. The study population comprised 28 patients, aged from 5.8 to 43.0 months, who had challenge-proven CMA, manifested either cutaneously (n = 17) or gastrointestinally (n = 11). A clinical cow's milk challenge was performed in hospital after a 4 week cow's milk elimination period. Eosinophil activation in vivo was studied by measuring the serum level of eosinophil cationic protein (ECP) before the oral cow's milk challenge, mean (SD) 27 (12) hours after commencing the challenge and one week later. These results were compared to those of 80 non-allergic age-matched controls. During the challenge, the level of ECP increased from 6.2 (4.5, 8.0) micrograms/L to 20.0 (9.5, 30, 4) micrograms/L in CMA patients with skin manifestations but not in those with gastrointestinal symptoms. The increase was shown to be transient. We conclude that eosinophil degranulation is an important immunologic mechanism leading to allergic inflammation in cutaneously manifested CMA.

The enzymic composition of the isolated cell wall and plasma membrane of baker's yeast.

A study was made of the enzyme content of the isolated cell walls and of a plasma-membrane preparation obtained by centrifugation after enzymic digestion of the cell walls of baker's yeast. The isolated cell walls showed no hexokinase, alkaline phosphatase, esterase or NADH oxidase activity. It was concluded that these enzymes exist only in the interior of the cell. Further, only a negligible activity of deamidase was detectable in the cell walls. Noticeable amounts of saccharase, phosphatases hydrolysing p-nitrophenyl phosphate, ATP, ADP, thiamin pyrophosphate and PP(i), with optimum activity at pH3-4, and an activity of Mg(2+)-dependent adenosine triphosphatase at neutral pH, were found in the isolated cell walls. During enzymic digestion, the other activities appearing in the cell walls were mostly released into the medium, but the bulk of the Mg(2+)-dependent adenosine triphosphatase remained in the plasma-membrane preparation. Accordingly, it may be assumed that the enzymes released into the medium during digestion are located in the cell wall outside the plasma membrane, whereas the Mg(2+)-dependent adenosine triphosphatase is an enzyme of the plasma membrane. This enzyme differs from the phosphatases with pH optima in the range pH3-4 with regard to location, pH optimum, substrate specificity and different requirement of activators.

Immunologic disturbances in cow's milk allergy, 2: Evidence for defective interferon-gamma generation.

We have investigated the role of interferon-gamma (IFN-gamma) in the regulation of antigen-specific T-cell function in patients with cow's milk allergy. The study population consisted of 22 patients, aged from 7.6 to 56.9 months, who had challenge-proven cow's milk allergy (CMA) manifested with either skin (n = 9) or gastrointestinal (n = 13) symptoms. In addition, 11 age-matched children and 6 adults, mean (SD) age 31 (7) years, were studied as controls. Patients with challenge-proven CMA were rechallenged to establish whether they had acquired clinical tolerance to cow's milk. The spontaneous and mitogen-induced IFN-gamma and interleukin-4 (IL-4) generation of isolated lymphocytes was evaluated in vitro with commercial ELISA Kits at diagnosis and at reassessment. At diagnosis, the IFN-gamma production was not detectable in patients with CMA as compared with control children. IL-4 production was almost undetectable in all subjects in this study. However, at reassessment the CMA patients who had acquired clinical tolerance to cow's milk (n = 16) showed enhanced IFN-gamma production, when compared with that of control children, but still lower when compared with that of healthy adults. Our results indicate that the maturation of IFN-gamma producing T-cells is delayed in CMA, which could lead to a disturbance in the regulation of T-cell function. This defect might be an important etiologic factor for CMA.

Diagnostic value of skin-prick and patch tests and serum eosinophil cationic protein and cow's milk-specific IgE in infants with cow's milk allergy.

The diagnosis of cow's milk allergy is based on a clinical response to an elimination-challenge test with cow's milk. We studied the usefulness of the skin-prick and patch tests and measurement of cow's milk-specific IgE and eosinophil cationic protein in serum as diagnostic tools for cow's milk allergy in a cohort of 6209 unselected infants followed from birth for the development of cow's milk allergy. Of the 239 infants challenged with cow's milk, 118 showed a positive and 121 a negative response at a mean age of 6.9 months. A positive reaction to a skin-prick test with cow's milk (> or = 3 mm) was seen in 72 (61%) and 29 (24%) infants with positive and negative challenges, elevated serum cow's milk-specific IgE (> or = 0.7 kU/L) in 52 (45%) and 15 (13%) infants, a positive reaction to patch test with cow's milk protein fractions in 26 (26%) and eight (8%) infants, and elevated serum eosinophil cationic protein (> or = 20 microg/L) in 22 (21%) and seven (13%) infants, respectively. Parallel use of the four tests with the above-mentioned cut-off values correctly classified 73% of the infants with a sensitivity of 0.76 and a specificity of 0.67. An immediate reaction to cow's milk challenge correlated with skin prick test positivity and elevated serum milk-specific IgE, and tended to correlate with patch test positivity. No single test or parallel use of the four tests could predict the challenge outcome acceptably in this prospectively followed, unselected cohort of 6209 infants. A positive reaction to one or more tests needs to be confirmed by a challenge test and a negative response to all four tests does not rule out the possibility of cow's milk allergy.

Neuropeptides in cutaneous neurofibromas of von Recklinghausen's disease.

The occurrence of neuropeptides was studied in neurofibromas of von Recklinghausen's disease by indirect immunofluorescence. All non-plexiform cutaneous neurofibromas contained abundant vasoactive intestinal polypeptide, peptide histidine-isoleucine and calcitonin gene-related peptide immunoreactive nerves. The nerves were small and unmyelinated. Neuropeptides might be responsible for itch that occurs especially in small cutaneous neurofibromas. Neuropeptides are also suggested to act as modulators and/or trophic factors for neurofibroma growth.

Promotion of IgA immune response in patients with Crohn's disease by oral bacteriotherapy with Lactobacillus GG.

The effect of oral bacteriotherapy with human Lactobacillus casei strain GG (10(10) colony-forming units twice daily for 10 days) was investigated in Crohn's disease and in juvenile chronic arthritis which are chronic inflammatory diseases associated with impaired mucosal barrier function. During oral bacteriotherapy, the gut immune response was indirectly assessed by solid-phase enzyme-linked immunoassay in 14 children with Crohn's disease, in 9 with juvenile chronic arthritis, and in 7 controls. The immunostimulatory effect of Lactobacillus GG was specific for Crohn's disease, irrespective of its activity: the mean (95% confidence interval) number of specific antibody secreting cells in the IgA class to beta-lactoglobulin increased significantly from 0.2 (0.04-1.3) to 1.4 (0.3-6.0)/10(6) cells and to casein from 0.3 (0.1-1.4) to 1.0 (0.2-4.8)/10(6) cells. The results indicate that orally administered Lactobacillus GG has the potential to increase the gut IgA immune response and thereby to promote the gut immunological barrier. Consequently, Lactobacillus GG could provide an adjunct nutritional therapy for Crohn's disease.