Yeast Opsonisation Research Articles

Release of phagocytosis-stimulating factor(s) by morula cells in a colonial ascidian

In vitro yeast phagocytosis by haemocytes of the compound ascidian Botryllus schlosseri was studied, with particular attention to interactions among different immunocyte types. It is demonstrated that the supernatant from haemocyte cultures matched with yeast cells contains factor(s) able to enhance yeast ingestion by Botryllus phagocytes. The increase in phagocytosis is not the consequence of yeast opsonisation, as the phagocytic index does not significantly increase when yeast cells, previously incubated in the culture media, are washed and re-suspended in filtered sea water. When haemocytes were fractionated by density gradient centrifugation and each band was incubated with yeast, the ability to stimulate phagocytosis was found in the supernatants from haemocyte cultures of fractions rich in morula cells (MC). Previous studies have demonstrated that MC express molecules recognised by anti-cytokine antibodies, as a consequence of the recognition of foreign molecules or cells. Our results indicate that molecules immunoreactive with anti-cytokine antibodies are required for modulating phagocyte activity, as the above-reported enhancing effect is completely absent in the presence of anti-IL-1-α and anti-TNF-α, but not of anti-rabbit-IgG antibodies, and they also highlight the presence of ‘cross-talk‘ between MC and phagocytes. A new scenario is therefore sketched, in which MC actively recognise non-self molecular patterns and, upon this recognition, release humoural factor(s) recognised by phagocytes, which modulate phagocytosis.

Therapeutic applications of mannan-binding lectin

Mannan-binding lectin (MBL) is perhaps the best known of the collectins, a subfamily of C-type lectins possessing an additional collagen-like domain. MBL is thought to be an important component of innate immunity. Since the discovery, around 15 years ago, that MBL was identical to a factor responsible for the opsonization of baker's yeast in vitro, a large number of disease-association studies have been conducted. Most of these investigations support the view that MBL can influence susceptibility to, or affect the course of, many diseases. These include bacterial infections, rheumatoid arthritis, cystic fibrosis and recurrent miscarriage. This meeting was arranged to bring together scientists and clinicians to review the clinical significance of MBL, and to consider the most appropriate target disorders for MBL-replacement therapy.

Phagocytosis of Candida albicans induces apoptosis of human neutrophils.

Neutrophil-mediated inflammation is terminated through the programmed cell death or apoptosis of the neutrophil, a process that can be inhibited by soluble mediators released during an inflammatory response. It has been reported, however, that the phagocytosis of intact bacteria can accelerate apoptosis. We evaluated the effects of the phagocytosis of a common nosocomial pathogen, Candida albicans, on the expression of apoptosis. Phagocytosis of killed Candida induced a dose-dependent increase in the apoptosis of normal neutrophils after 18 h of in vitro culture, from 40.7+/-9.1% to 81.7+/-4.5%, while supernatants from neutrophil:Candida co-cultures actually inhibited apoptosis. Induction of apoptosis was not dependent on phagocytosis, since opsonization of yeast with serum failed to increase apoptosis, while inhibition of phagocytosis with latrunculin B resulted in a slightly increased apoptotic rate. Increased apoptosis induced by Candida was associated with increased activity of the membrane-associated apoptotic enzyme, caspase 8, and with increased expression of the active form of the key executioner caspase, caspase 3. Increased apoptosis was associated with depletion of intracellular glutathione (GSH), and could be inhibited by the addition of exogenous GSH. These data demonstrate an important physiologic role for host-pathogen interactions in the resolution of inflammation and suggest that the response to an invading pathogen is an important stimulus to the restoration of normal immunologic homeostasis.

Phagocytosis of viable Candida albicans by alveolar macrophages: flow cytometric quantification

The phagocytic capacity of blood leukocytes may be assessed by flow cytometric techniques using fluorochrome-labeled particles including viable microorganisms. Application of this approach to alveolar macrophages (AM) is hampered or even rendered impossible by the strong autofluorescence of this cell type, superimposing the fluorescence intensity of the labeled phagocytic targets. Viable Candida albicans were loaded with the membrane-permeable fluorescent dye carboxy-seminaphtorhodafluor 2/acetoxymethylester (carboxy-SNARF 2-AM), which is cleaved intracellularly to generate the membrane-impermeable derivative carboxy-SNARF 2. Fluorescence was excited with the 488-nm line of an argon-ion laser, and the emission peak at 633 nm was used for quantification of dye-associated fluorescence. Rabbit and human AM were labeled with fluorescein isothiocyanate-coupled monoclonal mouse anti-macrophage antibodies. After coincubation of macrophages and yeast, 4% paraformaldehyde plus 0.5% EDTA in phosphate-buffered saline was used to stop the phagocytic process and detach adherent yeast from the AM surface. Macrophages loaded with yeast displayed a shift from monochromatic (green) to dual (green and red) fluorescence. The percentage of yeast-positive AM and red fluorescence intensity of phagocytosing macrophages were quantified. Yeast opsonization with serum or anti-Candida immunoglobulins was a prerequisite for phagocytosis. Under optimized conditions (0.5-10% serum; 60 min yeast-AM incubation; yeast-AM ratio 8:1 to 12:1), 71-91% of the AM were involved in the phagocytic process. Yeast engulfment was completely inhibited by N-ethylmaleimide and iodoacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

YEAST OPSONISATION IN CHILDREN WITH CHRONIC DIARRHOEA

Children with defective yeast opsonisation are more likely to have chronic diarrhoea and this can be halted by infusion of fresh plasma. It is not known whether these children have diarrhoea because of persistence of known stool pathogens, development of an enteropathy or some other mechanism. This study seeks to explore the association of stool pathogens and small intestinal enteropathy with defects in opsonisation. Thirty-two children (10F;22H, aged 3 mo to 7 yr) with chronic diarrhoea were investigated for yeast opsonisation, stool pathogens and an enteropathy. Thirteen (3F:10M) had severe opsonisation defects, of whom two had stool pathogens and six had an enteropathy, which appeared to account for the idarrhoea; however in six stool bacteriology an small bowel biopsy were normal and no other diagnosis was reached. Conversely, in those with normal opsonisation who had a normal small bowel biopsy and were free of stool pathogens a diagnosis was reached in all but one case. This included toddler's diarrhoea, response to food elimination. Severe atopy, IgE subclass deficiency and small intestinal bacterial overgrowth. The chronic diarrhoea found in children with defective yeast opsonisation often occurs independently of the presence of stool pathogens or an enteropathy. 1. Candy DCA et al. ARch Dis Chil 1980; 55 : 189.

Changes in plasma opsonization of yeast after isolation of Biomphalaria glabrata in small volumes of water

Changes in plasma opsonization of yeast after isolation of Biomphalaria glabrata in small volumes of water

Opsonization of yeast by the plasma of Biomphalaria glabrata (Gastropoda): a strain-specific, time-dependent process.

Phagocytosis of yeast by haemocytes of Biomphalaria glabrata, intermediate host of Schistosoma mansoni, is influenced strongly by host plasma components, although it can occur without involvement of such factors. Plasma from two strains of B. glabrata which are resistant to S. mansoni differs in its opsonic properties from the plasma of a susceptible strain. This may reflect the principles which determine compatibility or incompatibility in this host-parasite system. Opsonization is a time-dependent process: short periods of incubation in plasma from all strains reduces subsequent phagocytosis in the absence of plasma factors, whereas longer incubation in resistant strain plasma is markedly opsonic. Haemocytes from all strains examined are equally competent in their recognition of either native or opsonized yeast.

IgG subclass antibody production to milk and egg antigens during infancy

The type of antibody produced to foods during infancy may predict or influence the subsequent development of food intolerance and atopic disease. We therefore studied IgG antibody production to two common food antigens in 191 unselected healthy term Infants. IgG1, 2, 3 and 4 subclass antibodies to bovine casein and to ovalbumin were measured by an enzyme linked immunosorbent assay on sera collected at 7 days, 3 months and 1 year of age.Antibodies to casein were detected in all IgG subclasses. They were low at 7 days[mean IgG1 261, IgG2 52, IgG3 54, IgG4 125U/ml]and had risen by 3 months[mean IgG1 2325, IgG2 95, IgG3 130, IgG4 376] with similar levels at lyear. In contrast ovalbumin antibodies were restricted to IgGl & 4. The levels on day 7[mean IgG1 1001, IgG4 716 U/ml] had fallen by 3 months[mean IgG1 287,IgG4 108]but had risen again by 12 months[mean IgGl 836, IgG4 135]. The predominant subclass to both antigens was IgG1.We have investigated the relationship of IgG antibody production to maternal antibody levels and to infant feeding practice. Seventeen infants showed defective yeast opsonisation, an abnormality associated with atopy. IgG subclass production in these infants was similar to the group as a whole and to matched controls.

Inhibition of human neutrophil phagocytosis and intracellular killing of yeast cells by fluoride

These processes depend upon the catabolism of glucose, which can be inhibited by fluoride (F −). We studied them with a radiolabelling technique using [ 3H]-uridine, which is incorporated into viable yeast cells, but hardly at all into neutrophils and dead or phagocytosed yeast cells. Lysis of neutrophils and treatment of the lysate with [ 3H]-uridine allowed estimation of intracellular killing of yeast cells. F − inhibited neutrophil phagocytosis and intracellular killing in a dose-related manner. Intracellular killing was significantly more sensitive to environmental F − than was phagocytosis. This effect on phagocytosis was not because of inhibition of opsonization of yeast, nor was it related to an effect on medium Ca 2−.

Clinical and immunological characteristics of pre-school asthma.

Ninety-two children, sixty-two boys and thirty girls, aged 18 months to 6 years with frequent asthma were studied. Nine were born prematurely and two were ventilated in the neonatal period; two had been admitted to hospital with acute bronchiolitis. Most had an early onset of symptoms, forty-two developing asthma in infancy. Although overall growth was normal, 63% had some chest deformity. Half had past or present eczema and just over half had chronic rhinitis. Upper respiratory infection appeared to be an important precipitant of attacks in all, and 88% had exercise-induced asthma. The majority were atopic, 74% had elevated serum IgE levels, skin-prick tests were positive in 61 of 77 tested and half had eosinophilia. However, allergic factors were clearly identified as causing asthma attacks in only 24%. Immunoglobulin deficiencies were found in thirteen and defective yeast opsonization in eighteen; these children showed no other obvious differences from the remainder. Eighty-two children were followed up and after 1 year fifty-one were receiving inhaled steroids. Measurement of serum immunoglobulin, eosinophil count and skin-prick testing had little influence on management.

Spontaneous bacterial peritonitis in children with chronic liver disease: Clinical features and etiologic factors

We analyzed the clinical and bacteriologic features of 12 episodes of spontaneous bacterial peritonitis (SBP) in 11 children (four boys, median age 5.5 years) with chronic liver disease. All patients had cirrhosis and ascites; four had hypersplenism, and one was asplenic. Symptoms included increasing abdominal distention, pyrexia, abdominal pain, gastrointestinal disturbance, and encephalopathy. Nine had rebound tenderness on abdominal palpation, and 12 had reduced bowel sounds. The most frequent organisms isolated from culture of ascitic fluid were Streptococcus pneumoniae (nine). Klebsiella (two), and Haemophilus influenzae (one); blood cultures grew identical organisms in nine. Seven patients died despite intensive antibiotic therapy. In the 3 months prior to onset of SBP, defective yeast opsonization and reduced serum concentration of C4 were found in all nine children tested; eight had reduced concentration of C3. Functional deficiency of all complement components was present in four tested within 1 to 5 months of the onset. In contrast, only eight of 59 cirrhotic children without SBP had low C3, and eight had defective yeast opsonization, although 35 had low C4 values. Four of the patients with SBP and low C3 and C4 concentrations had normal concentrations at the time of diagnosis of liver disease 2 to 5 years previously. Opsonization of type III pneumococci was reduced in sera from three patients who subsequently developed pneumococcal peritonitis. The incidence of SBP in children with chronic liver disease is similar to that in adults, as are the clinical features. Our observations suggest that complement deficiency induced by chronic liver disease may be important in the pathogenesis of SBP.

995 EFFECT OF VITAMIN E ON PHAGOCYTOSIS IN CHILDREN WITH CYSTIC FIBROSIS (CF)

Phagocytic function, mainly polymorphonuclear intracellular bactericidal activity (PIBA), was investigated in relation to serum vitamin E level in children with CF and normal individuals. Twenty three patients with CF and pancreatic insufficiency aged 3 months to 14 years were compared to 23 age and sex matched controls. The following parameters were studied: serum vitamin E level, chemotaxis, yeast opsonization and PIBA against staphylococus aureus. In children with CF, both vitamin E levels and PIBA were significantly (P < 0.001) lower (0,65 ± 0,65 and 0.13 ± 0,09, respectively) as compared to values found in the controls ( 8,96± 2,86 and 0.06 ± 0.04, respectively). No further phagocytic impairment was detected in either group. These data support the concept that vitamin E deficiency in CF may contribute to infections with staphylococcus aureus and/or pseudomonas aeruginosa, the most frequent bacterial causes of infection in individuals with deficient bactericidal activity.

Immunological studies in asthmatic children undergoing antigen provocation in the skin, lung and nose.

In children with perennial asthma, dual (immediate and late) reactions in the skin to D. pteronyssinus and Timothy grass pollen were more frequent with high doses of antigen and were associated with large immediate reactions. The frequency of dual bronchial or nasal reactions was not related to the size of the immediate reaction and dual reactions were commonly elicited to the lowest antigen dose which would elicit an immediate reaction. Serum IgG, IgA, IgM or IgE concentrations, IgE or IgG antibodies to the antigens and defective yeast opsonization did not differ in children with dual or immediate only reactions in skin, nose or lung. In five patients undergoing bronchial provocation tests with D. pteronyssinus there was no fever, no fine crepitations in the lungs and no significant change in the levels of C3.

Yeast opsonisation and complement in children with liver disease. Analysis of 69 cases.

Opsonisation of heat-killed bakers yeast and C3 and C4 concentrations were determined in sera from 69 children with liver disease and 39 controls as simple screening procedures for abnormalities in the complement system. Where significant defects in these moieties were encountered, the activity of the following was measured: total haemolytic complement, C4, C5, total alternative pathway, factor B and D activities were measured. Complement activation was detected by the presence of C3d in EDTA plasma samples. Yeast opsonisation, C3 and C4 concentrations and activities of all complement components measured were significantly (P less than 0.001) reduced in all of 10 children with fulminant hepatic failure (FHF) and six with chronic active hepatitis (CAH). There was no consistent relationship between complement deficiency and standard tests of liver function. C3 breakdown products were identified in plasma from five patients with CAH and complement deficiency but not in three patients with complement deficiencies associated with FHF. Complement concentration and activity improved in children recovering from FHF and in CAH treated by immunosuppressants. Yeast opsonisation index was raised significantly without parallel increase in C3 or C4 concentration in eight of nine children with biliary atresia before surgery and 11 of 13 with alpha-1-antitrypsin deficiency regardless of age or the presence of active liver disease. Yeast opsonisation indices became normal after successful surgery in patients with biliary atresia.

Bacterial and fungal infection in children with fulminant hepatic failure: possible role of opsonisation and complement deficiency.

Serious bacterial infection, including eight episodes of bacteraemia, developed in seven of 15 (47%) children with fulminant hepatic failure. Those with infections had a slightly higher leucocyte response than those who did not. Serum immunoglobulin concentrations were normal or raised in all patients. Opsonisation of heat-killed baker's yeast, functionally measured total haemolytic complement, C4, C5, total alternative pathway activity, factor B and D activity, and C3 concentrations were all significantly (p less than 0.005) reduced at presentation but returned to normal in those who survived. The severity of defects in yeast opsonisation, C4, and factor B activity at presentation were significantly correlated with the subsequent development of infection. In five patients bacteraemia occurred at a time when opsonisation and complement components were defective. Plasma infusions in vivo improved opsonisation in vitro and only one bacterial isolate was obtained within four days of such an infusion. Those patients who developed infection had received significantly (p less than 0.05) fewer plasma infusions than those who did not. Our findings suggest that both alternative and classical pathways of complement are defective in children with severe liver disease and may contribute to the susceptibility of such patients to infections. Plasma infusions might be useful in reducing the incidence of bacterial infection in such conditions.

Defective yeast opsonisation and functional deficiency of complement in sickle cell disease.

Opsonisation of heat-killed baker's yeast, functional activity of the total alternative pathway of complement, and factor B detected functionally and immunochemically were significantly reduced in 72 children with sickle cell disease compared with 40 age-matched black control children. There was significant correlation between functional activity of the total alternative pathway and functionally measured factor B, but not between factor B measured functionally and immunochemically. The opsonisation defect could be corrected in vitro by normal serum, and factor B-depleted serum, and was qualitatively similar to that seen in patients with primary yeast opsonisation deficiency. Serial studies showed that these serum defects were persistent. Reduction in the activity of components of the alternative pathway of complement and opsonisation was found in 4 patients who had recovered from pneumococcal meningitis and in one who developed osteomyelitis. Defects of yeast opsonisation and complement which are common in patients with sickle cell disease, may partly explain the children's increased susceptibility to infection, and might help to identify individuals especially at risk.

Atopy does not predispose to RSV bronchiolitis or postbronchiolitic wheezing.

Twenty-six 8-year-old children who had had respiratory syncytial virus (RSV) bronchiolitis in infancy and their paired controls underwent skin and blood tests to assess the role of immunodeficiency and atopy in the pathogenesis of RSV bronchiolitis and the wheezing that may follow it. There was no difference between patients and controls in prevalence of atopy; positive results of prick tests to common antigens; eosinophil counts; yeast opsonisation defect; C2 deficiency; IgG, IgA, IgM, and IgE concentrations; or IgE antibody to dermatophagoides, timothy-grass pollen, and cat fur. Those of the children who had had RSV bronchiolitis and who continued to wheeze had a slightly higher mean eosinophil count and levels of IgE antibody to dermatophagoides than those who did not wheeze. Exercise-induced bronchial lability, though higher in patients than controls, did not correlate significantly with eosinophil counts or IgE concentrations. The genetic factors predisposing to RSV bronchiolitis and postbronchiolitic wheezing may differ from those predisposing to atopic asthma, though exclusive breast feeding may protect against both.

Atopy in parents of children dying with sudden infant death syndrome.

36 parents of infants who had died suddenly did not differ in frequency of atopic symptoms, immediate skin tests, IgE, IgE antibody, immunoglobulin G, A, and M, or yeast opsonisation, from 36 matched controls, although atopy was common (about half had atopy in both groups.

The atopic syndrome: in vitro immunological characteristics of clinically defined subgroups of atopic subjects.

Total IgE, selected specific IgE antibody levels, C2 and yeast opsonization were measured in four clinically defined subgroups of atopic patients who had previously been HLA-typed. Each group was characterized by a distinctive mean total IgE level and profile of specific IgE responses. All groups gave comparable levels of IgE antibodies to Timothy grass pollen but in patients initially presenting with infantile eczema and who subsequently developed asthma and/or hay fever, IgE antibodies to bovine milk proteins, egg ovalbumin and cat dander were frequently observed whereas in patients initially presenting with hay fever later in life these antibodies were usually absent. Computer assisted analysis failed to show any association between IgE antibody responses and (a) the HLA phenotypes A1,B8 and A3,B7 (b) C2 levels and (c) the yeast opsonization index.

Defective yeast opsonisation of serum in tuberculosis.

We have studied the opsonising ability of the sera of 92 patients with tuberculosis. Fourteen per cent of these patients showed defective opsonising ability compared with 4% in a clinic control group. This increased frequency in TB was accounted for by the greatly increased proportion with defects found in patients with intrathoracic TB (21%). We may have identified a section of the population with a specific genetically linked abnormality of a host defence mechanism which renders them more susceptible to intrathoracic TB. Further population studies are required.