In vitro cross-linking of gluten into high-molecular-weight polymers with transglutaminase.

From the amino acid composition of gluten proteins and the substrate specificity of transglutaminases (TGase) we concluded that gluten proteins can be favourable substrates for TGases due to their high glutamine content. By use of sodium dodecyl sulfate polyacrylamide gel-electrophoresis it was demonstrated that from gluten-ES and gluten-TS high-molecular-weight proteins developed in the presence of Ca2+ and red blood cell lysate containing TGase. When ovalbumin or deamidated gluten were applied as substrates no high-molecular-weight products were formed. Upon spectrophotometric measurements we found that covalent cross-links (isopeptide bonds) formed under the effect of TGases presumably cause a change in the position of chromophore groups in the substrates. Absorption decrease was detected between 274-276 nm as a result in the case of gluten-TS and gluten-ES used as substrates for TGase. No such change occurred in ovalbumin and deamidated gluten, applied as controls, under the influence of TGase. On the basis of our experiments it is postulated that the first step in gluten toxicity is presumably the binding of gluten to the intestine mucosa. In this binding the high transglutaminase activity in the intestines of coeliac patients and the high glutamine content of gluten may have an important role.

Effect of a calcium-binding gluten fraction on the superprecipitation of actomyosin.

The Ca2+-binding gluten fraction isolated by the authors has been shown to prolong the clearing phase and to shorten the physiological contraction phase of ATP activated natural actomyosin suspension in the presence of physiological potassium chloride in a dose dependent manner. The mean ATPase activity of myosin and actomyosin was calculated for each phase from the actual ATP concentrations, measured at 30 sec intervals, and from the phase lengths. The preparation was found markedly to inhibit the ATPase activity during the clearing and physiological contraction phase. Since both myosin and actomyosin ATPase is Ca2+-dependent, it is assumed that the inhibitory effect of the gluten fraction on ATPase activity may be mediated by its free Ca2+-binding capacity, resulting in modified phase lengths of actomyosin superprecipitation. On the basis of these experimental results a hypothesis is put forward of the part of the Ca2+-binding gluten fraction played in the pathomechanism of coeliac disease.

Isolation and physicochemical and functional properties of a calcium binding gluten fraction.

Each milligram gluten protein isolated from bread contains 0.03-0.06 mumol calcium. On theoretical grounds we have concluded that this calcium quantity is bound to the free carboxyl groups not participating in peptide bonds of dicarbonic aminoacids, especially glutaminic acid, making up a large proportion within the aminoacids of gluten. After treatment with EGTA, a well-known calcium complex forming compound, two gluten fractions can be distinguished: water-soluble gluten-ES, and gluten-EP soluble in acetic acid. The aminoacid composition of gluten-ES is similar to that of unfractionated gluten. It is rich in aminodicarbonic acid (glu), aminodicarbonic acid amide (gln) and proline. Further properties of gluten-ES are: immunological similarity to gluten; a molecular mass of 36 000 dalton; an absorption maximum at 275.6 nm; a Ca2+-binding capacity of 0.72 mumol Ca2+/mg protein as measured by atomic absorption spectrophotometry and by Ca2+ ion selective electrode; inhibitory effect of a small quantity (25-30 micrograms) of the compound on the Ca2+-Mg2+ dependent ATPase and Ca2+-uptake of fragmented sarcoplasmatic reticulum. Preliminary experiments have demonstrated that gluten-ES has an influence on other calcium ion mediated systems like actomyosin superprecipitation. We put forward the hypothesis that by its Ca2+-binding capacity, gluten-ES is capable of influencing the level of free calcium and may thus play a part in the pathomechanism of coeliac disease.

Relationship of maternal and newborn (cord) serum ferritin concentrations measured by immunoradiometry.

Serum ferritin concentration was determined by immunoradiometry in venous blood samples of 45 pregnant women at term, in their babies' cord blood samples, and blood specimens obtained from 43 infants aged 3-12 months. The concentration of ferritin was higher in cord serum than in respective maternal samples and infant specimens. Low values were found in more than half of the maternal venous samples. Iron stores of newborns delivered by mothers with low serum ferritin concentration were lower than in newborns of mothers having normal ferritin levels. Serum ferritin measurement is a sensitive method to determine iron deficiency in pregnancy.

Chemotactic and random movement of cord-blood granulocytes.

Chemotactic responsiveness and random movement of cord-blood granulocytes were studied with a modified Boyden's method. Cord-blood granulocytes were less active chemotactically than granulocytes from healthy children and adults, whereas the random filter movement of the cells from all three sources was about the same. In cord sera, concentrations of cell directed chemotaxis inhibitors were equal to those in sera from other age groups. Compared with the situation in healthy children and adults, the generation of chemotactic factors in cord-blood sera was impaired. This impairment was not related to an increased activity of chemotactic factor inactivators. Measurement of the cyclic nucleotide levels in granulocytes from cord-blood and from children belonging to various age groups revealed that the cord granulocytes have significantly lower concentrations of cAMP and cGMP, which could have been responsible for the decreased chemotactic responsiveness.

Activity and characteristics of inflammatory effector cells in newborns.

To investigate the aspecific activity of inflammatory effector cells (mononuclear and polymorphonuclear leukocytes) of newborn babies, a comparative study was performed of chemotactic and random filter motility, phagocytic activity, and bactericidal capacity of these cells, of the electron microscopic counterparts of these activities, the serum factors influencing motility and of the content and ratio of cyclic nucleotides present in the cells of subjects of different ages. It was found that chemotaxis of the mononuclear and polymorphonuclear leukocytes is a gradually maturing function; its deficiency observed during the neonatal period is not caused by a depressed excitability by chemical agents, by the presence of inactivators of chemotactic factors or of chemotaxis inhibitors. It is the low serum levels of total complement, C 3, IgG, IgM and properdin that explain the reduced chemotactic mobility. In addition to these factors reduced leukocyte flexibility, condition and activity of the flexibility, condition and activity of the microtubular and microfilamentary system depending on calcium ion and cyclic nucleotide concentrations, and an eventual immaturity or exhaustion of certain intracellular enzymes also play a part. In respect to orientation towards chemical stimuli, the leukocytes of the newborn largely differ from those of children or adults. Similarly, a difference in uptake and elimination of bacteria and in ultrastructural changes accompanying these processes can be demonstrated between newborn and adult cells. The aspecific cellular functions, the reduction of which plays an important part in the weak resistance of the newborn, also depend on certain properties of the infective agents.

Serum ferritin level in infants and children with anaemia and malignant disease.

Serum ferritin was determined by immunoradiometry in children aged 6 months to 3 years, immediately before leaving hospital where they had been treated for various acute, non-haematological diseases. A low value was found in 13% of them. Serum ferritin concentration is a sensitive method in differentiating between iron deficiency and infectious anaemia. A significantly higher mean value was found in children affected by malignant disease (acute lymphoid or myeloid leukaemia, various solid tumours).

Isolation and physiochemical properties of an adenosine-rich gluten fraction.

Gluten proteins were isolated from the 0.01 mol acetic acid extract of bread. It was observed that precipitation of gluten provoked by 200 mM NaCl could partly be inhibited by adenosine. Based on this finding a method for isolation of the gluten fraction resisting saline precipitation in the presence of adenosine was elaborated. This fraction termed by us gluten-A-S, was found to have a lower glutamine + glutaminic acid and a higher proline and phenylalanine content than gluten. By sodium dodecylsulphate polyacrylamide gel electrophoresis gluten-A-S was shown to contain components of 58 000 and 34 000 dalton molecular weight after mercaptoethanol treatment while without the latter it contained a component of 74 000 dalton. The absorption maximum of the compound is at 260 mm; E280nm/E260nm = 0.5. In accordance with previous findings, gluten-A-S was found to contain 50-100 nmol adenosine per mg protein in a strong binding. It seems that in addition to the small amount of tightly bound adenosine, gluten-A-S contains a larger quantity of adenosine loosely bound to the protein. The physiological effects of dissociable adenosine bound to gluten and its possible role in the pathomechanism of gluten sensitive enteropathy is discussed in detail.

Chemotactic and random movement of human newborn monocytes.

Boyden's method was used to assess the chemotactic and random mobility of monocytes from healthy newborns, children aged 3--8 years, and adults aged 20--28. Monocyte chemotaxis in the newborn was decreased in comparison with that of control groups. No difference was observed in random cell mobility in the groups examined. Monocyte chemotactic activity in infants over 3 years of age was the same as in adults. According to our results, the leading-front and lower-surface evaluation methods, in newborns at least, do not give the same information on chemotaxis.

[Leukocyte function in colostrum (author's transl)]. / Untersuchungen zur Leukozytenfunktion im Kolostrum.

Breast milk, especially colostrum was analysed quantitatively and qualitatively as well as the function of various cell types in it. The phagocyte activity of the mass of leukocytes consisting predominantly of macro- and microphages approaches the phagocyte index of leukocytes of the newborn infant, however, neither of them reaches the level of adults.--The lymphocytes traceable in a minor proportion produce secretory IgA as well as IgG.--On the basis of literary data the authors discuss the literature and their own results regarding the immunobiological significance of breast milk.

IgE levels of infants with complications after pertussis vaccination.

IgE levels of 31 infants who had severe complications after whooping cough vaccination, were determined by RIST. The patients had convulsions, encephalitis or anaphylactic shock symptoms. All but two had elevated IgE levels for their age. It is suggested that, in certain genetically predisposed human beings, pertussis antigen can also induce IgE synthesis, as has already been established in rodents.