[Celiac disease and "gluten sensitivity"]. / Malattia celiaca e "gluten sensitivity".

It is known that celiac disease is characterized by a huge variety of clinical forms ranging from classical ones to silent forms, potential ones and to an increased number of cases of gluten-sensitivity. The latter is an abnormal non-allergic sensibility to gluten. Clinical manifestations can be very different without a severe intestinal damage (Marsh/Oberhuber 0-I) and this condition seems to benefit from a gluten free diet. Cases of gluten-sensitivity appear very interesting in the search of histological markers with elevated specificity, which are able to identify slight and early gluten dependent enteropathy, especially in at risk patients for celiac disease even before classical autoantibodies appear: for instance, this is the case of intraepithelial lymphocytes T-cell receptor gamma delta and mucosal deposits of class IgA anti transglutaminase antibodies. Other studies are investigating transglutaminase isoenzimes (different from tissue one), that can be identified in patients with gluten dependent symptoms without classical autoantibodies. Forms of gluten allergy have a different pathogenesis from celiac disease and are represented by "backer's asthma" or by classical allergy to wheat proteins. Clinical manifestations can vary from anaphylactic reactions to dermatological, respiratory and intestinal symptoms. Also in these cases the therapeutic approach is based on gluten free diet.

Optical coherence tomography in pediatric patients: a feasible technique for diagnosing celiac disease in children with villous atrophy.

BACKGROUND AND AIM: Celiac disease is a common condition with many atypical manifestations, where histology serves as the "gold standard" for diagnosis. A useful new technique, optical coherence tomography, can depict villous morphology in detail, using light waves. This study examined the correlation between the sensitivity and specificity of optical coherence tomography in pediatric patients undergoing esophago-gastro-duodenoscopy for the diagnosis of celiac disease. MATERIALS AND METHODS: A total of 134 children were prospectively enrolled, 67 with a serological suspicion of celiac disease (group 1) and 67 with negative histology for celiac disease (group 2). During a diagnostic esophago-gastro-duodenoscopy we acquired multiple images and films in the four quadrants of the second part of the duodenum, and biopsies were taken in the area where optical coherence tomography had been done. Three patterns of villous morphology were considered: pattern 1=no atrophy (types 0, 1 or 2 of the Marsh classification); pattern 2=mild atrophy (type 3a or 3b); pattern 3=marked atrophy (type 3c). RESULTS: The diagnosis of celiac disease was histologically confirmed in all 67 children with positive antiendomysium and/or antitransglutaminase antibodies. Optical coherence tomography correlated with pattern 1 histology in 11/11 cases, pattern 2 in 30/32 (93.8%) and pattern 3 in 22/24 (91.6%). Sensitivity and specificity were 82% and 100%. In the control group there was 100% concordance between optical coherence tomography and histology. The overall concordance between optical coherence tomography and histology in determining patchy lesions was 75%. CONCLUSION: Optical coherence tomography could be a helpful diagnostic tool in children with mild or marked villous atrophy for diagnosing celiac disease during upper gastrointestinal (GI) endoscopy, avoiding biopsies. However, duodenal biopsies are mandatory if the optical coherence tomography shows normal villous morphology in patients with positive antibodies.

Antibodies to recombinant human tissue-transglutaminase in coeliac disease: diagnostic effectiveness and decline pattern after gluten-free diet.

BACKGROUND/AIMS: To assess the sensitivity and specificity of IgA and IgG tissue-transglutaminase antibodies assay, the pattern of antibody decline after gluten withdrawal and their modifications with reference to dietary compliance. SUBJECTS: We studied sera from 143 untreated coeliac children and adolescents (8.8+/-6.1 years), 212 sera from 97 of those patients after gluten withdrawal, and 64 control subjects with non-coeliac intestinal disorders (6.8+/-4.8 years). METHODS: Samples were tested for IgA and IgG class tissue-transglutaminase antibodies by radiobinding assay, using human-derived tissue-transglutaminase, and for IgA anti-endomysium antibodies by indirect immunofluorescence on monkey oesophagus. RESULTS: Untreated coeliac patients had significantly higher titres of IgA and IgG tissue-transglutaminase antibodies than controls (p<0.00001); the diagnostic sensitivity was 95.8% and 99.3%, respectively, and the specificity was 95.3%. Three patients with selective IgA deficiency were positive for IgG tissue-transglutaminase antibodies. The concordance rate between IgA tissue-transglutaminase antibodies and anti-endomysium antibodies was 98.1%. Patients on gluten-free diet showed a significant decrease in IgA and IgG tissue-transglutaminase antibodies with respect to untreated patients (p<0.0001). Tissue-transglutaminase was more sensible than anti-endomysium antibodies to detect small amounts of gluten intake when the compliance was poor. CONCLUSIONS: The recombinant human tissue-transglutaminase antibodies assay is a highly sensitive and specific test for diagnosis of coeliac disease, and it is useful in monitoring the compliance to gluten-free diet.

Silica xerogels as a delivery system for the controlled release of different molecular weight heparins.

In this work, we investigated a sol-gel derived silica matrix as a delivery system for the prolonged release of different molecular weight heparins, which allows the glycosaminoglicons to retain their whole biological activity. Several xerogels were obtained by embedding different molecular weight heparins into matrices prepared by using different amount of NH4OH as a catalyst during gel formation. Gel synthesis parameters, drug release properties, and xerogels surface area were evaluated. Unfractionated, low and oligo-molecular weight heparins were embedded into xerogels and the effect of the molecular weight on the release kinetics and the retained biological activity has been investigated. The results show that the surface area of the matrix is a determinant parameter affecting drug release kinetics. This structural feature can be modified by varying the catalyst tetraethoxysilane molar ratio used during the matrix synthesis. In most cases release kinetics fitted the Higuchi diffusive model and a lower diffusion rate was observed from silica matrices characterized by a smaller surface area. In the case of matrices with lower surface area, loaded with unfractionated heparin, zero order kinetics was observed. In this paper, we have defined a heparin release silica xerogel system and we have pointed out how modulation of its synthesis parameters allows adjusting the release of heparin according to therapeutic needs.

Immunological safety evaluation of a horse collagen haemostatic pad.

The safety of a haemostatic and wound dressing collagen pad (Antema) obtained from horse Achilles tendon treated with pepsin, has been studied from the immunological point of view. A sensitizing activity test and maximization test in guinea pig has been performed and its capacity of inducing antibodies has been tested in rabbit and man. In guinea pig the collagen pad tested did not show any symptom of allergic reaction, in rabbit it induced a weak immunological response, without any clinical symptoms, detectable with the ELISA method only at very high serum concentration levels (1:20). In a clinical trial, 20 patients submitted to different types of surgery in which the product had been used as haemostatic pad and left in the surgery area until absorption, did not develop any immunological clinical reaction. One year after the operation no traces of anti-collagen antibodies have been found with the ELISA method in the sera of those patients.

Heterogeneity of unfractionated heparins studied in connection with species, source, and production processes.

The heterogeneity of unfractionated heparins (Hep) can be correlated to the species and organs of origin and to the process of production. Heparins, extracted by different, validated processes from different organs and/or tissues (mucosa, thymus, pancreas, placenta, lung, intestine) or mammals (pig, beef, sheep, man) and other vertebrates (chicken), have been examined by HPLC analysis of heparinase digests. By analysis of disaccharides many observations have been made. Porcine mucosa heparin (pm-Hep) was always found to contain higher amounts of the disaccharides delta UA-GlcNS,6S and delta UA-2S-GlcNS,6S, than did bovine mucosa heparin (bm-Hep), whereas bm-Hep always showed higher amounts of the sequence IdoA(2OSO3)-GlcNSO3 than did pm-Hep. These findings mean that the last step of the biosynthesis, the 6-O-sulfation of glucosamine-N-sulfate (GlcNSO3), is accomplished; in bm-Hep, to a lesser extent than in pm-Hep. The 6-O-sulfated molar fractions of pig mucosa, chicken intestine, beef pancreas, beef placenta, and beef lung heparins were higher than the corresponding molar fractions of beef mucosa and beef thymus Heps. Also the manufacturing processes can partially rearrange the heparin structure. Even 6-O-sulfation enrichment (by chromatographic purification) or base-catalyzed displacement of sulfate groups from IdoA2SO3 occurred. The resulting anticoagulant activity roughly correlated with the percentage of trisulfated disaccharide and the 6-O-sulfated molar fraction. The heparin from human placenta was similar to pm-Hep. The observed species- and organ-dependent structural characteristics support the suggestion by Nader and Dietrich (in Heparin, Chemical and Biological Properties, Lane DA, U Lindahl (Eds). Arnold, London, 1989, p 81) on the antipathogenic role of heparin. The 6-O-sulfation of glucosamine, present in higher amounts in organs that function as barriers against many foreign bodies, like lung, placenta, intestine of chicken and pig, may play an important role in this antipathogenic action of Hep.

Active site for heparin cofactor II in low molecular mass dermatan sulfate. Contribution to the antithrombotic activity of fractions with high affinity for heparin cofactor II.

Dermatan sulfate (DS) is currently under clinical investigation as new antithrombotic agent. Unlike heparin, DS does not act through Antithrombin III (ATIII) but primarily through thrombin on Heparin Cofactor II (HCII). HCII is activated by the oversulfated sequence (IdoA2SO3-GalNAc4SO3)4 or by both the sequences (IdoA2SO3-GalNAc4SO3)n and (IdoA-GalNAc-4,6SO3)n, [n > or = 2]. A Low Molecular Mass Dermatan Sulfate (LMM-DS), endowed with a bioavailability three-four times higher than DS, by subcutaneous route, was obtained by chemical depolymerization of DS. The LMM-DS was fractionated by anion exchange and size exclusion chromatography. Fractions with high and low charge densities, high and low molecular masses, and high (2.66) and low (0.07) potencies on HCII were isolated. A relationship between the in vitro HCII-mediated inhibition of thrombin and the chain length of DS fractions containing oversulfated sequences was found [by a multiple regression test]. The in vivo activity increased until it reached a plateau. The important influence on the HCII activity of natural IdoA-GalNAc-4,6SO3 disaccharide was confirmed by investigation on oversulfated DS obtained by a limited and selective chemical 6-O-sulfation in GalNAc4SO3 units of DS.

Relationship between plasma antifactor Xa activity and the antithrombotic activity of heparins of different molecular mass.

The relationship between the inhibition of venous thrombosis and antifactor Xa (AXa) plasma levels, measured as ex vivo AXa activity at the moment of experimental thrombosis induction, was evaluated in rats treated by different administration routes with different doses of heparins of various molecular masses: unfractionated heparin (UH, 13 kDa), low-molecular-mass heparin (LMM-H, 5kD) and oligo-heparin (OL-H, 2 kD). The AXa activity levels of plasma samples were measured by an amidolytic method and expressed in AXa U/ml. The antithrombotic effect was determined by a vena cava ligature model and expressed as the percent inhibition of thrombus weight. A correlation between the two parameters was determined, regardless of the administration route used. Every heparin requires different AXa plasma levels to develop the same antithrombotic activity: the plasma concentration inducing a 50% protection was 0.09, 0.12 and 0.15 AXa U/ml, for UH, LMM-H and OL-H, respectively. Oligo-H has a significant antithrombotic activity when delivered by the intraileal route and the time course pharmacodynamics showed two phases for the parameters considered: in the second phase, a dissociation between AXa plasma level and antithrombotic effect was observed.

Structure and contribution to the heparin cofactor II-mediated inhibition of thrombin of naturally oversulphated sequences of dermatan sulphate.

Dermatan sulphate (DS) obtained from bovine and pig mucosa and pig skin, and charge-enriched fractions of a selected DS preparation, were characterized in terms of charge density, M(r) and disaccharide composition of chondroitin ABC lyase digests, and by 13C-n.m.r. spectroscopy. Besides the major IdoA-GalNAc4SO3 sequences, all DS preparations contain about 10% disulphated disaccharide sequences (mostly IdoA2SO3-GalNAc4SO3, with minor amounts of IdoA-GalNAc4,6SO3). DS fragments (prepared by radical-catalysed depolymerization of DS and retaining the internal structure of the parent polysaccharide) as well as Smith degraded fragments [SD-DS, obtained by controlled degradation of periodate-oxidized and borohydride-reduced DS (RO-DS)] with the general structure GalNAc4SO3(IdoA2SO3-GalNAc4SO3)n-R (where R is the remnant of a glycol-split uronic acid, and n = 2-3 and 3-4) were characterized by one- and two-dimensional 1H-n.m.r., 13C-n.m.r. and disaccharide composition analysis. In accordance with previous findings [Maimone and Tollefsen (1990) J. Biol. Chem. 265, 18263-18271], only fragments with n > or = 3 significantly enhance the heparin cofactor II-mediated inhibition of thrombin. In natural DS preparations and their fractions, this activity (as well as the antithrombotic activity in an animal model) appears to require IdoA2SO3-containing sequences. The heparin cofactor II activity of DS, RO-DS and SD-DS fragments decreases with decreasing M(r). However, RO-DS fragments are more active than DS fragments of similar M(r), probably because of the extra flexibility endowed by glycol-split IdoA residues.

Different pharmacokinetics, antithrombotic activity and bleeding effects of heparin and two new fragments administered in rat by subcutaneous route.

Native heparin (CAS 9005-49-6) and its two new fragments, low molecular weight heparin (LMW-H, 5 kDa) and oligo-heparin (oligo-H, 2 kDa) obtained by radical degradation were characterized as to their physicochemical properties. Heparin fragments differ from unfractionated heparin only in molecular weight. The pharmacokinetics and some pharmacological effects, bleeding and antithrombotic activity, of the three different molecular weight heparins were investigated. The plasma concentrations were determined by an amidolytic method which measures inhibiting effect on factor Xa. The blood levels of each substance were derived from their in vitro calibration curves. The examination of the pharmacokinetics parameters allowed to evaluate the differences in the bioavailability, absorption rate and elimination mechanisms between the three different heparins. The bioavailability, the absorption rate and the distribution of the molecules of heparins in biological compartments depend on the molecular weight. LMW-H and oligo-H exhibit greater antithrombotic activity than unfractionated heparin when administered subcutaneously. The pharmacokinetic behaviour of oligo-H considerably differs from that of unfractionated heparin and LMW-H. This new drug is able to bind cells and plasma proteins differently from heparin and LMW-H. The capacity of oligo-H to bind smooth muscle cells and to interact with myosin is discussed in relation to the bleeding effect.

Lack of correlation between "in vitro" and "in vivo" antithrombotic activity of heparin fractions and related compounds. Heparan sulfate as an antithrombotic agent "in vivo".

The anticoagulant (U.S.P., APTT; "in vitro" and "in vivo") antithrombotic (aXa; Yin and Wessler and chromogenic), antilipemic (LPL) activities of heparin, heparin fractions and fragments, heparinoids, heparan sulfate and other sulfated glycosaminoglycans were compared with the activities of these compounds as antithrombotics "in vivo" by four different methods (vena cavae ligature, kaolin, collagen and steel coil). A lack of correlation was observed between the activities "in vitro" and the antithrombotic activity "in vivo". For instance heparan sulfate which shows negligible pharmacological activities "in vitro" is a potent antithrombotic agent "in vivo". Likewise, several heparin fractions and fragments have low aXa activity "in vitro" and high antithrombotic activity "in vivo". It is concluded from these results that the "in vitro" tests used cannot predict the antithrombotic activity "in vivo".

Structural studies and "in vivo" and "in vitro" pharmacological activities of heparin fractions and fragments prepared by chemical and enzymic depolimerization.

Two heparin families (SM- and FM- heparins) have been fractionated with barium by selective precipitation at different temperatures. Fragments were obtained from these two fractions by enzymic and chemical degradation. Some structural features and pharmacological activities of FM- and SM-heparins as well as the fragments are now reported. The fragments, prepared by ascorbate/H2O2 oxidation (M.W. 4,500) or heparitinase II (M.W. 5,500), are enriched with trisulfated disaccharide units whereas heparin (M.W.12,500) and FM-heparin (M.W. 10,200) contain also N-sulfated and N-acetylated disaccharides. The chemical and enzymic fragments show 1/3 to 1/5 of the anticoagulant activity of heparin by the USP and APTT methods. The LPL releasing activity is also low in the fragments as well as the AXa activity measured by the chromogenic method. On the other hand the AXa activity measured by the Yin and Wessler procedure is two times higher in the fragments when compared to heparin. The fragments resulting from heparin after heparitinase II degradation and ascorbate/H2O2 oxidation were rich in trisulfated disaccharide units whereas heparin and FM-heparin contained also measurable amounts of monosulfated and N-acetylated disaccharide units. The mechanism of ascorbate oxidation and the structural requirements for the pharmacological activities of heparin is discussed in view of these and other findings.

Fractionation and structural features of two heparin families with high antithrombotic, antilipemic and anticoagulant activities.

15 heparin preparations from bovine intestine, pancreas and lung and hog intestine were fractionated in two main components by selective barium precipitation. The ones that precipitated at room temperature with barium (slow moving (SM)-heparins) had a high anticoagulant activity measured by the USP and APTT (activated partial thromboplastin time) assay and low antithrombotic activity by the Yin and Wessler method. The fractions precipitated at 5 degrees C with barium (fast moving (FM)-heparins) had a low anticoagulant action and high antithrombotic activity. The maximum anti-Xa activity (chromogenic method) was present in heparins with molecular weights around 12-15 X 10(3) daltons whereas high APTT and LPL releasing activities were present in SM-heparins with molecular weights of 30-40 X 10(3) and 15-25 X 10(3) daltons, respectively. FM-heparins had a higher anti-Xa activity and lower lipoprotein lipase (LPL)-releasing activity when compared with the SM-heparins with the same molecular weights. Significant structural differences were observed between SM- and FM-heparins by 13C-NMR spectra and enzymatic degradation with heparinase and heparitinase from Flavobacterium heparinum. Also, significant differences were observed for anti-Xa and anticoagulant activities for the two types of heparins depending on the pharmacological assay used.

Pharmacological activities of heparins obtained from different tissues: enrichment of heparin fractions with high lipoprotein lipase, antihemolytic and anticoagulant activities by molecular sieving and antithrombin III affinity chromatography.

The pharmacological activities of 14 heparins prepared from bovine and hog tissues and heparin fractions obtained by molecular sieving on sephacryl S-200 and antithrombin III affinity chromatography are reported. Two groups of heparins can be distinguished regarding the lipoprotein lipase releasing activity (LPL) and the activated partial thromboplastin time (APTT). Heparins prepared from liver, lung, pancreas and thymus have a ratio of LPL/APTT of 3.6, whereas heparins prepared from placenta and intestine show a ratio of only 2.1. The heparin APTT, LPL, antihemolytic, antifactor Xa and anticoagulant activities were partially resolved by fractionation on Sephacryl S-200 chromatography and antithrombin III affinity chromatography. These results suggest the presence of a mixture of heparins with different pharmacological activities in a commercial preparation.