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Background & Aims: Refractory celiac disease type II (RCDII) and EATL (Enteropathy Associated T-cell Lymphoma) are (pre)malignant complications of celiac disease (CD). Data on malnutrition and intestinal absorption is lacking in these patients. Therefore, the aim of the study is to comprehensively assess nutritional status and intestinal absorption capacity of patients with RCDII and EATL, compared with data of newly diagnosed CD patients., Methods: Observational study in tertiary care setting in RCDII (n = 24, 63.8 ± 8.2 y), EATL (n = 25, 62.3 ± 5.7 y) and CD patients (n = 43, 45.6 ± 14.8 y). At diagnosis, anthropometry (BMI, unintentional weight loss, fat-free mass index (FFMI), handgrip strength (HGS), nutritional intake, fecal losses and Resting Energy Expenditure (REE)) were assessed., Results: Low BMI (<18.5) was more often observed in RCDII patients than in CD or EATL patients (in 33%, 12% and 12%, respectively, p = 0.029). EATL patients more frequently had unintentional weight loss (>10%) than CD or RCDII patients (in 58%, 19% and 39% of patients, respectively; p = 0.005/0.082). Energy malabsorption (<85%) was detected in 44% and 33% of RCDII and EATL patients, vs 21.6% in CD (NS). Fecal energy losses were higher in RCDII than in CD patients (589 ± 451 vs 277 ± 137 kcal/d, p = 0.017). REE was underestimated by predicted-REE with>10% in 60% of RCDII, 89% of EATL, and 38% of CD patients (p = 0.006). Low FFMI and HGS were detected in one third and two thirds of all patients, respectively., Conclusions: The nutritional status of patients with RCDII and EATL is inferior compared with untreated naïve CD patients at presentation. Both malabsorption as well as hypermetabolism contribute to malnutrition., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Malabsorption, weight loss and vitamin/mineral-deficiencies characterize classical celiac disease (CD). This study aimed to assess the nutritional and vitamin/mineral status of current "early diagnosed" untreated adult CD-patients in the Netherlands. Newly diagnosed adult CD-patients were included (n = 80, 42.8 ± 15.1 years) and a comparable sample of 24 healthy Dutch subjects was added to compare vitamin concentrations. Nutritional status and serum concentrations of folic acid, vitamin A, B₆, B₁₂, and (25-hydroxy) D, zinc, haemoglobin (Hb) and ferritin were determined (before prescribing gluten free diet). Almost all CD-patients (87%) had at least one value below the lower limit of reference. Specifically, for vitamin A, 7.5% of patients showed deficient levels, for vitamin B₆ 14.5%, folic acid 20%, and vitamin B₁₂ 19%. Likewise, zinc deficiency was observed in 67% of the CD-patients, 46% had decreased iron storage, and 32% had anaemia. Overall, 17% were malnourished (>10% undesired weight loss), 22% of the women were underweight (Body Mass Index (BMI) < 18.5), and 29% of the patients were overweight (BMI > 25). Vitamin deficiencies were barely seen in healthy controls, with the exception of vitamin B₁₂. Vitamin/mineral deficiencies were counter-intuitively not associated with a (higher) grade of histological intestinal damage or (impaired) nutritional status. In conclusion, vitamin/mineral deficiencies are still common in newly "early diagnosed" CD-patients, even though the prevalence of obesity at initial diagnosis is rising. Extensive nutritional assessments seem warranted to guide nutritional advices and follow-up in CD treatment.

Background: Plasminogen activator inhibitor 1 (PAI-1) is a serpin that has a key role in the control of fibrinolysis through proteinase inhibition. PAI-1 also has a role in regulating cell adhesion processes relevant to tissue remodeling and metastasis; this role is mediated by its binding to the adhesive glycoprotein vitronectin rather than by proteinase inhibition. Active PAI-1 is metastable and spontaneously transforms to an inactive latent conformation. Previous attempts to crystallize the active conformation of PAI-1 have failed., Results: The crystal structure of a stable quadruple mutant of PAI-1(Asn150-->His, Lys154-->Thr, Gln319-->Leu, Met354-->Ile) in its active conformation has been solved at a nominal 3 A resolution. In two of four independent molecules within the crystal, the flexible reactive center loop is unconstrained by crystal-packing contacts and is disordered. In the other two molecules, the reactive center loop forms intimate loop-sheet interactions with neighboring molecules, generating an infinite chain within the crystal. The overall conformation resembles that seen for other active inhibitory serpins., Conclusions: The structure clarifies the molecular basis of the stabilizing mutations and the reduced affinity of PAI-1, on cleavage or in the latent form, for vitronectin. The infinite chain of linked molecules also suggests a new mechanism for the serpin polymerization associated with certain diseases. The results support the concept that the reactive center loop of an active serpin is flexible and has no defined conformation in the absence of intermolecular contacts. The determination of the structure of the active form constitutes an essential step for the rational design of PAI-1 inhibitors.

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor (serpin) supergene family and a central regulatory protein in the blood coagulation system. PAI-1 is unique among serpins in exhibiting distinct active and inactive (latent) conformations in vivo. Though the structure of latent PAI-1 was recently solved, the structure of the short-lived, active form of PAI-1 is not known. In order to probe the structural basis for this unique conformational change, a randomly mutated recombinant PAI-1 expression library was constructed in bacteriophage and screened for increased functional stability. Fourteen unique clones were selected, and shown to exhibit functional half-lives (T1/2S) exceeding that of wild-type PAI-1 by up to 72-fold. The most stable variant (T1/2 = 145 h) contained four mutations. Detailed analysis of these four mutations, individually and in combination, demonstrated that the markedly enhanced functional stability of the parent compound mutant required contributions from all four substitutions, with no individual T1/2 exceeding 6.6 h. The functional stability of at least eight of the remaining 13 compound mutants also required interactions between two or more amino acid substitutions, with no single variant increasing the T1/2 by > 10-fold. The nature of the identified mutations implies that the unique instability of the PAI-1 active conformation evolved through global changes in protein packing and suggest a selective advantage for transient inhibitor function.

Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence. Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding. Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions. Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five point mutations that selectively disrupt vitronectin binding were identified. All 5 residues are located on the exterior of the PAI-1 structure. These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123. These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.

Cultures of transformed fibroblasts actively involved in extracellular matrix degradation have been examined for initial activation of serine and metallo protease cascade systems. Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF), in contrast to transformed mammalian cells, produce active, two chain urokinase-type plasminogen activator (tcu-PA). Active tcu-PA is found in serum-free, plasmin-free conditioned medium from RSVCEF cultures as determined by two independent methods, immunoprecipitation and differential DFP sensitivity. RSVCEF cultures synthesize and secrete inactive, single chain uPA (scu-PA) which is converted to tcu-PA in a time dependent manner by a catalytic mechanism that appears to involve a functioning uPA receptor on the surface of intact cells. The enzyme activity responsible for this conversion may represent the initiating catalytic event in the PA/plasminogen serine protease cascade system. A 70 kDa prometalloprotease capable of degrading denatured collagen following its activation also is significantly elevated in RSVCEF cultures over that of normal CEF. Trace amounts of the active 62 kDa form of the metalloprotease (gelatinase) is found in the transformed RSVCEF cultures indicating that these cultures produce a natural activator of the prometalloprotease. Plasmin and/or PA do not appear to be the activator of this enzyme as determined by indirect inhibition assays and direct assays employing purified enzymes. The possible central position of pro PA and the 70 kDa prometalloprotease in an interacting, complex protease cascade system involved in extracellular matrix degradation is discussed.