Profiling of proteome changes in plasma of HIV-infected patients receiving antiretroviral therapy.

PURPOSE: Antiretroviral therapy (ART) prevents human immunodeficiency virus (HIV)-1 onward transmission and disease progression, leading to excellent prognosis in people living with HIV-1 (PWH). However, side effects, complications, and impaired immune reconstitution persist in some patients treated with ART. We aimed to profile proteome changes in plasma before and after ART to identify the molecular pathways altered by ART. EXPERIMENTAL DESIGN: Quantitative proteomics analysis based on tandem mass tag (TMT) labeling was used to profile proteome changes of paired plasma samples from HIV-1 patients before receiving ART and after ART treatment. RESULTS: A total of 1398 protein groups (PGs) were identified, in which 18 proteins were downregulated and 50 were upregulated in plasma from ART treated patients. Based on Ingenuity Pathway analysis (IPA), gap junction signaling and actin cytoskeleton signaling were enriched among upregulated proteins, while downregulated proteins were mainly participated in IL-15 signaling pathway. Patients with the low level of CSF1R and the high levels of MINPP1 and TGM3 showed better CD4+ T-cell recovery. CONCLUSIONS: The present study provided plasma proteome changes after ART to elucidate the underlying mechanistic pathways in response to ART, and also identified potential targets to prompt immune reconstitution.

Microbial transglutaminase: A biotechnological tool to manage gluten intolerance.

Celiac disease (CD) is a chronic immune-mediated disease in which gluten ingestion leads to damage of the small intestinal mucosa in genetically susceptible individuals. The enteropathy is mainly induced by the production of IFN-γ from intestinal CD4+T cells that recognise gliadin peptides following deamidation by tissue transglutaminase. The only available therapy is a strict, lifelong gluten-free diet (GFD). This diet is strongly demanding for patients, which justifies the search for alternative strategies. The enzyme approach is one promising strategy to address this issue. In particular, transamidation of wheat gliadin by microbial transglutaminase (mTG) was fully effective at inhibiting gliadin-specific IFN-γ secretion in intestinal T cells from CD patients. Furthermore, transamidated gliadin induced higher levels of the anti-inflammatory IL-10 than native gliadin in different in vitro models. These data suggest that a more balanced immune response could be induced by mTG-treated gliadin in the small intestine of celiac patients. Furthermore, the highlighted biological property of mTG-treated gliadin could be exploited to induce tolerance to native gliadin in at-risk individuals.

Celiac Crisis: an unusual presentation of gluten-sensitive enteropathy

Celiac disease (CD)­also known as gluten-sensitive enteropathy­is a chronic, genetically predisposing and autoimmune entity with a wide range of clinical manifestations triggered by gluten ingestion, which affects 1% of the general population. Currently, up to 60% of the diagnosis of CD is in adults due to the atypical course of the disease. The severe acute onset of CD­also called celiac crisis­is very uncommon and is still not well documented in adults. We report the case of a 58-year-old man who presented a 45-day history of subtle-onset diarrhea followed by malabsorption syndrome with progressive weight loss, anasarca, and electrolyte disturbances. The diagnostic work-up included an upper digestive endoscopy, which showed scalloping of the duodenal mucosa with pathological features confirmed on biopsies. Specific antibodies were positive, and a satisfactory clinical response was obtained once a gluten-free diet was started. Celiac crisis is a rare initial presentation of CD characterized by severe diarrhea, dehydration, weight loss, hypoproteinemia, and metabolic and electrolyte disturbances. Although rare, it should be considered in patients with apparently unexplained chronic diarrhea.

Uso del HeberFast Line® anti-transglutaminasa para pesquisa de enfermedad celiaca en pacientes pediátricos / Use of HeberFast Line® antitransglutaminase antibodies for the celiac disease screening in pediatric patients

Introducción: la enfermedad celiaca es una enteropatía autoinmune sensible al gluten y otras prolaminas, con base genética, que se genera al contacto del paciente con esas proteínas presentes en el trigo, la cebada y el centeno, que provoca síntomas intestinales y extra intestinales. En Cuba su diagnóstico ha progresado, desde criterios clínicos a pruebas inmunológicas y genéticas, sumándose a la biopsia intestinal. Objetivo: realizar pesquisa de muestras de sangre, usando el test cubano de HeberFast Line® anti-transglutaminasa para conocer la frecuencia de este marcador en una población infantil atendida en nuestro laboratorio. Métodos: se estudiaron 850 muestras de sangre con la finalidad de detectar la presencia de anti-transglutaminasa tisular. Resultados: dentro de nuestra investigación resultaron positivas 10 muestras (1,18 por ciento), lo que concuerda con lo reportado en la literatura revisada. Otras 75 muestras (8,8 por ciento), fueron no válidas para el primer intento, fenómeno descrito por los productores del test y fácilmente superable, utilizando nuevamente la muestra en cuestión, y todas ellas, al ser analizadas por segunda vez, pasaron a la categoría de negativas. Finalmente, 98,82 por ciento de las muestras analizadas fueron negativas. Conclusiones: el 1,18 por ciento de las muestras analizadas contenían anticuerpos anti-transglutaminasa tisular, por lo tanto, ~1 de cada 100 niños pueden padecer de enfermedad celiaca; además, esta prueba constituye una herramienta útil en el diagnóstico precoz, que como se observa, no es tan infrecuente en nuestro país(AU)

Introduction: celiac disease is an autoinmune enteropathy sensitive to gluten and other prolamines, with genetic basis. It generates when the patient gets in contact with those proteins present in wheat, barley and rye and causes intestinal and extraintestinal symptoms. The diagnosis of this disease has advanced from clinical criteria to immune and genetic tests in addition to intestinal biopsy. Objective: to perform screening of blood samples by using the Cuban test called HeberFast Line® antitransglutaminase to find out the frequency of this marker in the infant population tested in our laboratory. Methods: fight hundred and fifty blood samples were studied to detect the presence of tissue antitransglutaminase antibodies. Results: in our research work, 10 samples were positive (1.18 percent), which agrees with the reports of the reviewed literatura. Other 75 samples (8.8 percent) were not valid for the fist attempt, an event described by the test manufacturers, which can be easily solved by using the sample again and then analyzed for the second time; they changed to the caterogy of negative samples. Finally, 98.82 percent of the analyzed samples were negative. Conclusions: in the study samples, 1.18 percent had tissue antitransglutaminase antibodies, so roughly 1 per 100 children may suffer from celiac disease; additionally, this test is a useful tool for an early diagnosis that is not so uncommon in our country(AU)

Feasibility of using gelatin-microbial transglutaminase complex to repair experimental retinal detachment in rabbit eyes.

BACKGROUND: To investigate the effect of using gelatin-microbial transglutaminase (gelatin-mTG) complex for treating experimental retinal detachment. METHODS: Vitrectomy with artificial posterior vitreous detachment (PVD) followed by induction of a retinal tear and detachment was performed in rabbit eyes. Gelatin-mTG complex or gelatin alone (control) was placed on the retinal tears. Fundus examination using optical coherence tomography (OCT) was performed after the surgery. Vitrectomy with PVD alone was also performed in additional rabbits. After application of the gelatin-mTG complex on the normal retinal surface, the electroretinogram (ERG) was measured 7 days after surgery. RESULTS: Gelatin-mTG complex covered the retinal tear for more than 7 days after the vitrectomy, with less prominent inflammation. Reattachment of the retina occurred in all treated eyes. In contrast, massive fibrin materials were observed at 1 day after the surgery in the control group. In addition, OCT showed that all of the gelatin disappeared by day 3. Local retinal detachment remained in three of the eyes. As demonstrated by the ERG, gelatin-mTG complex had no harmful effects on retinal function. CONCLUSIONS: The results indicate that gelatin-mTG complex continues to adhere and seal retinal tears for at least several days after administration without any inflammatory reaction.

Tissue transglutaminase: a novel therapeutic target in cerebral amyloid angiopathy.

Accumulation of amyloid-ß (Aß) in brain vessel walls, known as cerebral amyloid angiopathy (CAA), plays a key role in Alzheimer's disease pathogenesis. CAA might result from impaired transport of Aß out of the brain. Although the mechanisms underlying reduced Aß transport are largely unknown, thickening of basement membrane extracellular matrix (ECM) is likely involved. Tissue transglutaminase (tTG) is an enzyme capable of modulating the ECM by covalently cross-linking ECM proteins. Recently, our group found that tTG and its cross-linking activity are associated with CAA pathology, suggesting a role for tTG in ECM modulation in CAA. Therefore, inhibition of tTG activity might be a promising novel therapeutic target to counteract CAA.

Anemia y enfermedad celiaca en una paciente con síndrome de Down / Anaemia and celiac disease in a patient with Down syndrome

Las personas con síndrome de Down (SD) tienen más probabilidades de presentar enfermedad celiaca (EC) que la población general. Se presenta un caso de EC que se manifiesta con anemia importante, en una chica adolescente con SD. Paciente de sexo femenino de 13 años de edad, con SD, que ingresa por anemia acompañada de astenia, anorexia y alteración del ritmo intestinal de unas 4 semanas de evolución. Tiene antecedentes de menstruaciones abundantes y algún vómito antes del inicio de la enfermedad actual. Al constatarse la palidez cutánea sele practica analítica y ésta indica una importante anemia, con cifra de hemoglobina de 4,7 g/dL, por lo que la paciente es derivada al hospital. La exploración física muestra una frecuencia cardiaca de 106xminuto,tensión arterial de 112/48 mmHg, palidez cutánea y de mucosas y soplo sistólico. La anemia es normocítica hipocroma y con ferropenia. Los estudios de imagen descartan patología hemorrágica gastrointestinal. El estudio de médula ósea también es normal. Se inicia tratamiento con sulfato ferroso por vía oral. Un mes después, la paciente vuelve a ingresar por dolor abdominal, vómitos y diarrea. Durante el ingreso se practican determinación de anticuerpos antiendomisio y antitransglutaminasa tisular, que resultan positivos. La biopsia intestinal confirma EC. Se instaura una dieta sin gluten y se mantiene el tratamiento con hierro, tras lo cual sigue una evolución favorable y recuperación dela anemia. Hay autores e instituciones que recomiendan la práctica de cribado de EC en personas asintomáticas con SD. En cualquier caso, en las que presentan sintomatología gastrointestinal o de otros tipos –como puede ser una anemia no explicable por otros motivos–, debe descartarse la posibilidad de EC (AU)

Down syndrome (DS) is associated with an increase drisk of celiac disease (CD) than that found ingeneral population. An adolescent girl with DS and CD presenting with severe anaemia is reported. A 13 year-old girl was admitted to hospital for anaemia and a 4 week-history of asthenia, anorexia, and disturbed bowel habit. Her past medical history was remarkable for hypermenorrhea and occasional vomiting. Heart rate was 106xminute and blood pressure112/48 mmHg. On physical exam she was pale and a systolic murmur was heard. Blood tests depicted a severe hypochromic normocytic anaemia with haemoglobin values of 4,7 g/dL Gastrointestinal bleeding was ruled out on the basis of several image studies and a bone marrow study was also normal. Iron supplement with ferrous sulphate was prescribed. A month later she was readmitted to hospital for abdominal pain, vomiting and diarrhoea. Serum endomysium antibodies and tissue transglutaminase antibodies were found to be positive and an intestinal biopsy confirmed the diagnosis of CD. She was started on a gluten-free diet and the iron supplement was maintained. She subsequently followed a favourable clinical course with cessation of gastrointestinal symptoms and correction of the manaemia. Several authors have suggested that people with DS should be routinely screened for CD even if they area symptomatic. Moreover, the existence of CD should be specially considered in people with DS who present with gastrointestinal symptoms or anaemia of unclear etiologies (AU)

Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease.

BACKGROUND & AIMS: Celiac disease is characterized by activation of HLA-DQ2/DQ8-restricted intestinal gluten-specific CD4(+) T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. METHODS: Enzyme modifications of the immunodominant alpha-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Wheat flour was treated with microbial transglutaminase and lysine methyl ester; gliadin was subsequently extracted, digested, and deamidated. Gliadin-specific intestinal T-cell lines (iTCLs) were generated from biopsy specimens from 12 adult patients with celiac disease and challenged in vitro with different antigen preparations. RESULTS: Tissue transglutaminase-mediated transamidation with lysine or lysine methyl ester of p56-68 or gliadin in alkaline conditions inhibited the interferon gamma expression in iTCLs; also, binding to DQ2 was reduced but not abolished, as suggested by in silico analysis. Lysine methyl ester was particularly effective in abrogating the activity of gliadin. Notably, a block in the response was observed when iTCLs were challenged with gliadin extracted from flour pretreated with microbial transglutaminase and lysine methyl ester. CONCLUSIONS: Transamidation of wheat flour with a food-grade enzyme and an appropriate amine donor can be used to block the T cell-mediated gliadin activity. Considering the crucial role of adaptive immunity in celiac disease, our findings highlight the potential of the proposed treatment to prevent cereal toxicity.

Molecular mechanisms responsible for the involvement of tissue transglutaminase in human diseases: Celiac Disease.

Tissue transglutaminase (tTG or TG2; E.C. 2.3.2.13) belongs to the transglutaminase family, a group of closely related enzymes that share the ability to catalyze the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. tTG is a multifunctional enzyme since it is also capable of catalyzing other biochemical reactions. The distribution and physiological roles of tTG have been widely studied in numerous cell types and tissues, but only recently its role in human diseases has started to be clarified. For example, transglutaminase activity has been hypothesized to be involved in the pathogenetic mechanisms responsible for several human diseases, including neurodegenerative diseases, such as polyglutamine diseases hitherto identified. Among human diseases, a large and recent series of studies have clearly shown that the activity of the tTG is critical for a very diffuse human pathology known as Celiac Disease. This disease is due to intolerance to a food component, gliadin, and is characterized by a very complex clinical syndrome, including gastrointestinal pathological manifestations, often associated with extra-intestinal manifestations. Interestingly, a subset of celiac patients also develops certain neurological disorders. In this review we describe the roles played by tTG in the molecular mechanisms responsible for pathophysiology of Celiac Disease.

In vivo transplantation of neonatal ovine neocartilage allografts: determining the effectiveness of tissue transglutaminase.

Following transplantation of ovine neocartilage allografts, 26 sheep were divided into groups according to the following weight-bearing schedule: 8-week nonweight bearing (8NWB, n=14), and 8-week nonweight bearing+4-week weight bearing (8NWB+4WB, n=12). In addition, 7 and 6 sheep, respectively, in the 8NWB and 8NWB+4WB groups received tTG treatment after allograft transplantation, whereas the remaining 13 sheep in these groups did not receive tTG. Finally, 8 sheep served as sham-operated controls without allograft transplantation. After euthanasia, stifle joints were harvested for the analysis of gross appearance, chondrocyte viability, histology, and biomechanical testing. No significant differences were noted in macroscopic graft survival and union with host tissue in both 8NWB and 8NWB+4WB groups between the tTG treated and non-tTG treated animals. Analysis of histological scores demonstrated no significant difference between tTG and non-tTG treatments in both 8NWB and 8NWB+4WB groups. Confocal laser microscopic analysis of the explanted defects revealed 70%-100% cell viability in all treatment groups. This study shows that allogeneic chondrocytes harvested from neonatal donors provide sufficient metabolic activity to affect repair. Use of tTG to augment resorbable suture fixation of neocartilage grafts provided no advantage over suture alone in this pilot study.

Transglutaminases: crosslinking enzymes with pleiotropic functions.

Blood coagulation, skin-barrier formation, hardening of the fertilization envelope, extracellular-matrix assembly and other important biological processes are dependent on the rapid generation of covalent crosslinks between proteins. These reactions--which are catalysed by transglutaminases--endow the resulting supramolecular structure with extra rigidity and resistance against proteolytic degradation. Some transglutaminases function as molecular switches in cytoskeletal scaffolding and modulate protein-protein interactions. Having knowledge of these enzymes is essential for understanding the aetiologies of diverse hereditary diseases of the blood and skin, and various autoimmune, inflammatory and degenerative conditions.

Transglutaminases: nature's biological glues.

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.

The role of plasma transglutaminase (F XIII) in wound healing of complicated pressure sores after spinal cord injury.

STUDY DESIGN: A case report. OBJECTIVES: To demonstrate stimulating action of F XIII in wound healing of complicated pressure sores. SETTING: A Spinal Cord Injury Center in Germany. METHODS: Clinical exam, clinical and photographic wound control, biochemical serum monitoring. RESULTS: Recurrent pressure sores in plegic patients are common complications requiring long-standing conservative or operative therapy. Additional risk factors such as diabetes increase the complication rate for surgery. Surgery itself may be difficult in recurrent pressure sores due to limited remaining soft tissues. We report the case and treatment of a 47-year-old patient with long-standing and recurrent ulcers and complications after flap surgery. As a final option we added plasma transglutaminase (factor XIII) to our treatment scheme which changed the course of the disease dramatically and we achieved complete and rapid healing. CONCLUSION: Our experience suggests that F XIII has a positive role in treating pressure sores as shown already in several other surgical fields. Its use is giving the surgeon an additional tool in complicated cases.

Maternal blood coagulation factor XIII is associated with the development of cytotrophoblastic shell.

We analysed the early implantation tissues of normal women and of a patient with congenital factor XIII deficiency in order to study the role of maternal subunit A of factor XIII (XIIIA) in the development of extravillous cytotrophoblast. The patient had received adequate administration of factor XIIIA concentrate only up to 7 weeks of gestation (wG). Her pregnancy was maintained until the latter half of 8 wG, but was terminated by intrauterine fetal death at 9 wG. Immunohistochemical staining of cytokeratin, XIIIA and subunit S of factor XIII was performed in the early implantation tissues of normal women and of this patient. Numerous well-formed cytotrophoblastic shells and Nitabuch's layers were detected in implantation tissues at 7-8 wG in normal women, and XIIIA was present in the intercellular space in well-formed cytotrophoblastic shells, while the cytotrophoblastic shells and Nitabuch's layers in this patient's implantation tissue were poorly-formed. Furthermore, XIIIA was not detected around them. It is suggested that when the maternal plasma activity of factor XIII is low, the concentration of XIIIA at the placental bed is also low, leading to the insufficient formation of cytotrophoblastic shell and therefore an increased probability of miscarriage in patients with congenital factor XIII deficiency.

Cultured epithelial autografts: diving from surgery into matrix biology.

Cultured epithelial autografts offer an exciting approach to cover extensive skin wounds. The main problem of this method is mechanical instability during the first weeks after grafting. There is evidence that the shortcomings of autografting cultured keratinocytes result from the lack of a mature and functional dermo-epidermal junction. This article summarizes the current knowledge regarding the de novo formation of the dermo-epidermal junction and the dynamics of "take" and stabilization of cultured epithelial autografts. Future strategies are discussed of how to improve and accelerate the process conferring definitive stabilization of cultured epithelial autografts including the potential therapeutic use of transglutaminase as well as cocultivation of a dermo-epidermal equivalent in order to facilitate a permanent skin replacement.

A new biological glue for cartilage-cartilage interfaces: tissue transglutaminase.

In this study, we used an in vitro model to test the capacity of tissue transglutaminase to increase the adhesive strength at a cartilage-cartilage interface. Full-thickness cartilage-bone cylinders were prepared from fresh adult bovine shoulder joints, and the superficial half of the hyaline cartilage was then removed to provide a plane surface. Tissue transglutaminase was applied to the freshly cut surface of one cylinder, and a calcium-chloride solution (to act as an activating agent) was applied to that of the other. The cartilage surfaces were immediately apposed, one on top of the other, and an eighty-gram weight was applied to the upper cylinder for ten minutes at 37 degrees Celsius under defined humidity conditions. A measured force was then applied transversely to the upper cylinder until it was displaced from the lower one (which was clamped in a holding device), and the force recorded at this point was taken as a measure of the adhesive strength achieved at the cartilage-cartilage interface. The adhesive strength increased linearly with an increasing concentration of tissue transglutaminase (0.25 to 2.75 milligrams per milliliter) and was enhanced by increasing the duration of incubation, but it was not influenced by the level of humidity. The adhesive strength was improved by as much as 40 per cent when the cartilage surfaces had been pretreated with chondroitinase AC or hyaluronidase to remove glycosaminoglycan chains of proteoglycans, which are largely responsible for the intrinsic anti-adhesive properties of cartilage.

Factor XIII in scleroderma: in vitro studies.

The administration of Factor XIII (FXIII) produces a beneficial effect on the skin lesions in about 50% of the treated patients with progressive systemic sclerosis (PSS). The effect of FXIII on various skin fibroblast functions (proliferation, attachment, biosynthetic activity and mechanical properties) was investigated in vitro using normal and PSS strains. In cell culture, most of the PSS fibroblast strains synthesized excessive amounts of collagen. Other cell functions such as adhesion to collagen I or III, to fibronectin, retraction of collagen lattices, proliferation in low serum concentration and degradation of newly synthesized collagen were not significantly different. The addition of FXIII (I U/ml) inhibited the synthesis of collagen by normal fibroblasts and reduced it in PSS fibroblasts to a level similar to that of normal fibroblasts. This effect was observed for cells cultured on plastic or in a collagen lattice. In the latter, an increased amount of collagen degradation was observed. No significant effect of FXIII on the other cell functions was noted. Excessive collagen production by PSS fibroblasts can be repressed by FXIII in vitro by at least two distinct mechanisms: a reduction of collagen synthesis and an increased degradation of the newly synthesized collagen.