Modeling Dynamics of Human Gut Microbiota Derived from Gluten Metabolism: Obtention, Maintenance and Characterization of Complex Microbial Communities.

Western diets are rich in gluten-containing products, which are frequently poorly digested. The human large intestine harbors microorganisms able to metabolize undigested gluten fragments that have escaped digestion by human enzymatic activities. The aim of this work was obtaining and culturing complex human gut microbial communities derived from gluten metabolism to model the dynamics of healthy human large intestine microbiota associated with different gluten forms. For this purpose, stool samples from six healthy volunteers were inoculated in media containing predigested gluten or predigested gluten plus non-digested gluten. Passages were carried out every 24 h for 15 days in the same medium and community composition along time was studied via V3-V4 16S rDNA sequencing. Diverse microbial communities were successfully obtained. Moreover, communities were shown to be maintained in culture with stable composition for 14 days. Under non-digested gluten presence, communities were enriched in members of Bacillota, such as Lachnospiraceae, Clostridiaceae, Streptococcaceae, Peptoniphilaceae, Selenomonadaceae or Erysipelotrichaceae, and members of Actinomycetota, such as Bifidobacteriaceae and Eggerthellaceae. Contrarily, communities exposed to digested gluten were enriched in Pseudomonadota. Hence, this study shows a method for culture and stable maintenance of gut communities derived from gluten metabolism. This method enables the analysis of microbial metabolism of gluten in the gut from a community perspective.

The Human Digestive Tract Is Capable of Degrading Gluten from Birth.

The human gastrointestinal system has the capacity to metabolize dietary gluten. The capacity to degrade gliadin-derived peptide is present in humans from birth and increases during the first stages of life (up to 6-12 months of age). Fecal samples from 151 new-born and adult non-celiac disease (NCD) volunteers were collected, and glutenase and glianidase activities were evaluated. The capacity of total fecal proteins to metabolize 33-mer, 19-mer, and 13-mer gliadin peptides was also evaluated by high-performance liquid chromatography (HPLC). Feces from new-borns (meconium) showed glutenase and gliadinase activities, and peptidase activity against all three gliadin peptides. Maximal gluten degradative activity was observed in fecal samples from the youngest volunteers (0-12 months old). After the age of nine months, the gluten digestive capacity of gastrointestinal tract decreases and, from ±8 years old, individuals lose the ability to completely degrade toxic peptides. The gastrointestinal proteases involved in gluten digestion: elastase 2A, elastase 3B, and carboxipeptidase A1 are present from earlier stages of life. The human digestive tract contains the proteins capable of metabolizing gluten from birth, even before starting gluten intake. Humans are born with the ability to digest gluten and to completely degrade the potentially toxic gliadin-derived peptides (33-, 19-, and 13-mer).

Does an early mobilization and immediate home-based self-therapy exercise program displace proximal humeral fractures in conservative treatment? Observational study.

BACKGROUND: Nonoperative management of proximal humeral fractures (PHFs) is the most common treatment, but its functional outcome may improve with early mobilization. In frail osteoporotic patients, quick recovery of prefracture independency is mandatory. This study assessed fracture displacement in PHFs managed with conservative treatment after early mobilization and a home-based self-exercise program. METHODS: We retrospectively analyzed the radiologic displacement of fracture fragments of PHFs treated conservatively with early mobilization and a home-based self-exercise program. RESULTS: Included were 99 patients with 26 one-part, 32 two-part, 32 three-part, and 9 four-part PHFs managed conservatively, followed by early mobilization and a home-based self-exercise program. In the x-ray examinations, the head displaced from varus into valgus 55° ± 23° to 42° ± 22°, in the normal range of anatomic values. The medial hinge displaced from medial to the diaphysis (+1 ± 6 mm) to lateral to the head (-0.6 ± 6 mm). The greater tuberosity displaced cranially from -1 ± 7 mm to 2 ± 5 mm. The Constant score at the 1-year follow-up was 79.69 ± 16.3. DISCUSSION AND CONCLUSIONS: The home-based self-exercise program for conservative treatment of PHFs displaces the head-diaphysis angle and the medial hinge toward anatomic reduction, but there is a risk of greater tuberosity cranial displacement. Functional results are fairly good, allowing frail patients to keep on with their independency and life style. Because a large number of patients might need further physiotherapy, the quality of the home-based self-exercises should be supervised.

Factors influencing recurrence and progression of Dupuytren's disease treated by Collagenase Clostridium histolitycum.

AIM OF THE STUDY: The purpose of this study was to determine the recurrence rate, possible adverse reactions and factors influencing recurrence and progression of Dupuytren's disease (DD) treated with Collagenase from Clostridium histolyticum (CCH). METHOD: This was a prospective study of 71 patients with DD treated with CCH from 2011 to February 2013, with a minimum follow-up period of four years. Clinical, functional, patient satisfaction, drug safety and factors influencing recurrence and disease progression were evaluated. RESULTS: In all patients, the rupture of the cord was achieved after the injection, reducing joint contracture. In five patients (7%) we verified the existence of disease recurrence during the follow-up. In 11 patients (15.5%) there was a disease progression. Three patients have been surgically operated on, without added surgery difficulty; the rate of recurrence and progression was higher in grades III and IV of Tubiana, in proximal interphalangeal (PIP) punctures, and was earlier in patients younger than 60 years. DISCUSSION: No serious local complications or general complications were observed with this method. The recurrence of DD, following criteria of Felici, is mainly observed in young patients with greater severity of the disease and at the PIP level. Progression is influenced by the same factors. Patients operated on after recurrence have no added difficulty in the surgical technique, as it has also been published in other studies. CONCLUSIONS: Patients with the lowest rates of recurrence and progression were those with a single cord in the metacarpophalangeal (MCP), a grade II of Tubiana, and were older than 60 years.

The young patient and the medial unicompartmental knee replacement.

PURPOSE: Younger patients with unicompartmental degenerative knee joint diseases present a therapeutic dilemma. The purpose of this study is to evaluate the long-term results of the Oxford phase III unicompartmental knee replacement through a minimally invasive approach in the young patient. METHODS: We have studied a total of 51 patients (59 interventions) who underwent the Oxford Phase III Unicompartmental Knee Replacement. A clinical, radiographical and functional evaluation was performed twelve years after intervention. RESULTS: According to the American Knee Society Score using Insall's criteria overall results of unicompartmental knee arthroplasty showed an excellent or good outcome for 53 knees (96.36%), fair for 1 (1.81%) and poor for 1 (1.81%) in the postoperative long term. The survival rate of the implants was close to 95% at follow-up. CONCLUSIONS: Twelve years follow-up results of UKA through a minimally invasive exposure in young patients demonstrate predictably good outcomes.

Radial tunnel syndrome: results of surgical decompression by a postero-lateral approach.

PURPOSE: Our purpose is to describe the results obtained in surgical treatment of a series of patients with symptoms of radial tunnel syndrome. METHODS: We performed a prospective study on 42 patients (43 limbs) operated for radial tunnel syndrome between 1996 and 2010, using a posterior-external approach. RESULTS: Using the Roles and Maudsley criteria, 21 patients had excellent results (48.8%), 16 good (37.2%) and six fair results (13.9%). Most patients were satisfied with the surgery, reporting symptom relief and improved functionality. CONCLUSIONS: Radial tunnel syndrome consists of intermittent compression of the posterior interosseous nerve in the forearm, with pain and functional disability of the forearm, without motor or sensory electromyogram alterations. Because it is often confused with enthesitis of the epicondyle muscle insertions (an entity often occurring simultaneously), differential diagnosis is necessary with treatment-resistant epicondylitis. The most effective treatment is surgical, releasing all possible nerve compression sites.

Long-term results of the retrocapital metatarsal percutaneous osteotomy for hallux valgus.

PURPOSE: The current trend in hallux valgus surgery is directed toward percutaneous procedures. However, no evidence that any of these methods of treatment are superior to the others has been described, excepting studies in the long term. The aim of this study was to analyse a series of patients who had undergone a percutaneous distal retrocapital osteotomy of the first metatarsal, and had been followed up for ten years. METHODS: We carried out a clinical and radiological evaluation of 115 feet ten years after surgery. RESULTS: The AOFAS scale results in the tenth postoperative year remained significantly favourable compared to their corresponding values in the preoperative period, yielding an improvement of 42.2 points overall on average. In relation to radiological findings, the mean hallux angle was maintained below 20 °, with a mean intermetatarsal angle of 8.1 °. CONCLUSION: Percutaneous retrocapital metatarsal osteotomy for treatment of mild to moderate hallux valgus is effective in the long term, with the advantages of a minimally invasive procedure.

Oxford phase 3 unicondylar knee arthroplasty through a minimally invasive approach: long-term results.

PURPOSE: Surgical treatment options for medial compartment osteoarthritis of the knee include high tibial osteotomy, total knee arthroplasty or unicompartmental knee arthroplasty (UKA), depending on the patient's age, level of physical activity and the degree of deformity. METHODS: In this study, we evaluated the long-term results of patients who underwent the Oxford cemented meniscal-bearing unicondylar knee prosthesis through a minimally invasive approach including a clinical, functional and radiographic assessment. RESULTS: Favourable clinical and radiological outcomes were registered overall at ten years after surgery. Overall results of UKA according to the American Knee Society (AKS) using Insall's criteria showed an excellent or good outcome for 492 knees (96.28 %), fair for 11 (2.15 %) and poor for eight (1.57 %) in the post-operative long term. CONCLUSIONS: We believe that with appropriate surgical technique, patient selection, prosthetic design and specific training, surgeons should achieve good outcomes with the added advantages of a minimally invasive approach. High volume for this technique is important in our opinion.

Long-Term Results of Joint Arthroplasty with Total Prosthesis for Trapeziometacarpal Osteoarthritis in Patients over 65 Years of Age.

Trapeziometacarpal osteoarthritis (TMCOA) is a highly prevalent disease in the older population. Many different types of surgical treatments are possible, depending on the degree of joint involvement, the personal and professional circumstances of the patient and the preferences of the orthopedic surgeon. This paper evaluated the clinical and radiological results of consecutive cohorts of patients over 65 years old treated with total joint arthroplasties (TJA) of the ball and socket type (B&S) for TMCOA, with a minimum of 10 years follow-up. The survival rate (Kaplan-Meier) of the functional prostheses at 10 years was 92.2% (95% CI (89.1%, 96.1%). These functional arthroplasty patients, after 10 years of follow-up, showed little or no pain, good function and good key pinch, without radiological alterations. TJAs of the B&S type are a long lasting, effective and reliable alternative to surgical treatment of TMCOA in patients over 65 years of age, when they are performed with the patient selection criteria and surgical technique described throughout this study.

Total Knee Arthroplasty in Octogenarians: Should We Still Be so Restrictive?

Demand for total knee arthroplasty (TKA) in octogenarians will increase in subsequent years as society ages. We conducted a retrospective observational study in octogenarians operated on with TKA between 2015 and 2019, comparing preoperative and postoperative Knee Society Score (KSS), Knee Society Function Score (KSFS), extension and flexion balance, and radiologic alignment using a paired Student t-test. A chi-squared test was used to correlate mortality with Charlson comorbidities index score and with ASA scale. Kaplan-Meier analysis was performed to calculate patient survival. In this period 36 patients ≥80 years underwent TKA, with a mean age of 81.6 years. Of these, 24 patients (66.7%) were classified as ASA II and 12 (33.3%) as ASA III. Sixteen patients (44.4%) were Charlson 0, 14 (38.9%) Charlson 1, two (5.6%) Charlson 2, and four (11.1%) Charlson 3. KSS, KSFS, flexion and extension range, and radiologic alignment were statistically significant (p < 0.001) when comparing preoperatory and post-operatory data. No correlation (p > 0.05) was found between mortality and ASA or Charlson score. Seven patients (19.4%) suffered a medical complication and two patients experienced surgical complications. Four patient died (11.1%) during follow-up. The mean patient survival was 67.4 months. Patients ≥80 years achieve clinical improvement after TKA. Comorbidities, not age, are the burden for surgery in older patients.

Biosynthesis and production of polysialic acids in bacteria.

Polysialic acids (PA) are protective capsular sialohomopolymers present in some bacteria which can invade the mammalian host and cause lethal bacteremia and meningitis. Biosynthesis and translocation of PA to the cell surface are equivalent in different species and bacterial strains which are produced. The diversity in PA structure is derived from the PA linkages and is a consequence of the specific sialyltransferase activities. The monomer acetylation and the polymer length could be important factors in the potential virulence. In vivo PA production is affected by different physical and chemical factors. The temperature of cellular growth strictly regulates PA genesis through a molecular complex and multifactorial mechanism that operate to transcription level.

Three-sectioning method: A procedure for studying hard tissues and large pieces under light and electron microscopy.

The histological study of hard pieces such as tendons and calcified lesions and tissues is a field that has been gaining increased attention owing to the rapid development of implantable prostheses, among other factors. In these studies, serial sectioning is utilized to detect areas of interest throughout the entire piece, as it enables the application of the appropriate light and electron microscopy techniques in these areas. We propose the "three-sectioning method" that subjects the pieces to three consecutive cycles of embedding and sectioning to localize and study the areas of interest, as an efficient technique for these histological studies. The pieces were first embedded in epoxy resin and then cut into thick sections (approximately 300 µm) for the first cycle. Next, areas of interest selected on these thick sections were re-embedded in epoxy resin to be sectioned again (second sectioning) to obtain a series of semithin sections (1-3 µm). These semithin sections are usually studied using the most relevant techniques for light microscopy. Smaller areas of interest are selected to be cut into ultrathin sections (60-90 nm) for transmission electron microscopy. If necessary, the selected areas of the semithin sections can be embedded again, and then cut into new ultrathin sections. The different kinds of sections we have described here may also be studied using scanning electron microscopy. This systematic method facilitates correlative microscopy from lower to higher magnifications along with the usage of a broad variety of histological techniques including electron microscopy.

Temperature has reciprocal effects on colanic acid and polysialic acid biosynthesis in E. coli K92.

Escherichia coli K92 is an opportunistic pathogen bacterium able to produce polysialic acid (PA) capsules when grows at 37 degrees C. PA polysaccharides are cell-associated homopolymers tailored from acid sialic monomers that function as virulence factors in different neuroinvasive diseases caused by certain Enterobacteriaceae. Conversely, when grows at 19 degrees C (restrictive conditions), PA synthesis was negligible, whereas in such condition, a slimy substance started to be accumulated in the culture broths. Analysis by uronic acids colorimetric determinations, gas chromatography-mass spectrometry, and Fourier transform infrared spectroscopy allowed the isolation and identification of mucoid substance as colanic acid (CA). CA is a heteropolymer containing glucose, galactose, fucose, and glucuronic acid as monomers which seems to be involved in the protection of this bacterium against environment assaults. The study of physicochemical conditions required for CA synthesis revealed that in E. coli K92, nutrient (carbon and nitrogen sources) modulates CA production, reaching the maximal values when glucose and proline were as carbon and nitrogen sources, respectively. Furthermore, we have found that E. coli K92 is able to produce CA at all temperatures tested (from 42 degrees C to 15 degrees C), whereas PA synthesis only occurred when bacteria were cultured at temperatures higher than 25 degrees C. Additionally, genetic engineering approaches revealed that the CA cluster including several genes required for synthesis was placed into a DNA fragment of 100 kb using polymerase chain reaction methodology.

The role of RcsA in the adaptation and survival of Escherichia coli K92.

The Rcs phosphorelay is a two-component signal transduction system that senses stressful environmental signals such as desiccation or low temperatures, which serve as natural inducers in bacteria. RcsA is an important coregulator in this system involved in some functions regulated by the Rcs system, including biofilm formation and capsule synthesis. In this sense, we previously showed that RcsA is necessary for colanic acid synthesis in Escherichia coli K92. Here, using an E. coli K92ΔrcsA mutant lacking rcsA gene we further characterize the implications of RcsA on E. coli K92 survival under osmotic and oxidative stressful conditions, and bacterial attachment and biofilm formation on both biotic and abiotic surfaces. Our results show that RcsA protects E. coli K92 against osmotic and, especially, oxidative stress at low temperatures. In addition, RcsA did not interfere in biofilm formation in any surface tested, including polystyrene, stainless steel, silicone, Teflon, aluminum and glass. By contrast, deletion of rcsA increased bacterial attachment to the caco-2 cells monolayer used as biotic surface.

Effect of weight loss on bone metabolism in postmenopausal obese women with osteoarthritis.

BACKGROUND: The choice of hypocaloric diets in obesity can affect bone health. AIMS: The aim of this study is to assess the effect of a hypocaloric diet in postmenopausal obese women and to determine the influence of weight reduction on bone metabolism. METHODS: This was a non-randomised, single-treatment study in 96 postmenopausal women with a body mass index (BMI) greater than 35kg/m2 and osteoarthritis. The patients received a formula diet with two intake levels of a normocaloric hyperproteic formula (1035kcal (25% protein)). Anthropometry and biochemistry with CrossLaps, osteocalcin, parathyroid hormone (PTH) and 25-OH vitamin D were measured. Consumption of protein, calcium and vitamin D were determined at the beginning of and 3 and 6 months into the study. The response to treatment was compared (high-responder (HR): weight loss greater than 15%, and low-responder (LR): weight loss less than 15%). RESULTS: The mean age was 64.2 (7.5) years. After 6 months of treatment, a weight loss of 10.2% (8.2-13.8) was observed. There was a significant increase in vitamin D (HR: 21.8% (36.2) vs. LR: 22.7% (36.9), p=0.93) and CrossLaps (HR: 26.8% (19.5-35.2)) vs. LR: 13.3% (-6.1 to 27.9), p=0.01). The loss of more than 15% of initial body weight was an independent risk factor for an increase in CrossLaps (OR: 4.22 (1.1-16.8), p=0.04). CONCLUSIONS: In postmenopausal obese women, weight loss was associated with an increase in the biochemical parameters of bone resorption. The increase in resorption parameters was related to the magnitude of weight loss.

Influence of a meal-replacement diet on quality of life in women with obesity and knee osteoarthritis before orthopedic surgery.

BACKGROUND: Knee osteoarthritis is a disease with a high prevalence in our environment, especially in women. Weight loss can improve the quality of life of these patients before surgery. OBJECTIVES: To evaluate the effect of a meal-replacement diet on weight loss, body composition, and the improvement of the quality of life in obese women with knee osteoarthritis pending surgery. METHODS: One branch intervention study was performed over three months on 81 women with a body mass index greater than 30 kg/m2 with knee osteoarthritis before surgery. Patients received a hyperproteic meal-replacement diet with two bottles of an oral nutrition supplement in lunch and dinner (1,035 kcal). Anthropometric parameters, and body composition were measured. The quality of life was assessed by WOMAC and SF-36 test. RESULTS: The mean age of the patients was 62.23 (8.50) years. The percentage of weight loss was 8.23% (4.04). An improvement in the SF-36 total score was observed (basal: 49.35 [20.41], three months: 58.71 [17.07], p < 0.01). There was an improvement in WOMAC test (basal: 49.24% [25.53], three months: 40.59% [21.76], p < 0.01). It was observed that a 10% improvement in the SF-36 test was independently related to weight loss (OR: 1.2 [1.03-1.36], p < 0.02) adjusted by age and changes in body composition. CONCLUSIONS: In women with osteoarthritis of the knee treated with a meal-replacement diet, there is a significant decrease in weight and fat mass with a relative increase of the latter. There is an improvement in the quality of life according to SF-36 and WOMAC. There is an independent relationship between weight loss and SF-36 improvement.

Purification and characterization of GlcNAc-6-P 2-epimerase from Escherichia coli K92.

N-Acetylmannosamine (ManNAc) is the first committed intermediate in sialic acid metabolism. Thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. In prokaryotic organisms, UDP-N-acetylglucosamine (GlcNAc) 2-epimerase and GlcNAc-6-P 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc-6-P, respectively. We have purified for the first time native GlcNAc-6-P 2-epimerase from bacterial source to apparent homogeneity (1 200 fold) using Butyl-agarose, DEAE-FPLC and Mannose-6-P-agarose chromatography. By SDS/PAGE the pure enzyme showed a molecular mass of 38.4 +/- 0.2 kDa. The maximum activity was achieved at pH 7.8 and 37 degrees C. Under these conditions, the K(m) calculated for GlcNAc-6-P was 1.5 mM. The 2-epimerase activity was activated by Na(+) and inhibited by mannose-6-P but not mannose-1-P. Genetic analysis revealed high homology with bacterial isomerases. GlcNAc-6-P 2-epimerase from E. coli K92 is a ManNAc-inducible protein and is detected from the early logarithmic phase of growth. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes the biosynthesis of sialic acid, GlcNAc-6-P 2-epimerase plays a catabolic role. When E. coli grows using ManNAc as a carbon source, this enzyme converts the intracellular ManNAc-6-P generated into GlcNAc-6-P, diverting the metabolic flux of ManNAc to GlcNAc.

The human digestive tract has proteases capable of gluten hydrolysis.

OBJECTIVE: To identify, purify, and characterize the proteins responsible for glutenase activity in the feces of healthy subjects and patients with celiac disease (CD). METHODS: Sixteen subjects were included in this study; 8 were healthy with no known food intolerances, and 8 were treated CD patients on a gluten-free diet. Fecal samples were homogenized, and precipitated proteins were purified by chromatography. Glutenase activity was evaluated by bioassays, zymography, and high-performance liquid chromatography with immunogenic 33-mer, 19-mer, and 13-mer gliadin peptides. RESULTS: The gastrointestinal elastase 3B (CEL3B), elastase 2A (CEL2A), and carboxypeptidase A1 (CBPA1) enzymes degraded human gluten. These proteins fully hydrolyzed 13-mer and 19-mer gliadin peptides that trigger immune-mediated enteropathy in individuals genetically predisposed to CD and partially digested a 33-mer. Feces from patients with CD showed more glutenase activity than feces from individuals without CD (171-466% higher). Peptidase activity against the gliadin peptides also increased in patients with CD. CONCLUSION: The digestive tracts of patients with CD and healthy subjects have enzymatic machinery needed for gluten degradation. Patients with CD showed more gluten hydrolysis than did healthy individuals, although, in both cases, a fraction of 33-mer peptide remained intact. Gliadin peptides derived from gastrointestinal digestion, especially the 33-mer, can potentially be used by commensal microbiota from both CD-positive and CD-negative individuals, and differences in bacterial hydrolysis can modify its immunogenic capacity.

Intervertebral Disc Repair by Allogeneic Mesenchymal Bone Marrow Cells: A Randomized Controlled Trial.

BACKGROUND: Degenerative disc disease often causes severe low-back pain, a public health problem with huge economic and life quality impact. Chronic cases often require surgery, which may lead to biomechanical problems and accelerated degeneration of the adjacent segments. Autologous mesenchymal stromal cells (MSC) treatments have shown feasibility, safety and strong indications of clinical efficacy. We present here a randomized, controlled trial using allogeneic MSC, which are logistically more convenient than autologous cells. METHODS: We randomized 24 patients with chronic back pain diagnosed with lumbar disk degeneration and unresponsive to conservative treatments into 2 groups. The test group received allogeneic bone marrow MSCs by intradiscal injection of 25 × 10 cells per segment under local anesthesia. The control group received a sham infiltration of paravertebral musculature with the anesthetic. Clinical outcomes were followed up for 1 year and included evaluation of pain, disability, and quality of life. Disc quality was followed up by magnetic resonance imaging. RESULTS: Feasibility and safety were confirmed and indications of clinical efficacy were identified. MSC-treated patients displayed a quick and significant improvement in algofunctional indices versus the controls. This improvement seemed restricted to a group of responders that included 40% of the cohort. Degeneration, quantified by Pfirrmann grading, improved in the MSC-treated patients and worsened in the controls. CONCLUSIONS: Allogeneic MSC therapy may be a valid alternative for the treatment of degenerative disc disease that is more logistically convenient than the autologous MSC treatment. The intervention is simple, does not require surgery, provides pain relief, and significantly improves disc quality.