Epidemiology of small intestinal bacterial overgrowth

Small intestinal bacterial overgrowth(SIBO) is defined as an increase in the bacterial content of the small intestine above normal values. The presence of SIBO is detected in 33.8% of patients with gastroenterological complaints who underwent a breath test, and is significantly associated with smoking, bloating, abdominal pain, and anemia. Proton pump inhibitor therapy is a significant risk factor for SIBO. The risk of SIBO increases with age and does not depend on gender or race. SIBO complicat...     

Heart Rate Recovery After 6-min Walking Test Predicts Acute Exacerbation in COPD

Introduction

Abnormalities of autonomic function have been reported in patients with chronic obstructive pulmonary disease (COPD). Our objectives were to identify determinants of abnormal heart rate recovery at 1 min (HRR₁) following completion of the 6-min walk test (6MWT) in COPD and to establish whether abnormal HRR₁ predicts acute exacerbations (AECOPD).

Methods

Hundred one COPD patients (FEV₁ (SD) 53 (19)  % predicted) were prospectively recruited in a multi-center study. HRR₁ after the 6MWT was evaluated as the difference between heart rate at the end of the test and 1 min into the recovery (HRR₁). Linear and logistic regression was used to identify predictors of HRR₁ and AECOPD, respectively. The best HRR₁ cut-off point to predict AECOPD was selected using the receiver operating characteristics (ROC) curves. The follow-up period was 12 months.

Results

Distance covered during the 6MWT (m) and DLco (% predicted) were independently associated with HRR₁ (r ² = 0.51, p = 0.001). Among several potential covariates, HRR₁ emerged as the most significant predictor of AECOPD (Odds ratio [OR], 0.91 per beat of recovery; 95% confidence interval [CI], 0.85–0.97; p = 0.02). The ROC analysis indicated that subjects with HRR₁ less than 14 beats (AUC, 0.71 [CI] 0.60–0.80; p = 0.0001) were more likely to suffer an exacerbation during the follow-up period (for HRR₁, p = 0.004 [log-rank test]).

Conclusions

HRR₁ after the 6MWT is an independent predictor factor for AECOPD. Further studies are warranted to examine the physiological mechanisms associating a delayed HRR and acute exacerbations in COPD patients.

     

Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE^−/− mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Atherosclerotic plaque rupture is the leading cause of myocardial infarction and stroke (1, 2). Studies assessing the correlation between calcium scores and cardiovascular events have demonstrated a predictive power that is superior to and independent from that of lipid scores (3, 4). Additionally, clinical imaging studies have revealed that the risk of plaque rupture is further heightened by the presence of small, “spotty” calcifications, or microcalcifications (5, 6), and cardiovascular risk is inversely correlated with the size of calcific deposits, quantified as a calcium density score (7). Indeed, computational modeling has demonstrated that, while large calcifications can reinforce the fibrous cap (8), microcalcifications (typically 5 to 15 μm in diameter) uniquely mediate an increase in mechanical stress of the relatively soft, collagen-rich fibrous cap (9–12).Histologic studies have revealed the presence of cell-derived vesicles within calcifying atherosclerotic lesions (13–16). The inflammatory environment of the atherosclerotic lesion can induce vascular smooth muscle cells (vSMCs) to take on an osteochondrogenic phenotype and release calcifying extracellular vesicles (EVs) (17–19). Macrophages have also been shown to release procalcifying vesicles (20, 21). Thus, just as bone formation is hypothesized to be an active, cell-driven process (22, 23), mediated by calcifying matrix vesicles, atheroma-associated calcification may similarly be initiated by the production and aggregation of calcifying EVs (11, 20, 24–28).One proposed strategy for halting pathologic calcification has been the use of bisphosphonates (BiPs). BiPs are analogs of pyrophosphate (29), a naturally occurring compound derived in vivo from adenosine triphosphate (ATP) (30). Pyrophosphate binds to calcium phosphate and inhibits calcification via physicochemical mechanisms, namely, by blocking calcium and phosphate ions from forming crystals, preventing crystal aggregation, and preventing mineral transformation from amorphous calcium phosphate to hydroxyapatite (29). BiPs were identified as pyrophosphate analogs that, unlike pyrophosphate itself, resist enzymatic hydrolysis. A second, distinct property of BiPs is the ability to inhibit bone resorption via biological activity directed against osteoclasts following osteoclast endocytosis of the BiP molecule adsorbed to the surface of bone (29, 31). First-generation, or nonnitrogen-containing BiPs, are incorporated into nonhydrolyzable ATP analogs, and induce osteoclast apoptosis by limiting ATP-dependent enzymes. In contrast, nitrogen-containing BiPs inhibit farnesyl pyrophosphate synthetase and thereby induce osteoclast apoptosis (31).In vivo animal investigations have been performed to explore the potential for BiPs to inhibit cardiovascular calcification. Studies of first-generation BiPs revealed that the doses required to inhibit cardiovascular calcification also critically compromised normal bone mineralization (29, 32). However, newer, nitrogen-containing BiPs effectively arrested cardiovascular calcification in animal models at doses that did not compromise bone formation (32). Further, while it has been proposed that BiP treatment modifies cardiovascular calcification via its impact on bone-regulated circulating calcium and phosphate levels, a study in uremic rats demonstrated that BiP treatment inhibited medial aortic calcification with no significant change in plasma calcium and phosphate levels (33). The same study demonstrated that BiP treatment inhibited calcification of explanted rat aortas, indicating that BiPs can act directly on vascular tissue, independent of bone metabolism (33).Retrospective clinical data examining the effect of BiP therapy on cardiovascular calcification has demonstrated conflicting findings and intriguing paradoxes. In women with chronic kidney disease, BiP therapy decreased the mortality rate for patients without a prior history of cardiovascular disease (34), but for those patients with a history of prior cardiovascular events, BiP therapy was associated with an increased mortality rate (35). In another study, BiP therapy correlated with a lower rate of cardiovascular calcification in older patients (>65 y), but a greater rate in younger patients (<65 y) (36). These clinical findings motivated our study, in which we sought to further understand how BiP therapy impacts cardiovascular outcomes. Given that cardiovascular calcification, and especially the presence of microcalcification, is a strong and independent risk factor for adverse cardiac events, and BiPs are prescribed to modulate pathologies of mineralization, we hypothesize that BiPs modulate cardiovascular outcomes by altering the dynamics of cardiovascular calcification.EVs are smaller than the resolution limits of traditional microscopy techniques, hindering studies into the mechanisms of calcification nucleation and growth. We previously developed an in vitro collagen hydrogel platform that allowed the visualization of calcific mineral development mediated by EVs isolated from vSMCs (24). Using superresolution microscopy, confocal, and electron microscopy techniques, we showed that calcification requires the accumulation of EVs that aggregate and merge to build mineral. Collagen serves as a scaffold that promotes associations between EVs that spread into interfibrillar spaces. The resultant mineral that forms within the collagen hydrogel appears spectroscopically similar to microcalcifications in human tissues and allows the study of these structures on the time scale of 1 wk. In this study, we utilized this three-dimensional (3D) acellular platform to examine the direct effect of ibandronate, a nitrogen-containing BiP, on the EV-directed nucleation and growth of microcalcifications, a process that cannot be isolated from cellular and tissue-level mechanisms in a more complex, in vivo system. In parallel, we utilized a mouse model of atherosclerosis to assess the effect of ibandronate therapy on plaque-associated calcification, comparing mineral morphologies between the in vitro and in vivo samples. We hypothesize that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Understanding the EV-specific action of BiPs is imperative both to develop anticalcific therapeutics targeting EV mineralization and to understand one potential mechanism driving the cardiovascular impact of BiPs used in clinical settings.     

Corrosion of Steel Rebars in Anoxic Environments. Part I: Electrochemical Measurements

The number of reinforced concrete structures subject to anoxic conditions such as offshore platforms and geological storage facilities is growing steadily. This study explored the behaviour of embedded steel reinforcement corrosion under anoxic conditions in the presence of different chloride concentrations. Corrosion rate values were obtained by three electrochemical techniques: Linear polarization resistance, electrochemical impedance spectroscopy, and chronopotenciometry. The corrosion rate ceiling observed was 0.98 µA/cm², irrespective of the chloride content in the concrete. By means of an Evans diagram, it was possible to estimate the value of the cathodic Tafel constant (b_c) to be 180 mV dec⁻¹, and the current limit yielded an i_lim value of 0.98 µA/cm². On the other hand, the corrosion potential would lie most likely in the −900 mV_Ag/AgCl to −1000 mV_Ag/AgCl range, whilst the bounds for the most probable corrosion rate were 0.61 µA/cm² to 0.22 µA/cm². The experiments conducted revealed clear evidence of corrosion-induced pitting that will be assessed in subsequent research.     

Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αβ T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug–like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αβTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1–TRAJ6) that was conserved across donors. By trapping a CD1d–type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Natural killer T (NKT) cells are defined as T cells that are restricted to the lipid antigen-presenting molecule, CD1d. The most extensively studied are type I NKT cells, which typically express an invariant T cell receptor (TCR)-α chain consisting of TRAV10–TRAJ18 in humans (TRAV11–TRAJ18 in mice) paired with a constrained repertoire of TCR-β chains, enriched for TRBV25 in humans (TRBV13, 29, 1 in mice) (reviewed in ref. 1). Type I NKT cells are defined by their strong responses to α-galactosylceramide (α-GalCer) and structurally related hexosylceramides presented by CD1d, while in contrast, type II NKT cells are defined as CD1d-restricted T cells that express diverse TCRs and do not recognize α-GalCer (reviewed in refs. 1 and 2). Very little is known about the chemical identity of antigens for type II NKT cells; however, some studies suggest that these cells are abundant in humans (3–5), and, by virtue of their greater TCR diversity, they can interact with a broader range of antigens compared to type I NKT cells (2, 6–14).In 2004, a nonlipidic molecule, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), was described that stimulated a human TRAV10⁻ (type II) NKT cell line (clone ABd) in a CD1d-dependent manner (15). These observations were notable because PPBF resembles various sulfonamide drugs: furosemide (diuretic), sulfasalazine (disease-modifying antirheumatic), and celecoxib (anti-inflammatory) as well as “sulfa” antibiotics such as sulfonamide, sulfapyridine, sulfamethoxazole, sulfadiazine, and sulfadoxine (16). These drugs can cause systemic delayed-type hypersensitivity reactions, which are thought to be mediated by T cells (17–20). Most of our limited understanding of drug hypersensitivity reactions comes from work focusing on Human Leukocyte Antigen (HLA)-restricted conventional T cells, which has led to the proposal of four main mechanisms for small-drug immune activity as reviewed in ref. 21: 1) hapten/prohapten formation, whereby the drug reacts with self-Ags to generate a neo-product that undergoes processing and presentation to T cells; 2) noncovalent/labile pharmacological interaction with immune receptors on the cell surface; 3) superantigen mediating direct linkage of TCRs and Ag-presenting molecules; and 4) anchor site occupation by small molecules in Ag-presenting molecules inducing an altered self-Ag repertoire (22). Whether the CD1d-NKT cell axis is implicated in drug hypersensitivity remains unclear (23). Whereas most antigens in the CD1d system are lipids that use their alkyl chains to bind to CD1d, PPBF is a polycyclic small molecule and so would have to act through an atypical display mechanism. As previous attempts to stain the ABd clone with CD1d-PPBF tetramers were unsuccessful, the atypical drug-like structure of PPBF raised the possibility of direct pharmacological action on T cells rather than the presentation of CD1d-PPBF complexes to TCRs. However, the mechanism of PPBF-mediated type II NKT cell activation remains undefined (15).Here, using TCR-transduced cell lines, CD1d tetramers treated with PPBF, and new analogs in the pentamethylbenzofuransulfonate (PBF) family, we discovered that several molecules stimulate polyclonal NKT cells. Using CD1d tetramers treated with a newly identified and more potent analog of PPBF, we identified populations of type II NKT cells that comprise a polyclonal repertoire of both αβ and γδ T cells, including those with conserved TCR sequences. This enigmatic nature of T cell responses to PBF molecules was resolved using TCR trap technology (24, 25). Mass-spectrometric analysis of all molecules present in CD1d-PBF-TCR complexes indicates that CD1d binds PBFs and small self-lipids that promote CD1d-TCR binding. These data support a model of type II NKT cell recognition of small sulfa drug–like compounds in association with CD1d and flag a possible mechanism in which such cells may be involved in sulfa drug hypersensitivity.     

Comparison of combination therapies in the treatment of rheumatoid arthritis: leflunomide-anti-TNF-alpha versus methotrexate-anti-TNF-alpha

To compare the efficacy and safety of leflunomide (LEF)-anti-TNF-alpha combination therapy to methotrexate (MTX)-anti-TNF-alpha combination therapy in a group of patients with active rheumatoid arthritis (RA). We have recruited 120 patients with RA with a high disease activity despite being treated with MTX (15 mg/week) or LEF (20 mg/die) for 3 months, without side effects. In each of these patients, therapy with either MTX or LEF was continued and randomly combined with an anti-TNF-alpha drug: etanercept, infliximab, or adalimumab. Patients were assessed at study entry and at 4, 12, and at 24 weeks. The efficacy endpoints included variations in the DAS28-ESR and the ACR20, ACR50, and ACR70 responses. At each visit, any side-effect was recorded. There were no statistically significant differences in the DAS28 variations and in the ACR responses between the two groups or among the six subgroups. The number of discontinuation due to the appearance of serious side effects was higher, but not statistically significant, in the LEF-anti-TNF-alpha group than in the MTX-anti-TNF-alpha group. Other adverse events that did not necessitate the discontinuation of therapy occurred much more frequently in patients treated with MTX than in those treated with LEF. Anti-TNF-alpha drugs can be used in combination not only with MTX, but also with LEF, with the same probability of achieving significant clinical improvement in RA patients and without a significantly greater risk of serious adverse events. In contrast, it seems that combination therapy with LEF-anti-TNF-alpha is more readily tolerated than combination therapy with MTX-anti-TNF-alpha.     

Efficacy of traditional and biologic agents in different clinical phenotypes of adult‐onset Still's disease

Objective

To evaluate the efficacy of antiinflammatory agents, steroids, immunosuppressants, and biologic agents in patients with adult‐onset Still's disease (AOSD) who have either chronic articular disease or nonchronic disease.

Methods

Forty‐five patients with AOSD were seen and followed up for at least 2 years at our institution, from 1991 to 2008. The majority of patients were treated with several therapeutic regimens; a total of 152 efficacy trials were administered. Data regarding the type of medication, the dosage used, and the outcome of these trials were collected and analyzed.

Results

Our data showed that the efficacy of monotherapy with a nonsteroidal antiinflammatory drug was very low (16%) and confirmed good efficacy of steroid therapy (63%), particularly in patients without chronic articular disease (78%). Patients whose disease did not respond to steroid therapy at the time of disease onset were at risk of the subsequent development of chronic arthritis. Disease‐modifying antirheumatic drug (DMARD) monotherapy was successful in controlling steroid‐resistant or steroid‐dependent disease in 60% of patients. Methotrexate and cyclosporine showed the best response rates. The combination of high‐dose steroids and cyclosporine was administered to successfully control some acute life‐threatening complications. Only 6 patients had disease that was both steroid resistant and DMARD resistant. Treatment with biologic agents eventually led to satisfactory control of disease manifestations in 5 (83%) of these 6 patients.

Conclusion

Steroids were less effective in patients with chronic articular disease than in those with nonchronic disease. The administration of DMARDs early after disease onset could be beneficial in patients with steroid‐resistant disease who are at risk of the development of chronic articular disease. Biologic agents proved to be highly effective in both steroid‐resistant and DMARD‐resistant AOSD.