Orthogonal organic and aqueous-based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry.

Imaging mass spectrometry (IMS) is an emergent and innovative approach for measuring the composition, abundance and regioselectivity of molecules within an investigated area of fixed dimension. Although providing unprecedented molecular information compared with conventional MS techniques, enhancement of protein signature by IMS is still necessary and challenging. This paper demonstrates the combination of conventional organic washes with an optimized aqueous-based buffer for tissue section preparation before matrix-assisted laser desorption/ionization (MALDI) IMS of proteins. Based on a 500 mM ammonium formate in water-acetonitrile (9:1; v/v, 0.1% trifluororacetic acid, 0.1% Triton) solution, this buffer wash has shown to significantly enhance protein signature by profiling and IMS (~fourfold) when used after organic washes (70% EtOH followed by 90% EtOH), improving the quality and number of ion images obtained from mouse kidney and a 14-day mouse fetus whole-body tissue sections, while maintaining a similar reproducibility with conventional tissue rinsing. Even if some protein losses were observed, the data mining has demonstrated that it was primarily low abundant signals and that the number of new peaks found is greater with the described procedure. The proposed buffer has thus demonstrated to be of high efficiency for tissue section preparation providing novel and complementary information for direct on-tissue MALDI analysis compared with solely conventional organic rinsing.

Mycophenolic acid formulations in adult renal transplantation - update on efficacy and tolerability.

The description more than 30 years ago of the role of de novo purine synthesis in T and B lymphocytes clonal proliferation opened the possibility for selective immunosuppression by targeting specific enzymatic pathways. Mycophenolic acid (MPA) blocks the key enzyme inosine monophosphate dehydrogenase and the production of guanosine nucleotides required for DNA synthesis. Two MPA formulations are currently used in clinical transplantation as part of the maintenance immunosuppressive regimen. Mycophenolate mofetil (MMF) was the first MPA agent to be approved for the prevention of acute rejection following renal transplantation, in combination with cyclosporine and steroids. Enteric-coated mycophenolate sodium (EC-MPS) is an alternative MPA formulation available in clinical transplantation. In this review, we will discuss the clinical trials that have evaluated the efficacy and safety of MPA in adult kidney transplantation for the prevention of acute rejection and their use in new combination regimens aiming at minimizing calcineurin inhibitor toxicity and chronic allograft nephropathy. We will also discuss MPA pharmacokinetics and the rationale for therapeutic drug monitoring in optimizing the balance between efficacy and safety in individual patients.

Ten years of lung transplantation in Switzerland: results of the Swiss Lung Transplant Registry.

Lung transplantation has evolved from an experimental procedure to a viable therapeutic option in many countries. In Switzerland, the first lung transplant was performed in November 1992, more than ten years after the first successful procedure world-wide. Thenceforward, a prospective national lung transplant registry was established, principally to enable quality control. The data of all patients transplanted in the two Swiss Lung Transplant centres Zurich University Hospital and Centre de Romandie (Geneva-Lausanne) were analysed. In 10 years 242 lung transplants have been performed. Underlying lung diseases were cystic fibrosis including bronchiectasis (32%), emphysema (32%), parenchymal disorders (19%), pulmonary hypertension (11%) and lymphangioleiomyomatosis (3%). There were only 3% redo procedures. The 1, 5 and 9 year survival rates were 77% (95% CI 72-82), 64% (95% CI 57-71) and 56% (95% CI 45-67), respectively. The 5 year survival rate of patients transplanted since 1998 was 72% (95% CI 64-80). Multivariate Cox regression analysis revealed that survival was significantly better in this group compared to those transplanted before 1998 (HR 0.44, 0.26-0.75). Patients aged 60 years and older (HR 5.67, 95% CI 2.50-12.89) and those with pulmonary hypertension (HR 2.01, 95% CI 1.10-3.65) had a significantly worse prognosis The most frequent causes of death were infections (29%), bronchiolitis obliterans syndrome (25%) and multiple organ failure (14%). The 10-year Swiss experience of lung transplantation compares favourably with the international data. The best results are obtained in cystic fibrosis, pulmonary emphysema and parenchymal disorders.

Dual transplant of marginal kidneys. Case report and review of the literature.

INTRODUCTION: Double transplantation is one possible answer to the shortage of donor organs. While each donor kidney would be unsuitable when considered as a single allograft, use of both kidneys should provide sufficient nephron mass for effective glomerular filtration. CASE REPORT: This is the first Swiss report of a dual adult transplant of marginal kidneys in a 46-year-old man, who was transplanted for the fourth time. Follow-up at 6 months is excellent without acute rejection. CONCLUSION: Recent analysis of dual marginal versus single ideal transplant outcomes, found a comparable 1-yr graft survival in both of the procedures. Long term results are still lacking and guidelines to decide between single, double or no transplantation are emerging.

Secretory IgA-mediated neutralization of Shigella flexneri prevents intestinal tissue destruction by down-regulating inflammatory circuits.

Shigella, a Gram-negative invasive enteropathogenic bacterium responsible for bacillary dysentery, causes the rupture, invasion, and inflammatory destruction of the human colonic mucosa. We explored the mechanisms of protection mediated by Shigella LPS-specific secretory IgA (SIgA), the major mucosal Ab induced upon natural infection. Bacteria, SIgA, or SIgA-S. flexneri immune complexes were administered into rabbit ligated intestinal loops containing a Peyer's patch. After 8 h, localizations of bacteria, SIgA, and SIgA-S. flexneri immune complexes were examined by immunohistochemistry and confocal microscopy imaging. We found that anti-Shigella LPS SIgA, mainly via immune exclusion, prevented Shigella-induced inflammation responsible for the destruction of the intestinal barrier. Besides this luminal trapping, a small proportion of SIgA-S. flexneri immune complexes were shown to enter the rabbit Peyer's patch and were internalized by dendritic cells of the subepithelial dome region. Local inflammatory status was analyzed by quantitative RT-PCR using newly designed primers for rabbit pro- and anti-inflammatory mediator genes. In Peyer's patches exposed to immune complexes, limited up-regulation of the expression of proinflammatory genes, including TNF-alpha, IL-6, Cox-2, and IFN-gamma, was observed, consistent with preserved morphology. In contrast, in Peyer's patches exposed to Shigella alone, high expression of the same mediators was measured, indicating that neutralizing SIgA dampens the proinflammatory properties of Shigella. These results show that in the form of immune complexes, SIgA guarantees both immune exclusion and neutralization of translocated bacteria, thus preserving the intestinal barrier integrity by preventing bacterial-induced inflammation. These findings add to the multiple facets of the noninflammatory properties of SIgA.

Kidney transplantation in patients with Fabry disease

Introduction and Aims: Fabry disease is an X-linked lysosomal storage disorder caused by absence or deficient activity of the lysosomal enzyme alpha-galactosidase A. Renal manifestations occur early in life in a significant proportion of children, in many women and in almost all men with Fabry disease. These manifestations ultimately progress to end-stage renal disease in nearly all males and in some female patients. Data on kidney transplantation in patients with Fabry disease who are receiving enzyme replacement therapy (ERT), however, are scarce. Methods: We examined the clinical characteristics of kidney transplant recipients (KTRs) in the Fabry Outcome Survey (FOS) - a European database of patients with Fabry disease that was established to monitor the safety and outcome of ERT. Results: Of the 752 patients enrolled in FOS up to October 2005, 34 (4.5%) were reported to be KTRs. The mean age of these 32 male and 2 female patients was 45 ± 9 years, the median time since the transplant was 9 years, the median estimated glomerular filtration rate (eGFR) was 46 mL/min/1.73 m2 and the median level of proteinuria was 180 mg/24 hours. ERT was well tolerated, with mild infusion-related reactions reported in only one patient. Amongst these patients, 53% were reported to have hypertension, 71% left ventricular hypertrophy, 27% cardiac valve disease and 27% arrhythmia. A total of 23 (68%) of the patients (1 female, 22 males) were receiving ERT with agalsidase alfa (Replagal; Shire Human Genetic Therapies, UK), with a median duration of treatment of 2.5 years. There were no differences in age or time since transplantation between treated and untreated patients. The median eGFRs were 46 and 49 mL/min/1.73 m2 and the median levels of proteinuria were 200 and 160 mg/24 hours, respectively. Conclusions: KTRs represent a significant minority of individuals enrolled in a large international registry of patients with Fabry disease (FOS). Approximately two-thirds of KTRs with Fabry disease enrolled in FOS receive ERT with agalsidase alfa, which is well tolerated. Comparison of treated and untreated patients has the potential to examine effects of ERT on the progression of renal and cardiovascular disease.

Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion.

Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA-) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.

Patients' spontaneous discourse on the importance of significant others in the course of transplantation: A qualitative study

Background: This study explores significant ones' implication before and after transplantation. Methods: Longitudinal semi-structured interviews were conducted in 64 patients awaiting all-organ transplantation. Among them, 58 patients spontaneously discussed the importance of their significant other in their daily support. Discourse analysis was applied. Findings: During the pre-transplantation period renal patients reported that significant others took part in dialysis treatment and participated to regimen adherence. After transplantation, quality of life improved and the couple dynamics returned to normal. Patients awaiting lung or heart transplantation were more heavily impaired. Significant others had to take over abandoned roles. After transplantation resuming normal life became gradually possible, but after one year either transplantation health benefits relieved physical, emotional and social loads, or complications maintained the level of stress on significant others. Discussion: Patients reported that significant others had to take over various responsibilities and were concerned about long-term stress that should be adequately supported.

Tacrolimus and cyclosporine have differential effects on the risk of development of bronchiolitis obliterans syndrome: results of a prospective, randomized international trial in lung transplantation.

BACKGROUND: Chronic lung allograft dysfunction, which manifests as bronchiolitis obliterans syndrome (BOS), is recognized as the primary cause of morbidity and mortality after lung transplantation. In this study we assessed the efficacy and safety of two de novo immunosuppression protocols to prevent BOS. METHODS: Our study approach was a multicenter, prospective, randomized (1:1) open-label superiority investigation of de novo tacrolimus vs cyclosporine, with both study arms given mycophenolate mofetil and prednisolone after lung transplantation. Cytolytic induction therapy was not employed. Patients were stratified at entry for cystic fibrosis. Primary outcome was incidence of BOS 3 years after transplant (intention-to-treat analysis). Secondary outcomes were survival and incidence of acute rejection, infection and other adverse events. RESULTS: Group demographic data were well matched: 110 of 124 tacrolimus vs 74 of 125 cyclosporine patients were treated per protocol (p < 0.01 by chi-square test). Cumulative incidence of BOS Grade ≥1 at 3 years was 11.6% (tacrolimus) vs 21.3% (cyclosporine) (cumulative incidence curves, p = 0.037 by Gray's test, pooled over strata). Univariate proportional sub-distribution hazards regression confirmed cyclosporine as a risk for BOS (HR 1.97, 95% CI 1.04 to 3.77, p = 0.039). Three-year cumulative incidence of acute rejection was 67.4% (tacrolimus) vs 74.9% (cyclosporine) (p = 0.118 by Gray's test). One- and 3-year survival rates were 84.6% and 78.7% (tacrolimus) vs 88.6% and 82.8% (cyclosporine) (p = 0.382 by log-rank test). Cumulative infection rates were similar (p = 0.91), but there was a trend toward new-onset renal failure with tacrolimus (p = 0.09). CONCLUSIONS: Compared with cyclosporine, de novo tacrolimus use was found to be associated with a significantly reduced risk for BOS Grade ≥1 at 3 years despite a similar rate of acute rejection. However, no survival advantage was detected.

Advances in tissue section preparation for MALDI imaging MS

Enriched by a decade of remarkable developments, matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has witnessed a phenomenal expansion. Initially introduced for the mapping of peptides and intact proteins from mammalian tissue sections, MALDI IMS applications now extend to a wide range of molecules including peptides, lipids, metabolites and xenobiotics. Technology and methodology are quickly evolving to push the limits of the technique forward. Within a short period of time, numerous protocols and concepts have been developed and introduced in tissue section preparation, nonexhaustively including in situ tissue chemistries and solvent-free matrix depositions. Considering the past progress and current capabilities, this Review aims to cover the different aspects and challenges of tissue section preparation for MALDI IMS.

Impact of reduced cerebral perfusion pressure on outcome after severe traumatic brain injury is dependent on brain tissue oxygen pressure

INTRODUCTION. Reduced cerebral perfusion pressure (CPP) may worsen secondary damage and outcome after severe traumatic brain injury (TBI), however the optimal management of CPP is still debated. STUDY HYPOTHESIS: We hypothesized that the impact of CPP on outcome is related to brain tissue oxygen tension (PbtO2) level and that reduced CPP may worsen TBI prognosis when it is associated with brain hypoxia. DESIGN. Retrospective analysis of prospective database. METHODS. We analyzed 103 patients with severe TBI who underwent continuous PbtO2 and CPP monitoring for an average of 5 days. For each patient, duration of reduced CPP (\60 mm Hg) and brain hypoxia (PbtO2\15 mm Hg for[30 min [1]) was calculated with linear interpolation method and the relationship between CPP and PbtO2 was analyzed with Pearson's linear correlation coefficient. Outcome at 30 days was assessed with the Glasgow Outcome Score (GOS), dichotomized as good (GOS 4-5) versus poor (GOS 1-3). Multivariable associations with outcome were analyzed with stepwise forward logistic regression. RESULTS. Reduced CPP (n=790 episodes; mean duration 10.2 ± 12.3 h) was observed in 75 (74%) patients and was frequently associated with brain hypoxia (46/75; 61%). Episodes where reduced CPP were associated with normal brain oxygen did not differ significantly between patients with poor versus those with good outcome (8.2 ± 8.3 vs. 6.5 ± 9.7 h; P=0.35). In contrast, time where reduced CPP occurred simultaneously with brain hypoxia was longer in patients with poor than in those with good outcome (3.3±7.4 vs. 0.8±2.3 h; P=0.02). Outcome was significantly worse in patients who had both reduced CPP and brain hypoxia (61% had GOS 1-3 vs. 17% in those with reduced CPP but no brain hypoxia; P\0.01). Patients in whom a positive CPP-PbtO2 correlation (r[0.3) was found also were more likely to have poor outcome (69 vs. 31% in patients with no CPP-PbtO2 correlation; P\0.01). Brain hypoxia was an independent risk factor of poor prognosis (odds ratio for favorable outcome of 0.89 [95% CI 0.79-1.00] per hour spent with a PbtO2\15 mm Hg; P=0.05, adjusted for CPP, age, GCS, Marshall CT and APACHE II). CONCLUSIONS. Low CPP may significantly worsen outcome after severe TBI when it is associated with brain tissue hypoxia. PbtO2-targeted management of CPP may optimize TBI therapy and improve outcome of head-injured patients.

Effects of immunosuppressive drugs on T helper cells subsets and potential to promote tolerance in a stringent experimental transplantation model.

To achieve the goal of sustained donor-specifi c transplantation (Tx) tolerance, research efforts are now focusing on therapies based on specifi c cell subsets with regulatory properties. We and others have previously highlighted the therapeutic potential of naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTreg) in promoting long-term graft acceptance. Using more stringent experimental Tx models, we were however confronted to limitations. Indeed, while the transfer of antigenspecifi c nTreg promoted long-term MHC-mismatched skin allograft acceptance in lymphopenic mice in the absence of any immunosuppressive drug, allograft survival was only slightly prolonged when nTreg were transferred alone into non-lymphopenic mice. This suggested that in more stringent conditions, adjuvant therapies may be needed to effectively control alloreactive T cells (Teff). Whether and how the expansion of the Treg pool could be best combined with current immunosuppressive regimens in clinical settings remains to be defi ned. In this study, we have used in vitro assays and an in vivo skin Tx model to investigate the effects of various immunosuppressive drugs on the survival, proliferation and effector function of Teff and nTreg in response to alloantigens. Teff proliferation was inhibited in a dose-dependent manner by rapamycin and cyclosporine A, while anti-CD154 mAb only marginally affected Teff survival, proliferation and effector fucntion in vitro. Rapamycin promoted apoptosis of Teff as compared to nTreg that were more resistant in the presence of IL-2. In vivo, the transfer and/or expansion of Treg could be advantageously combined with rapamycin and anti-CD154 mAb treatment to signifi cantly prolong MHC-mismatched skin allografts survival in non-lymphopenic recipients. Taken together our data indicate that immunosuppressive drugs differentially target T-cell subsets and that some regimens could promote Treg expansion while controlling the Teff pool in response to alloantigens.

Aganirsen Antisense Oligonucleotide Eye Drops Inhibit Keratitis-Induced Corneal Neovascularization and Reduce Need for Transplantation: The I-CAN Study.

OBJECTIVE: Eye drops of aganirsen, an antisense oligonucleotide preventing insulin receptor substrate-1 expression, inhibited corneal neovascularization in a previous dose-finding phase II study. We aimed to confirm these results in a phase III study and investigated a potential clinical benefit on visual acuity (VA), quality of life (QoL), and need for transplantation. DESIGN: Multicenter, double-masked, randomized, placebo-controlled phase III study. PARTICIPANTS: Analysis of 69 patients with keratitis-related progressive corneal neovascularization randomized to aganirsen (34 patients) or placebo (35 patients). Patients applied aganirsen eye drops (86 μg/day/eye) or placebo twice daily for 90 days and were followed up to day 180. MAIN OUTCOME MEASURES: The primary end point was VA. Secondary end points included area of pathologic corneal neovascularization, need for transplantation, risk of graft rejection, and QoL. RESULTS: Although no significant differences in VA scores between groups were observed, aganirsen significantly reduced the relative corneal neovascularization area after 90 days by 26.20% (P = 0.014). This improvement persisted after 180 days (26.67%, P = 0.012). Aganirsen tended to lower the transplantation need in the intent-to-treat (ITT) population at day 180 (P = 0.087). In patients with viral keratitis and central neovascularization, a significant reduction in transplantation need was achieved (P = 0.048). No significant differences between groups were observed in the risk of graft rejection. However, aganirsen tended to decrease this risk in patients with traumatic/viral keratitis (P = 0.162) at day 90. The QoL analyses revealed a significant improvement with aganirsen in composite and near activity subscores (P = 0.039 and 0.026, respectively) at day 90 in the per protocol population. Ocular and treatment-related treatment-emergent adverse events (TEAEs) were reported in a lower percentage with aganirsen compared with placebo. Only 3 serious TEAEs (2 with aganirsen and 1 with placebo) were considered treatment-related. CONCLUSIONS: This first phase III study on a topical inhibitor of corneal angiogenesis showed that aganirsen eye drops significantly inhibited corneal neovascularization in patients with keratitis. The need for transplantation was significantly reduced in patients with viral keratitis and central neovascularization. Topical application of aganirsen was safe and well tolerated.

Involvement of PPAR nuclear receptors in tissue injury and wound repair.

Tissue damage resulting from chemical, mechanical, and biological injury, or from interrupted blood flow and reperfusion, is often life threatening. The subsequent tissue response involves an intricate series of events including inflammation, oxidative stress, immune cell recruitment, and cell survival, proliferation, migration, and differentiation. In addition, fibrotic repair characterized by myofibroblast transdifferentiation and the deposition of ECM proteins is activated. Failure to initiate, maintain, or stop this repair program has dramatic consequences, such as cell death and associated tissue necrosis or carcinogenesis. In this sense, inflammation and oxidative stress, which are beneficial defense processes, can become harmful if they do not resolve in time. This repair program is largely based on rapid and specific changes in gene expression controlled by transcription factors that sense injury. PPARs are such factors and are activated by lipid mediators produced after wounding. Here we highlight advances in our understanding of PPAR action during tissue repair and discuss the potential for these nuclear receptors as therapeutic targets for tissue injury.