Multiphoton morpho-functional imaging of healthy colon mucosa,adenomatous polyp and adenocarcinoma

Two-photon spectral resolved imaging was used to image fresh human biopsies of colon tissue and to characterize healthy colon mucosa, adenomatous polyp and adenocarcinoma by means of a morpho-functional analysis. Morphological examination, performed using endogenous tissue fluorescence, discriminated adenomatous and adenocarcinoma tissues from normal mucosa in terms of cellular asymmetry and nucleus-to-cytoplasm ratio. Good agreement was found between multiphoton images and histological examination performed on the same samples. Further characterization, performed by means of spectral-resolved analysis of NADH and FAD fluorescence, demonstrated an altered metabolic activity in both adenomatous and adenocarcinoma tissues compared to healthy mucosa. This morpho-functional approach may represent a powerful method to be used in combination with endoscopy for in vivo optical diagnosis of colon cancer and may be extended to other tissues.OCIS codes: (180.4315) Nonlinear microscopy, (170.3880) Medical and biological imaging     

Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation

LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.LPS is a glycolipid located in the outer membrane of Gram-negative bacteria. It is composed of three covalently linked domains: lipid A, which is embedded in the outer membrane; the oligosaccharide core; and the O-polysaccharide or O-antigen, which cover the bacterial surface. During infections sustained by Gram-negative bacteria, detection of LPS initiates an acute inflammatory response as LPS, mainly by the lipid A, which is the real pathogen-associated molecular pattern (PAMP), is sensed by the innate immune system, through the binding to the pattern recognition receptor (PRR) complex of myeloid differentiation protein 2 (MD-2) and Toll-like receptor (TLR) 4 (TLR4) (1–3). The downstream effects of LPS recognition elicit effector mechanisms aimed at pathogen eradication. However, LPS can also elicit an host reaction because it is a major mediator of pathologic processes (4). The strength of the innate immune response to LPS can be modulated by its chemical structure; specifically, a fine tuning of the lipid A structure can significantly affect the immunostimulatory properties of the whole LPS molecule (5, 6). There is a strong correlation between the number of acyl chains of lipid A and the immunological response via the TLR4 pathway. In general, hexaacylated lipid A species are agonists, whereas tetraacylated species are antagonists with a weak inflammatory potential (7). Gram-negative bacteria can synthesize a range of differentially acylated LPSs as a result of the LPS biosynthesis. Changes in lipid A acylation underlie the adaptation of pathogens to different hosts, such as Yersinia pestis (8), or to different phases of pathogenesis such as Salmonella typhimurium (9) or in the establishment of chronic infection such as Pseudomonas aeruginosa (10, 11).Shigella flexneri is a Gram-negative pathogen that infects humans. The ingestion of as few as 100 bacteria is sufficient to cause bacillary dysentery, a severe rectocolitis caused by the dramatic inflammatory reaction induced by Shigella invasion on the colonic and rectal mucosa (12). Shigella enters epithelial cells by injecting effectors via a type III secretion system (T3SS) (13), escapes from the phagocytic vacuole, and actively proliferates within the cytosol of infected cells (14, 15). Bacterial proliferation is a potent signal to initiate inflammation because intracellular shigellae activate NF-κB following recognition of peptidoglycan (PGN) by the PRR Nod1, leading to IL-8 production (16, 17). IL-8 attracts neutrophils that are required for the clearance of shigellae, but also participates in epithelial barrier destruction (18). In macrophages, Shigella is able to trigger the assembly of the inflammasome, an important defense mechanism that is part of the innate immune system (19). The inflammasome is a multiprotein complex that mediates activation of caspase-1, which promotes the secretion of the proinflammatory cytokines IL-1β and IL-18 as well as a cell death process called pyroptosis (20, 21). Different PRRs, i.e., TLRs and nucleotide-binding oligomerization domain-like receptors (NLRs) contribute to the inflammasome assembly (22). In Shigella-infected macrophages, the activation of the NLRC4-mediated inflammasome triggers cell death and release of IL-1β and IL-18 (19, 23). Indeed, production of IL-1β is a paradigm of shigellosis: the chief role of this cytokine has been highlighted in vivo in several studies (24–26).In tissues of animals and in ex vivo human samples infected with Shigella (27), a huge amount of LPS is usually observed, reflecting the presence of living bacteria and/or of processed molecules. However, whether, how, and at to what extent this mass of LPS present in Shigella-infected tissues could play a role in the inflammation remains largely unknown.In 2002, D’Hauteville et al. reported that, in S. flexneri, the lack of msbB genes, msbB1 and msbB2, both encoding the enzyme myristoyl transferase, reduces lipid A acylation degree along with TNF-α production and epithelial lining inflammatory destruction in a rabbit model of Shigella infection (28, 29). This study suggests that LPS composition can greatly influence the degree of inflammation induced by Shigella.In line with these issues, here we intend to contribute to the understanding of the role played by LPS in Shigella pathogenesis. Hence, we addressed the question of whether Shigella could adapt the LPS structure to the host thereby exploiting the mechanism of LPS modification to hijack the innate immune response. With this aim, we extracted, purified, and analyzed the LPS of shigellae resident in epithelial cells. We detailed the immunopotential of this structure and compared it to that of conventionally grown bacteria. Together our results point to a key role for LPS during the Shigella invasive process.We report that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria conventionally grown (aLPS) differ in the number of acyl chains in lipid A, with iLPS being hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of aLPS; (iv) both LPS forms signal mainly through the TLR4/MyD88 pathway; (v) iLPS influences the inflammasome-mediated production of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime PMNs for an oxidative burst.     

Microbiota and Metabolomic Patterns in the Breast Milk of Subjects with Celiac Disease on a Gluten-Free Diet

The intestinal microbiome may trigger celiac disease (CD) in individuals with a genetic disposition when exposed to dietary gluten. Research demonstrates that nutrition during infancy is crucial to the intestinal microbiome engraftment. Very few studies to date have focused on the breast milk composition of subjects with a history of CD on a gluten-free diet. Here, we utilize a multi-omics approach with shotgun metagenomics to analyze the breast milk microbiome integrated with metabolome profiling of 36 subjects, 20 with CD on a gluten-free diet and 16 healthy controls. These analyses identified significant differences in bacterial and viral species/strains and functional pathways but no difference in metabolite abundance. Specifically, three bacterial strains with increased abundance were identified in subjects with CD on a gluten-free diet of which one (Rothia mucilaginosa) has been previously linked to autoimmune conditions. We also identified five pathways with increased abundance in subjects with CD on a gluten-free diet. We additionally found four bacterial and two viral species/strains with increased abundance in healthy controls. Overall, the differences observed in bacterial and viral species/strains and in functional pathways observed in our analysis may influence microbiome engraftment in neonates, which may impact their future clinical outcomes.     

Hematological improvement during iron-chelation therapy in myelodysplastic syndromes: The experience of the “Rete Ematologica Lombarda”

To analyze the unpredicted event of hematological improvement (HI) during iron-chelation therapy (ICT), we reviewed a series of 53 myelodysplastic patients with transfusion dependency in a retrospective study involving 8 centers afferent to the “Rete Ematologica Lombarda”. According to the IWG response criteria published in the year 2000, we observed erythroid responses in 19 patients (35.1%), 5 major (9.2%) and 14 minor (25.9%). In the assessable patients, platelet response was 8/13 (61%) and neutrophil response was 13/17 (76.4%). Only in patients with erythroid improvement, multilineage responses were observed. Apparently, patients with greater erythropoiesis dysfunction may take more advantage.     

Amyotrophic lateral sclerosis-associated mutant VAPBP56S perturbs calcium homeostasis to disrupt axonal transport of mitochondria

A proline-to-serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes some dominantly inherited familial forms of motor neuron disease, including amyotrophic lateral sclerosis (ALS) type-8. Here, we show that expression of ALS mutant VAPBP56S but not wild-type VAPB in neurons selectively disrupts anterograde axonal transport of mitochondria. VAPBP56S-induced disruption of mitochondrial transport involved reductions in the frequency, velocity and persistence of anterograde mitochondrial movement. Anterograde axonal transport of mitochondria is mediated by the microtubule-based molecular motor kinesin-1. Attachment of kinesin-1 to mitochondria involves the outer mitochondrial membrane protein mitochondrial Rho GTPase-1 (Miro1) which acts as a sensor for cytosolic calcium levels ([Ca(2+)]c); elevated [Ca(2+)]c disrupts mitochondrial transport via an effect on Miro1. To gain insight into the mechanisms underlying the VAPBP56S effect on mitochondrial transport, we monitored [Ca(2+)]c levels in VAPBP56S-expressing neurons. Expression of VAPBP56S but not VAPB increased resting [Ca(2+)]c and this was associated with a reduction in the amounts of tubulin but not kinesin-1 that were associated with Miro1. Moreover, expression of a Ca(2+) insensitive mutant of Miro1 rescued defective mitochondrial axonal transport and restored the amounts of tubulin associated with the Miro1/kinesin-1 complex to normal in VAPBP56S-expressing cells. Our results suggest that ALS mutant VAPBP56S perturbs anterograde mitochondrial axonal transport by disrupting Ca(2+) homeostasis and effecting the Miro1/kinesin-1 interaction with tubulin.     

Calsyntenin-1 mediates axonal transport of the amyloid precursor protein and regulates Aβ production

Understanding the mechanisms that control processing of the amyloid precursor protein (APP) to produce amyloid-β (Aβ) peptide represents a key area of Alzheimer's disease research. Here, we show that siRNA-mediated loss of calsyntenin-1 in cultured neurons alters APP processing to increase production of Aβ. We also show that calsyntenin-1 is reduced in Alzheimer's disease brains and that the extent of this reduction correlates with increased Aβ levels. Calsyntenin-1 is a ligand for kinesin-1 light chains and APP is transported through axons on kinesin-1 molecular motors. Defects in axonal transport are an early pathological feature in Alzheimer's disease and defective APP transport is known to increase Aβ production. We show that calsyntenin-1 and APP are co-transported through axons and that siRNA-induced loss of calsyntenin-1 markedly disrupts axonal transport of APP. Thus, perturbation to axonal transport of APP on calsyntenin-1 containing carriers induces alterations to APP processing that increase production of Aβ. Together, our findings suggest that disruption of calsyntenin-1-associated axonal transport of APP is a pathogenic mechanism in Alzheimer's disease.     

Near-miss events are really missed! Reflections on incident reporting in a department of pediatric surgery

Purpose

The aim of this study was to evaluate the frequency of surgical and organizational events that occurred in the whole Department of Paediatric Surgery at Gaslini Children’s Hospital through an incident-reporting system in order to identify the vulnerabilities of this system and improve it.

Materials and methods

This is a 6-month prospective observational study (1st January–1st July 2010) of all events (including surgical and organizational events, and near misses) that occurred in our department of surgery (pediatric surgery, orthopedics and neurosurgery units).

Results

Over a 6-month study period, 3,635 children were admitted: 1,904 out of 3,635 (52.4%) children underwent a surgical procedure. A total number of 111 adverse events and 4 near misses were recorded in 100 patients. A total of 108 (97.3%) adverse events occurred following a surgical procedure. Of 111 adverse events, 34 (30.6%) required re-intervention. Eighteen of 100 patients (18%) required a re-admission, and 18 of 111 adverse events (16.2%) were classified as organizational. Infection represented the most common event.

Conclusions

An electronic physician-reported event tracking system should be incorporated into all surgery departments to report more accurately adverse events and near misses. In this system, all definitions must be standardized and near misses should be considered as important as the other events, being a rich source of learning.     

Overcoming a “Probable” Diagnosis in Antimitochondrial Antibody Negative Primary Biliary Cirrhosis: Study of 100 Sera and Review of the Literature

Serum anti-mitochondrial antibodies (AMA) are the serological hallmark of primary biliary cirrhosis (PBC), yet up to 15% of PBC sera are AMA negative at routine indirect immunofluorescence (IIF) while being referred to as ??probable?? cases. The diagnostic role of PBC-specific antinuclear antibodies (ANA) remains to be determined. We will report herein data on the accuracy of new laboratory tools for AMA and PBC-specific ANA in a large series of PBC sera that were AMA-negative at IIF. We will also provide a discussion of the history and current status of AMA detection methods. We included IIF AMA-negative PBC sera (n?=?100) and sera from patients with other chronic liver diseases (n?=?104) that had been independently tested for IIF AMA and ANA; sera were blindly tested with an ELISA PBC screening test including two ANA (gp210, sp100) and a triple (pMIT3) AMA recombinant antigens. Among IIF AMA-negative sera, 43/100 (43%) manifested reactivity using the PBC screening test. The same test was positive for 6/104 (5.8%) control sera. IIF AMA-negative/PBC screen-positive sera reacted against pMIT3 (11/43), gp210 (8/43), Sp100 (17/43), both pMIT3 and gp210 (1/43), or both pMIT3 and Sp100 (6/43). Concordance rates between the ANA pattern on HEp-2 cells and specific Sp100 and gp210 ELISA results in AMA-negative subjects were 92% for nuclear dots and Sp100 and 99% for nuclear rim and gp210. Our data confirm the hypothesis that a substantial part of IIF AMA-negative (formerly coined ??probable??) PBC cases manifest disease-specific autoantibodies when tested using newly available tools and thus overcome the previously suggested diagnostic classification. As suggested by the recent literature, we are convinced that the proportion of AMA-negative PBC cases will be significantly minimized by the use of new laboratory methods and recombinant antigens.