Culture-independent characterization of the digestive-tract microbiota of the medicinal leech reveals a tripartite symbiosis

Culture-based studies of the microbial community within the gut of the medicinal leech have typically been focused on various Aeromonas species, which were believed to be the sole symbiont of the leech digestive tract. In this study, analysis of 16S rRNA gene clone libraries confirmed the presence of Aeromonas veronii and revealed a second symbiont, clone PW3, a novel member of the Rikenellaceae, within the crop, a large compartment where ingested blood is stored prior to digestion. The diversity of the bacterial community in the leech intestinum was determined, and additional symbionts were detected, including members of the alpha-, gamma-, and delta-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The relative abundances of the clones suggested that A. veronii and the novel clone, PW3, also dominate the intestinum community, while other clones, representing transient organisms, were typically present in low numbers. The identities of these transients varied greatly between individual leeches. Neither time after feeding nor feeding on defibrinated blood caused a change in identity of the dominant members of the microbial communities. Terminal restriction fragment length polymorphism analysis was used to verify that the results from the clone libraries were representative of a larger data set. The presence of a two-member bacterial community in the crop provides a unique opportunity to investigate both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations.     

PslD is a secreted protein required for biofilm formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.     

Regulation of cardiac hypertrophy by intracellular signalling pathways

The mammalian heart is a dynamic organ that can grow and change to accommodate alterations in its workload. During development and in response to physiological stimuli or pathological insults, the heart undergoes hypertrophic enlargement, which is characterized by an increase in the size of individual cardiac myocytes. Recent findings in genetically modified animal models implicate important intermediate signal-transduction pathways in the coordination of heart growth following physiological and pathological stimulation.     

Sustained inflation and incremental mean airway pressure trial during conventional and high-frequency oscillatory ventilation in a large porcine model of acute respiratory distress syndrome

Background

To compare the effect of a sustained inflation followed by an incremental mean airway pressure trial during conventional and high-frequency oscillatory ventilation on oxygenation and hemodynamics in a large porcine model of early acute respiratory distress syndrome.

Methods

Severe lung injury (Ali) was induced in 18 healthy pigs (55.3 ± 3.9 kg, mean ± SD) by repeated saline lung lavage until PaO₂ decreased to less than 60 mmHg. After a stabilisation period of 60 minutes, the animals were randomly assigned to two groups: Group 1 (Pressure controlled ventilation; PCV): FIO₂ = 1.0, PEEP = 5 cmH₂O, V_T = 6 ml/kg, respiratory rate = 30/min, I:E = 1:1; group 2 (High-frequency oscillatory ventilation; HFOV): FIO₂ = 1.0, Bias flow = 30 l/min, Amplitude = 60 cmH₂O, Frequency = 6 Hz, I:E = 1:1. A sustained inflation (SI; 50 cmH₂O for 60s) followed by an incremental mean airway pressure (mPaw) trial (steps of 3 cmH₂O every 15 minutes) were performed in both groups until PaO₂ no longer increased. This was regarded as full lung inflation. The mPaw was decreased by 3 cmH₂O and the animals reached the end of the study protocol. Gas exchange and hemodynamic data were collected at each step.

Results

The SI led to a significant improvement of the PaO₂/FiO₂-Index (HFOV: 200 ± 100 vs. PCV: 58 ± 15 and T_Ali: 57 ± 12; p < 0.001) and PaCO₂-reduction (HFOV: 42 ± 5 vs. PCV: 62 ± 13 and T_Ali: 55 ± 9; p < 0.001) during HFOV compared to lung injury and PCV. Augmentation of mPaw improved gas exchange and pulmonary shunt fraction in both groups, but at a significant lower mPaw in the HFOV treated animals. Cardiac output was continuously deteriorating during the recruitment manoeuvre in both study groups (HFOV: T_Ali: 6.1 ± 1 vs. T₇₅: 3.4 ± 0.4; PCV: T_Ali: 6.7 ± 2.4 vs. T₇₅: 4 ± 0.5; p < 0.001).

Conclusion

A sustained inflation followed by an incremental mean airway pressure trial in HFOV improved oxygenation at a lower mPaw than during conventional lung protective ventilation. HFOV but not PCV resulted in normocapnia, suggesting that during HFOV there are alternatives to tidal ventilation to achieve CO₂-elimination in an "open lung" approach.     

Initial clinical trial of epratuzumab (humanized anti-CD22 antibody) for immunotherapy of systemic lupus erythematosus

B cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE), so the safety and activity of anti-B cell immunotherapy with the humanized anti-CD22 antibody epratuzumab was evaluated in SLE patients. An open-label, single-center study of 14 patients with moderately active SLE (total British Isles Lupus Assessment Group (BILAG) score 6 to 12) was conducted. Patients received 360 mg/m² epratuzumab intravenously every 2 weeks for 4 doses with analgesic/antihistamine premedication (but no steroids) prior to each dose. Evaluations at 6, 10, 18 and 32 weeks (6 months post-treatment) follow-up included safety, SLE activity (BILAG score), blood levels of epratuzumab, B and T cells, immunoglobulins, and human anti-epratuzumab antibody (HAHA) titers. Total BILAG scores decreased by ≥ 50% in all 14 patients at some point during the study (including 77% with a ≥ 50% decrease at 6 weeks), with 92% having decreases of various amounts continuing to at least 18 weeks (where 38% showed a ≥ 50% decrease). Almost all patients (93%) experienced improvements in at least one BILAG B- or C-level disease activity at 6, 10 and 18 weeks. Additionally, 3 patients with multiple BILAG B involvement at baseline had completely resolved all B-level disease activities by 18 weeks. Epratuzumab was well tolerated, with a median infusion time of 32 minutes. Drug serum levels were measurable for at least 4 weeks post-treatment and detectable in most samples at 18 weeks. B cell levels decreased by an average of 35% at 18 weeks and remained depressed at 6 months post-treatment. Changes in routine safety laboratory tests were infrequent and without any consistent pattern, and there was no evidence of immunogenicity or significant changes in T cells, immunoglobulins, or autoantibody levels. In patients with mild to moderate active lupus, 360 mg/m² epratuzumab was well tolerated, with evidence of clinical improvement after the first infusion and durable clinical benefit across most body systems. As such, multicenter controlled studies are being conducted in broader patient populations.     

Cloning and molecular characterization of a unique hemolysin gene of Vibrio pommerensis sp. nov.: development of a DNA probe for the detection of the hemolysin gene and its use in identification of related Vibrio spp. from the Baltic Sea

A group of hemolytic Vibrio strains was isolated from surface water of the Baltic Sea in 1995. A typical representative strain, CH-291, was found to lyse washed human and animal erythrocytes. Hemolysis was found to be calcium-dependent and occurred over a temperature range from 25 to 37 degrees C. The hemolysin-encoding genes were identified by screening a genomic library of total DNA from strain CH-291. A cloned chromosomal DNA fragment of 15.6 kb conferred to Escherichia coli DH5alpha a hemolytic phenotype. Hybridization and sequence analysis showed the cloned sequence to be unique to these Baltic Sea Vibrio isolates and therefore provides a useful marker for their identification. Moreover, the cloned 15.6-kb DNA fragment possessed structural features typical for genetic islands, including a decreased GC content and a flanking cryptic insertion sequence element.     

Triacylglycerol-rich lipoproteins protect lipoprotein lipase from inactivation by ANGPTL3 and ANGPTL4

Lipoprotein lipase (LPL) is important for clearance of triacylglycerols (TG) from plasma both as an enzyme and as a bridging factor between lipoproteins and receptors for endocytosis. The amount of LPL at the luminal side of the capillary endothelium determines to what extent lipids are taken up. Mechanisms to control both the activity of LPL and its transport to the endothelial sites are regulated, but poorly understood. Angiopoietin-like proteins (ANGPTLs) 3 and 4 are potential control proteins for LPL, but plasma concentrations of ANGPTLs do not correlate with plasma TG levels. We investigated the effects of recombinant human N-terminal (NT) ANGPTLs3 and 4 on LPL-mediated bridging of TG-rich lipoproteins to primary mouse hepatocytes and found that the NT-ANGPTLs, in concentrations sufficient to cause inactivation of LPL in vitro, were unable to prevent LPL-mediated lipoprotein uptake. We therefore investigated the effects of lipoproteins (chylomicrons, VLDL and LDL) on the inactivation of LPL in vitro by NT-ANGPTLs3 and 4 and found that LPL activity was protected by TG-rich lipoproteins. In vivo, postprandial TG protected LPL from inactivation by recombinant NT-ANGPTL4 injected to mice. We conclude that lipoprotein-bound LPL is stabilized against inactivation by ANGPTLs. The levels of ANGPTLs found in blood may not be sufficient to overcome this stabilization. Therefore it is likely that the prime site of action of ANGPTLs on LPL is in subendothelial compartments where TG-rich lipoprotein concentration is lower than in blood. This could explain why the plasma levels of TG and ANGPTLs do not correlate.     

Lrig1: a new master regulator of epithelial stem cells

Nat Cell Biol 14 4, 401–408 March042012The intestine represents the most vigorously renewing, adult epithelial tissue that makes maintenance of its homeostasis a delicate balance between proliferation, cell cycle arrest, migration, differentiation, and cell death. These processes are precisely controlled by a network of developmental signalling cascades, which include Wnt, Notch, BMP/TGFβ, and Hedgehog pathways. A new, elegant study by Wong et al (2012) now adds Lrig1 as a key player in the control of intestinal homeostasis. As for epidermal stem cells, Lrig1 limits the size of the intestinal progenitor compartment by dampening EGF/ErbB-triggered stem cell expansion.The epithelium of the small intestine is separated into two distinct compartments: a proliferative crypt, containing tissue-specific stem cells, and a villus with differentiated, short-lived cells, which are replenished by a constant stream of cell migration from the underlying crypt (Scoville et al, 2008). In particular, the canonical Wnt pathway in combination with Notch signals control stem cell maintenance and proliferation in the crypt. In addition, both pathways direct differentiation into the Paneth and the absorptive cell lineage, respectively. Intensive cross-talk between the epithelium and the underlying mesenchyme helps to define the crypt–villus boundary. This relies on epithelial-derived Hedgehog and Wnt ligands that trigger stromal BMP production, which in turn signals back to the epithelium to restrict proliferation to the crypt. A gradient of BMP antagonists produced by mesenchymal cells at the bottom of the crypts supports compartmentalization. In addition, a Wnt gradient in the crypt defines EphB expression and establishes repulsion-mediated separation into Paneth cell, proliferative, and differentiation zones along the crypt–villus axis (Figure 1A).Open in a separate windowFigure 1(A) The epithelium of the small intestine contains two populations of multipotent stem cells that reside at the bottom of the crypts. These give rise to transit-amplifying progenitors, which rapidly divide while migrating upwards. Cell cycle arrest and functional differentiation occur when these cells pass from the upper part of the crypt into the villus where they continue their upward movement until they finally undergo apoptosis. Only long-living Paneth cells follow a different path as they migrate downwards to populate the base of the crypt. Control of proliferation and lineage specification of all intestinal epithelial cells is directed in a self-organizing, dynamically regulated process based on cell–cell and cell–environment interactions. Among them, Wnt and Notch signalling have been defined as major determinants for stem cell maintenance, for proliferation of stem cells in the crypt and lineage specification. Epithelial-derived Hedgehog ligands and reciprocal stromal BMP ligands establish a connection between the epithelium and the stroma that regulates the crypt–villus boundary. In addition, repulsive interactions mediated by the Eph/ephrin family allow establishment of stable compartments. Importantly, ErbB signalling, which is partially suppressed by Lrig1 at the base of the crypt, is now shown to be a new key player in the control of stem and progenitor cell expansion. (B) Cross-talk of signalling pathways in intestinal homeostasis with an emphasis on ErbB signalling. A negative feedback loop via Lrig1 helps to fine-tune population size and proliferative activity of intestinal progenitor cells. Lrig1 has been identified as a direct target of Myc and is known to repress ErbB signalling. Myc itself is a main target of the ErbB and Wnt pathways implicated in intestinal stem and progenitor cell expansion. Moreover, Lrig1 has been found to promote BMP signalling, which interferes with intestinal proliferation by restricting AKT activation via PTEN.In the small intestine, two stem cell (SC) populations coexist: Lgr5⁺crypt base columnar cells (CBCs) that cycle every 24 h and are interspersed between Paneth cells, and slower dividing SCs concentrated above (around position +4 relative to the crypt bottom) the Lgr5⁺position (Takeda et al, 2011). The localization of these Hopx⁺mTert⁺slowly cycling SCs partly overlaps with that of quiescent cells, which show long-term label retention upon irradiation damage and pulse labelling with BrdU. Lgr5⁺CBCs are, however, dispensable (Tian et al, 2008) and can be replaced by the second stem cell population, which also shows greater activity during damage repair. The relationship between these two stem cell populations, which can reciprocally generate each other, and the mechanisms that govern quiescence are being elucidated. Importantly, leucine-rich repeats and Ig-like domains 1 (Lrig1), a transmembrane protein that interacts with ErbBs and promotes its degradation, has now been found to be enriched at the crypt base and in the progenitor compartment of the small intestine and colon (Wong et al, 2012). Lrig1 is highly expressed in Lgr5⁺, Musashi1⁺, Ascl2⁺, and Olfm4⁺CBCs, and shows an inverse relation to the pattern of activated, phosphorylated EGFR above the crypt base (Figure 1A). In line with these patterns, deletion of Lrig1 in the mouse causes a dramatic crypt expansion and increased numbers of CBCs, transit-amplifying and Paneth cells. Whether the increase of Paneth cells, which actually do not express Lrig1, is a secondary effect due to the progenitor expansion remains open. Importantly, reduction of EGFR signalling by pharmacological (Gefitinib) and genetic modulation (Egfr^wa-2 mice) is able to partially normalize all Lrig1 phenotypes. These data establish EGF/ErbB signalling, as an important regulator of the crypt compartment, and suggest Lrig1 as a central control that dampens the expansion of stem cells during normal intestinal homeostasis.Lrig1 was initially identified in the skin and proposed to maintain epidermal stem cells in a quiescent state (Watt and Jensen, 2009). Lrig1 marks human interfollicular epidermal stem cells, which can give rise to all epithelial lineages including hair follicle cells in skin reconstitution assays. However, during normal homeostasis, these cells are only bipotent, contributing to the sebaceous gland and the interfollicular epidermis. In contrast to quiescent Lrig1⁺SCs in the skin, Lrig1⁺ intestinal SCs are rapidly dividing and Lrig1 appears to only reduce their proliferative capacity. However, similar to the situation in the skin, Lrig1 and EGF signalling may play an important role during damage repair. Earlier experiments analysed the phenotype of mice lacking major EGF family members (Egger et al, 1997; Troyer et al, 2001). While these mice display some duodenal lesions during normal homeostasis, further experiments established EGF signalling as a key protective component that ameliorates mucosal damage. It remains to be seen whether activation of intestinal SCs during damage repair involves mitigation of Lrig1 dampening.Lrig1 is known to repress ErbB signalling by mediating ubiquitinylation and degradation of activated receptors, thereby limiting the amplitude of EGF signalling (Watt and Jensen, 2009). Consequently, Lrig1 deletion in the intestine induced upregulation of EGFR, ErbB2, and ErbB3, promoting downstream activation of c-Myc within intestinal stem and progenitor cells (Wong et al, 2012). Importantly, Lrig1 is a direct Myc target gene, and thereby part of a negative feedback loop that helps to fine-tune the population size and proliferative activity of intestinal progenitor cells (Figure 1B).Since the rescue of the Lrig1^−/− phenotype by EGFR deficiency was only partial (Wong et al, 2012), other mechanisms may contribute. Intriguingly, Lrig1 has been shown to promote BMP signalling by direct binding to Type I (ALK6) and Type II (ALK1, ALK2, ALK3, and ActRIB) BMP receptors (Gumienny et al, 2010). BMPR1A inactivation, deficiency of its downstream effector PTEN, and transgenic overexpression of the BMP inhibitor Noggin display crypt expansion and increased SC numbers. Inhibition of BMP signalling in these genetic models enhanced AKT activation and increased Wnt signalling, promoting proliferation and adenoma formation (Figure 1B; Scoville et al, 2008). Future work will reveal a potential involvement of BMP and Wnt signalling in the Lrig1 knockout phenotype.The ErbB pathway has been linked to inflammatory bowel disease, and progression and metastatic potential of colorectal cancer. EGFR inhibition blocks adenoma formation in preclinical models, and ErbB pathway inhibition is currently being evaluated in clinical trials with colorectal cancer patients, where promising results have been reported (Cunningham et al, 2004). In contrast, Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumours (Hedman and Henriksson, 2007). Given this heterogeneity, the Lrig1 function in tumours appears to be cell- and context-dependent. Due to early postnatal lethality of Lrig1 knockout mice, the exciting possibility that Lrig1 may act as an intestinal tumour suppressor could not be answered by the current study but clearly deserves further attention.