A strategy to obtain axenic cultures of <Emphasis Type="Italic">Arthrospira</Emphasis> spp. cyanobacteria

A strategy to obtain axenic cultures of the cyanobacterium Arthrospira sp. (‘platensis’) Lefevre 1963/M-132-1 strain, consisting of a series of physical and chemical procedures, and the application of an optimized pool of antibiotics, is described in this paper. This strategy, which is an inexpensive and fast way to obtain axenic cultures, can be applied to Arthrospira spp. from culture collections or samples from their natural habitats to eliminate a wide spectrum of contaminants. A high alkaline treatment (pH 12, using KOH) of 72 h is a determinant initial procedure applied to eliminate protozoa and Microcystis sp. Bacteria were eliminated by an optimal antibiotic pool treatment, and Chroococcus sp. residuals were discarded by serial dilution. Optimal concentrations of the antibiotics composing the pool were obtained by a 2⁴ factorial central composite rotatable design (CCRD) and Response Surface Methodology (RSM), resulting in: ampicillin 61.6 μg/ml, penicillin 85.8 μg/ml, cefoxitin 76.9 μg/ml, and meropenem 38.9 μg/ml. The results also indicate that cefoxitin was the most effective antibiotic of this pool. After obtaining the axenic culture, identification of Lefevre 1963/M-132-1 strain was performed using amplification and sequencing of the ITS region (including part of 16S rRNA, tRNA Ile, ITS, tRNA Ala and part of 23S rRNA region) and fatty acid composition data. Data base comparison revealed that Lefevre strain is closely related to A. platensis species (99% identity), while fatty acid composition data suggested A. maxima. These seemingly contradictory results are discussed.     

B cell response during infection with the MAT a and MAT alpha mating types of Cryptococcus neoformans

In the present study, we compared the B cell response of BALB/c and C57Bl/6 mice during Cryptococcus neoformans infection. This response was investigated using virulent serotype D forms of mating types alpha and a (MAT alpha and MAT a). C57Bl/6 mice showed massive (mainly cerebral) infection by both types, while BALB/c were resistant to infection. Some resistance of C57Bl/6 mice was induced by previous immunization with the capsular polysaccharide from MAT alpha. Passive immunization of C57Bl/6 mice with purified antibody (Ab) obtained from capsular polysaccharide-immunized mice also increased resistance to infection. Both mouse strains showed comparable low IgM response to the capsular polysaccharide from MAT alpha, and only C57Bl/6 mice produced IgM to the polysaccharide of MAT a. Comparable levels of different immunoglobulin (Ig) isotypes against capsular components of MAT alpha and MAT a were detected, and the response of C57Bl/6 mice was higher when compared to that of BALB/c mice. FACS analysis indicated an increase in the percentage of a high-granulosity (side-scatter) splenic subpopulation and in the percentage of splenic Gr-1+ cells in infected C57Bl/6 mice. In addition, the percentage of follicular splenic B cells was decreased after C. neoformans infection of C57Bl/6 mice. This response was more pronounced when we investigated infection induced by the MAT a mating type. Taken together, our results indicate that capsular polysaccharide derived from MAT alpha and MAT a types of C. neoformans have a stimulatory effect upon B cells but that there is no correlation between resistance of BALB/c mice and Ab production. However, the increase in resistance of C57Bl/6 mice parallels the production of Abs and a major change in splenic cell populations.     

Translation initiator EIF4G1 mutations in familial Parkinson disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.     

P311-induced myofibroblasts exhibit ameboid-like migration through RalA activation

We previously showed that P311, an intracellular protein involved in cell migration, is found in human wound myofibroblast precursors (proto-myofibroblasts) and myofibroblasts. Furthermore, by binding to the TGF-beta1 latency associated protein (LAP), P311 induced NIH 3T3 cells to transform into non-fibrogenic myofibroblasts characterized by lack of TGF-beta1 production. Here we demonstrate that P311-induced myofibroblasts migrate in an ameboid rather than a mesenchymal pattern. Ameboid migration is characterized by lack of focal adhesions and stress fibers, absence of integrins and MMPs clustering/activation and changes in small GTPases activity, all leading to increased cell motility. P311-induced ameboid migration depended on activation of the GTPase RalA and was reverted to mesenchymal-type migration by RalA RNA interference. Ameboid migration was conserved in cells plated on fibrin, the initial wound matrix, but was switched back to mesenchymal-type migration by collagen I, the main ECM component in late stages of wound healing. TGF-beta1, the major stimulus of collagen production during wound repair, also reversed the ameboid phenotype to mesenchymal. Our studies therefore suggest that, by inducing RalA activity, P311 promotes a motile proto-myofibroblast and myofibroblast phenotype specifically adapted to rapidly populate the initial wound matrix.     

Evaluation of the phenotypic characteristics of coliform bacteria recovered with two methods: the European Drinking Water Directive reference method and the Colilert 18/Quanti-Tray™ system

Summary The taxonomy of the species belonging to Enterobacteriaceae has undergone a series of revisions. As a consequence, the new classification has caused the analytical detection methods to be updated. According to the European Drinking Water Directive 98/83/CE coliforms/Escherichia coli have to be determined with the ISO 9308-1 reference method. Many technical drawbacks of the procedure as well as limitations regarding the recent taxonomy of coliforms have been pointed out by laboratories working in water quality control. In our investigation, water was analyzed in parallel with the reference method and the rapid Colilert 18/Quanti-Tray™ system. Phenotypic characteristics of isolates were recorded for the verification of the response of the two methods to the new taxonomic approach. Results obtained with the ISO standard confirmed the limitations of the test. In fact, in addition to difficulties linked to the readability of results, it failed to detect a significant proportion of coliforms and E. coli in water. Furthermore, it allowed the growth of microorganisms belonging to other groups. The Colilert 18/Quanti-Tray™ system detected a qualitatively and quantitatively higher number of target microorganisms. It also provided results in a shorter time, allowing the simultaneous detection of E. coli and coliforms with no further confirmation steps.     

Effect of novel non-peptidic delta opioid receptor antagonists on human T and B cell activation

The effects of the antagonist naltrindole (NTI) on cells of the immune system have been largely studied although the mechanisms of action are still unclear. The aim of this study is to evaluate, in vitro, the immunomodulatory activity of four new delta-selective opioid compounds structurally related to naltrindole. The effects at different concentrations of these opioid antagonists on proliferative response were studied on normal human peripheral blood mononuclear cells (PBMC) stimulated with different stimuli: mitogens, the antigen PPD, the anti-CD3 monoclonal antibodies (mAb), the superantigen Staphylococcus aureus Cowan strain 1 (SAC) and alloantigens in the mixed lymphocyte cultures (MLR). The immunomodulatory capacity of these compounds was evaluated by determining the interleukin-2 (IL-2) release in mitogen activated PBMC. The present study shows that all the new delta opioid antagonists at 10(-5) M concentration are immunosuppressive. The inhibitory action is also evident at lower concentrations when anti-CD3 mAb and SAC were used as stimulators. In addition, the production of IL-2 was inhibited by the opioid treatment, but this might not be the only mechanism of action.     

Apolipoprotein M modulates erythrocyte efflux and tubular reabsorption of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) mediates several cytoprotective functions of HDL. apoM acts as a S1P binding protein in HDL. Erythrocytes are the major source of S1P in plasma. After glomerular filtration, apoM is endocytosed in the proximal renal tubules. Human or murine HDL elicited time- and dose-dependent S1P efflux from erythrocytes. Compared with HDL of wild-type (wt) mice, S1P efflux was enhanced in the presence of HDL from apoM transgenic mice, but not diminished in the presence of HDL from apoM knockout (Apom^−/−) mice. Artificially reconstituted and apoM-free HDL also effectively induced S1P efflux from erythrocytes. S1P and apoM were not measurable in the urine of wt mice. Apom^−/− mice excreted significant amounts of S1P. apoM was detected in the urine of mice with defective tubular endocytosis because of knockout of the LDL receptor-related protein, chloride-proton exchanger ClC-5 (Clcn5^−/−), or the cysteine transporter cystinosin. Urinary levels of S1P were significantly elevated in Clcn5^−/− mice. In contrast to Apom^−/− mice, these mice showed normal plasma concentrations for apoM and S1P. In conclusion, HDL facilitates S1P efflux from erythrocytes by both apoM-dependent and apoM-independent mechanisms. Moreover, apoM facilitates tubular reabsorption of S1P from the urine, however, with no impact on S1P plasma concentrations.     

Immune Regulatory Neural Stem/Precursor Cells Protect from Central Nervous System Autoimmunity by Restraining Dendritic Cell Function

Background

The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinico-pathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs.

Methodology/Principal Findings

To achieve the exclusive targeting of the peripheral immune system, SJL mice with PLP-induced EAE were injected subcutaneously with NPCs and the treatment commenced prior to disease onset. NPC-injected EAE mice showed significant clinical improvement, as compared to controls. Exogenous NPCs lacking the expression of major neural antigens were reliably (and for long-term) found at the level of draining lymph nodes, while establishing sophisticated anatomical interactions with lymph node cells. Importantly, injected NPCs were never found in organs other than lymph nodes, including the brain and the spinal cord. Draining lymph nodes from transplanted mice showed focal up-regulation of major developmental stem cell regulators, such as BMP-4, Noggin and Sonic hedgehog. In lymph nodes, injected NPCs hampered the activation of myeloid dendritic cells (DCs) and steadily restrained the expansion of antigen-specific encephalitogenic T cells. Both ex vivo and in vitro experiments identified a novel highly NPC-specific–BMP-4-dependent–mechanism hindering the DC maturation.

Conclusion/Significance

The study described herein, identifies the first member of the TGF β/BMP family of stem cell regulators as a novel tolerogenic factor released by NPCs. Full exploitation of this pathway as an efficient tool for vaccination therapy in autoimmune inflammatory conditions is underway.     

Concomitant chemo-radiotherapy for unresectable oesophageal cancer: A mono-institutional study on 40 patients

Background/AimTo analyse clinical response, overall (OS) and disease free survival (DFS) and toxicity in patients with unresectable oesophageal cancer treated by concomitant chemo-radiotherapy (CRT).Materials and methodsForty patients with stage IIa–IVa biopsy proven oesophageal carcinoma were treated with CRT. All patients were studied with endoscopy and CT and judged unresectable after multidisciplinary discussion. CRT consisted of 3 cycles of cisplatin 100 mg/m² or carboplatin 300 mg/m² on day 1 and 5-fluorouracil 1000 mg/m² as a continuous infusion of 96 h associated with concurrent 3D-conformal RT. By using 15 MeV X-rays, a total dose of 60–66 Gy was delivered with daily fractions of 1.8–2.0 Gy.ResultsComplete response (CR), partial response (PR) and no response (NR) were observed in 50%, 20% and 20% of cases, respectively. Of the 20 patients with CR, 15 developed loco-regional recurrent disease. OS and DFS rates at 3 and 5 years were 38%, 8%, 49% and 10%, respectively. Total radiation dose ≥60 Gy improved loco-regional control and complete response (CR vs. PR + NR; p = 0.004) influenced both DFS and loco-regional control. Grade 3 gastrointestinal and haematological acute toxicity occurred in 3/40 patients (7.5%). One patient developed grade 4 renal failure. Late toxicity was reported in 2/40 patients (5.0%), consisting of grade 3 radiation pneumonitis.ConclusionsConcomitant CRT for unresectable oesophageal cancer can result in an acceptable loco-regional control with limited toxicity. Response after treatment and total radiation dose influenced the outcome.     

Microbial carbon limitation: The need for integrating microorganisms into our understanding of ecosystem carbon cycling

Numerous studies have demonstrated that fertilization with nutrients such as nitrogen, phosphorus, and potassium increases plant productivity in both natural and managed ecosystems, demonstrating that primary productivity is nutrient limited in most terrestrial ecosystems. In contrast, it has been demonstrated that heterotrophic microbial communities in soil are primarily limited by organic carbon or energy. While this concept of contrasting limitations, that is, microbial carbon and plant nutrient limitation, is based on strong evidence that we review in this paper, it is often ignored in discussions of ecosystem response to global environment changes. The plant‐centric perspective has equated plant nutrient limitations with those of whole ecosystems, thereby ignoring the important role of the heterotrophs responsible for soil decomposition in driving ecosystem carbon storage. To truly integrate carbon and nutrient cycles in ecosystem science, we must account for the fact that while plant productivity may be nutrient limited, the secondary productivity by heterotrophic communities is inherently carbon limited. Ecosystem carbon cycling integrates the independent physiological responses of its individual components, as well as tightly coupled exchanges between autotrophs and heterotrophs. To the extent that the interacting autotrophic and heterotrophic processes are controlled by organisms that are limited by nutrient versus carbon accessibility, respectively, we propose that ecosystems by definition cannot be ‘limited’ by nutrients or carbon alone. Here, we outline how models aimed at predicting non‐steady state ecosystem responses over time can benefit from dissecting ecosystems into the organismal components and their inherent limitations to better represent plant–microbe interactions in coupled carbon and nutrient models.