AAV-Mediated CAG-Targeting Selectively Reduces Polyglutamine-Expanded Protein and Attenuates Disease Phenotypes in a Spinocerebellar Ataxia Mouse Model.

Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs). PolyQ-expanded SCA proteins are toxic for cerebellar neurons, with Purkinje cells (PCs) being the most vulnerable. RNA interference (RNAi) reagents targeting transcripts with expanded CAG reduce the level of various mutant SCA proteins in an allele-selective manner in vitro and represent promising universal tools for treating multiple CAG/polyQ SCAs. However, it remains unclear whether the therapeutic targeting of CAG expansion can be achieved in vivo and if it can ameliorate cerebellar functions. Here, using a mouse model of SCA7 expressing a mutant Atxn7 allele with 140 CAGs, we examined the efficacy of short hairpin RNAs (shRNAs) targeting CAG repeats expressed from PHP.eB adeno-associated virus vectors (AAVs), which were introduced into the brain via intravascular injection. We demonstrated that shRNAs carrying various mismatches with the CAG target sequence reduced the level of polyQ-expanded ATXN7 in the cerebellum, albeit with varying degrees of allele selectivity and safety profile. An shRNA named A4 potently reduced the level of polyQ-expanded ATXN7, with no effect on normal ATXN7 levels and no adverse side effects. Furthermore, A4 shRNA treatment improved a range of motor and behavioral parameters 23 weeks after AAV injection and attenuated the disease burden of PCs by preventing the downregulation of several PC-type-specific genes. Our results show the feasibility of the selective targeting of CAG expansion in the cerebellum using a blood-brain barrier-permeable vector to attenuate the disease phenotype in an SCA mouse model. Our study represents a significant advancement in developing CAG-targeting strategies as a potential therapy for SCA7 and possibly other CAG/polyQ SCAs.

The Novel Alpha-2 Adrenoceptor Inhibitor Beditin Reduces Cytotoxicity and Huntingtin Aggregates in Cell Models of Huntington's Disease.

Huntington's disease (HD) is a monogenetic neurodegenerative disorder characterized by the accumulation of polyglutamine-expanded huntingtin (mHTT). There is currently no cure, and therefore disease-slowing remedies are sought to alleviate symptoms of the multifaceted disorder. Encouraging findings in Alzheimer's and Parkinson's disease on alpha-2 adrenoceptor (α2-AR) inhibition have shown neuroprotective and aggregation-reducing effects in cell and animal models. Here, we analyzed the effect of beditin, a novel α2- adrenoceptor (AR) antagonist, on cell viability and mHTT protein levels in cell models of HD using Western blot, time-resolved Foerster resonance energy transfer (TR-FRET), lactate dehydrogenase (LDH) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) cytotoxicity assays. Beditin decreases cytotoxicity, as measured by TUNEL staining and LDH release, in a neuronal progenitor cell model (STHdh cells) of HD and decreases the aggregation propensity of HTT exon 1 fragments in an overexpression model using human embryonic kidney (HEK) 293T cells. α2-AR is a promising therapeutic target for further characterization in HD models. Our data allow us to suggest beditin as a valuable candidate for the pharmaceutical manipulation of α2-AR, as it is capable of modulating neuronal cell survival and the level of mHTT.

Brain-penetrant PQR620 mTOR and PQR530 PI3K/mTOR inhibitor reduce huntingtin levels in cell models of HD.

One of the pathological hallmarks of Huntington disease (HD) is accumulation of the disease-causing mutant huntingtin (mHTT), which leads to the disruption of a variety of cellular functions, ultimately resulting in cell death. Induction of autophagy, for example by the inhibition of mechanistic target of rapamycin (mTOR) signaling, has been shown to reduce HTT levels and aggregates. While rapalogs like rapamycin allosterically inhibit the mTOR complex 1 (TORC1), ATP-competitive mTOR inhibitors suppress activities of TORC1 and TORC2 and have been shown to be more efficient in inducing autophagy and reducing protein levels and aggregates than rapalogs. The ability to cross the blood-brain barrier of first generation catalytic mTOR inhibitors has so far been limited, and therefore sufficient target coverage in the brain could not be reached. Two novel, brain penetrant compounds - the mTORC1/2 inhibitor PQR620, and the dual pan-phosphoinositide 3-kinase (PI3K) and mTORC1/2 kinase inhibitor PQR530 - were evaluated by assessing their potential to induce autophagy and reducing mHTT levels. For this purpose, expression levels of autophagic markers and well-defined mTOR targets were analyzed in STHdh cells and HEK293T cells and in mouse brains. Both compounds potently inhibited mTOR signaling in cell models as well as in mouse brain. As proof of principle, reduction of aggregates and levels of soluble mHTT were demonstrated upon treatment with both compounds. Originally developed for cancer treatment, these second generation mTORC1/2 and PI3K/mTOR inhibitors show brain penetrance and efficacy in cell models of HD, making them candidate molecules for further investigations in HD.

Contribution of the Gut Microbiota in P28GST-Mediated Anti-Inflammatory Effects: Experimental and Clinical Insights.

An original immuno-regulatory strategy against inflammatory bowel diseases based on the use of 28 kDa glutathione S-transferase (P28GST), a unique schistosome protein, was recently proposed. Improvement of intestinal inflammation occurs through restoration of the immunological balance between pro-inflammatory T-helper 1 (Th1) responses and both T-helper 2 (Th2) and regulatory responses. However, detailed mechanisms explaining how P28GST prevents colitis and promotes gut homeostasis remain unknown. Considering the complex interplay between the adaptive and innate immune system and the intestinal microbiota, we raised the question of the possible role of the microbial ecosystem in the anti-inflammatory effects mediated by the helminth-derived P28GST protein. We first analyzed, by 16S rRNA sequencing, the bacterial profiles of mice fecal microbiota at several time points of the P28GST-immunomodulation period prior to trinitrobenzene sulfonic acid (TNBS)-colitis. The influence of gut microbiota in the P28GST-mediated anti-inflammatory effects was then assessed by fecal microbiota transplantation experiments from P28GST-immunized mice to either conventional or microbiota depleted naïve recipient mice. Finally, the experimental data were supplemented by the temporal fecal microbiota compositions of P28GST-treated Crohn's disease patients from a pilot clinical study (NCT02281916). The P28GST administration slightly modulated the diversity and composition of mouse fecal microbiota while it significantly reduced experimental colitis in mice. Fecal microbiota transplantation experiments failed to restore the P28GST-induced anti-inflammatory effects. In Crohn's disease patients, P28GST also induced slight changes in their overall fecal bacterial composition. Collectively, these results provide key elements in both the anti-inflammatory mechanisms and the safe therapeutic use of immunomodulation with such promising helminth-derived molecules.

Intranasal Administration of Mesenchymal Stem Cells Ameliorates the Abnormal Dopamine Transmission System and Inflammatory Reaction in the R6/2 Mouse Model of Huntington Disease.

Intrastriatal administration of mesenchymal stem cells (MSCs) has shown beneficial effects in rodent models of Huntington disease (HD). However, the invasive nature of surgical procedure and its potential to trigger the host immune response may limit its clinical use. Hence, we sought to evaluate the non-invasive intranasal administration (INA) of MSC delivery as an effective alternative route in HD. GFP-expressing MSCs derived from bone marrow were intranasally administered to 4-week-old R6/2 HD transgenic mice. MSCs were detected in the olfactory bulb, midbrain and striatum five days post-delivery. Compared to phosphate-buffered saline (PBS)-treated littermates, MSC-treated R6/2 mice showed an increased survival rate and attenuated circadian activity disruption assessed by locomotor activity. MSCs increased the protein expression of DARPP-32 and tyrosine hydroxylase (TH) and downregulated gene expression of inflammatory modulators in the brain 7.5 weeks after INA. While vehicle treated R6/2 mice displayed decreased Iba1 expression and altered microglial morphology in comparison to the wild type littermates, MSCs restored both, Iba1 level and the thickness of microglial processes in the striatum of R6/2 mice. Our results demonstrate significantly ameliorated phenotypes of R6/2 mice after MSCs administration via INA, suggesting this method as an effective delivering route of cells to the brain for HD therapy.

Killing Two Angry Birds with One Stone: Autophagy Activation by Inhibiting Calpains in Neurodegenerative Diseases and Beyond.

Proteolytic machineries execute vital cellular functions and their disturbances are implicated in diverse medical conditions, including neurodegenerative diseases. Interestingly, calpains, a class of Ca2+-dependent regulatory proteases, can modulate the degradational system of autophagy by cleaving proteins involved in this pathway. Moreover, both machineries are common players in many molecular pathomechanisms and have been targeted individually or together, as a therapeutic strategy in experimental setups. In this review, we briefly introduce calpains and autophagy, with their roles in health and disease, and focus on their direct pathologically relevant interplay in neurodegeneration and beyond. The modulation of calpain activity may comprise a promising treatment approach to attenuate the deregulation of these two essential mechanisms.

Occurrence and Dynamism of Lactic Acid Bacteria in Distinct Ecological Niches: A Multifaceted Functional Health Perspective.

Lactic acid bacteria (LAB) are representative members of multiple ecosystems on earth, displaying dynamic interactions within animal and plant kingdoms in respect with other microbes. This highly heterogeneous phylogenetic group has coevolved with plants, invertebrates, and vertebrates, establishing either mutualism, symbiosis, commensalism, or even parasitism-like behavior with their hosts. Depending on their location and environment conditions, LAB can be dominant or sometimes in minority within ecosystems. Whatever their origins and relative abundance in specific anatomic sites, LAB exhibit multifaceted ecological and functional properties. While some resident LAB permanently inhabit distinct animal mucosal cavities, others are provided by food and may transiently occupy the gastrointestinal tract. It is admitted that the overall gut microbiome has a deep impact on health and diseases. Here, we examined the presence and the physiological role of LAB in the healthy human and several animal microbiome. Moreover, we also highlighted some dysbiotic states and related consequences for health, considering both the resident and the so-called "transionts" microorganisms. Whether LAB-related health effects act collectively or follow a strain-specificity dogma is also addressed. Besides the highly suggested contribution of LAB to interplay with immune, metabolic, and even brain-axis regulation, the possible involvement of LAB in xenobiotic detoxification processes and metal equilibrium is also tackled. Recent technological developments such as functional metagenomics, metabolomics, high-content screening and design in vitro and in vivo experimental models now open new horizons for LAB as markers applied for disease diagnosis, susceptibility, and follow-up. Moreover, identification of general and more specific molecular mechanisms based on antioxidant, antimicrobial, anti-inflammatory, and detoxifying properties of LAB currently extends their selection and promising use, either as probiotics, in traditional and functional foods, for dedicated treatments and mostly for maintenance of normobiosis and homeostasis.

[Bilateral Medial Recession for Infantile Esotropia]. / Bilaterale Medialis-Rücklagerung bei frühkindlicher Esotropie.

OBJECTIVE: Bilateral medial rectus recession (BMR) is a common surgical treatment of infantile esotropia with smaller and larger deviations. The aim of this study is to evaluate the dose-effect relationship and the success of the therapy. DESIGN: The study is a retrospective, monocentric clinical study with one surgeon and 60 patients with infantile esotropia. METHODS: 60 patients with infantile esotropia were included, who were treated with BMR using microinvasive technique between April 2012 and September 2015. We evaluated pre- and post-operative data in three follow-ups, at 3, 12 and 36 months. A residual deviation ≤ ± 10 PD (prism dioptres) at 12 month follow-up or a measurable positive binocular vision was defined as a positive outcome. RESULTS: Age at surgery is 5.2 ± 4.6 (median ± SD). Pre-operative deviations at distance were 41 ± 10.5 PD and for near vision 44 ± 11 PD. Spherical equivalent refractive errors were + 2.25 dpt. Post-operative deviations at distance (n = 58) at 12 month follow-up were 6 ± 9.6 PD and at 36 month follow-up (n = 38) were 4 ± 10.5 PD. The mean reduction in deviation in 12 month follow-up were 3.08 ± 1.20 PD per mm and at 36 months 3.18 ± 1.27 PD. For children ≤ 4 years, the value was 3.93 ± 1.86 PD and for children > 4 years 2.81 ± 0.98 PD. The therapeutic success at 12 month follow-up was 67%. Positive binocular vision was proved post-operative in 62% of patients. CONCLUSION: BMR is a long-term and efficient surgical treatment of infantile esotropia. The average dose-effect relationship at 36 month follow-up was 3.1 PD per mm.

Reduced cell size, chromosomal aberration and altered proliferation rates are characteristics and confounding factors in the STHdh cell model of Huntington disease.

Huntington disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Expression of the mutant protein disrupts various intracellular pathways and impairs overall cell function. In particular striatal neurons seem to be most vulnerable to mutant huntingtin-related changes. A well-known and commonly used model to study molecular aspects of Huntington disease are the striatum-derived STHdh cell lines generated from wild type and huntingtin knock-in mouse embryos. However, obvious morphological differences between wild type and mutant cell lines exist, which have rarely been described and might not have always been considered when designing experiments or interpreting results. Here, we demonstrate that STHdh cell lines display differences in cell size, proliferation rate and chromosomal content. While the chromosomal divergence is considered to be a result of the cells' tumour characteristics, differences in size and proliferation, however, were confirmed in a second non-immortalized Huntington disease cell model. Importantly, our results further suggest that the reported phenotypes can confound other study outcomes and lead to false conclusions. Thus, careful experimental design and data analysis are advised when using these cell models.

Delivery of a mucin domain enriched in cysteine residues strengthens the intestinal mucous barrier.

A weakening of the gut mucous barrier permits an increase in the access of intestinal luminal contents to the epithelial cells, which will trigger the inflammatory response. In inflammatory bowel diseases, there is an inappropriate and ongoing activation of the immune system, possibly because the intestinal mucus is less protective against the endogenous microflora. General strategies aimed at improving the protection of the intestinal epithelium are still missing. We generated a transgenic mouse that secreted a molecule consisting of 12 consecutive copies of a mucin domain into its intestinal mucus, which is believed to modify the mucus layer by establishing reversible interactions. We showed that the mucus gel was more robust and that mucin O-glycosylation was altered. Notably, the gut epithelium of transgenic mice housed a greater abundance of beneficial Lactobacillus spp. These modifications were associated with a reduced susceptibility of transgenic mice to chemically induced colitis. Furthermore, transgenic mice cleared faster Citrobacter rodentium bacteria which were orally given and mice were more protected against bacterial translocation induced by gavage with adherent-invasive Escherichia coli. Our data show that delivering the mucin CYS domain into the gut lumen strengthens the intestinal mucus blanket which is impaired in inflammatory bowel diseases.

Effect of aspirin versus clopidogrel on walking exercise performance in intermittent claudication-a double-blind randomized multicenter trial.

BACKGROUND: This study sought to determine possible effects of different antiplatelet therapies on walking exercise performance in intermittent claudication. Aspirin, in contrast to clopidogrel, interferes with processes that increase collateral conductance in an ischemic animal model. METHODS AND RESULTS: Patients with stable intermittent claudication were recruited from 21 centers in Switzerland and Germany and randomized to either aspirin or clopidogrel treatment. They participated in a 3-month rehabilitation program (electronically monitored, home-based, 1-hour daily walking sessions at a speed of approximately 120 steps/min). Walking distance was assessed by treadmill tests (3.2 km/h; 12% grade) at baseline and after 12 weeks. A total of 229 of 259 patients with a mean age of 66.2±7.7 years completed the study according to the protocol. A total of 24.5% were females, 20.1% diabetics, and 85.6% were active/ex-smokers. The baseline characteristics were a median (interquartile range) ankle/brachial index of 0.69 (0.57±0.8), an initial claudication distance (ICD) of 98 m (70 to 151 m), and an absolute claudication distance (ACD) of 162 m (113 to 302 m). Training resulted in a median increase of initial claudication distance by 33.5 m (33.3%) in the clopidogrel group and 29 m (33.9%) in the aspirin group. The values for absolute claudication distance were 60.5 m (34.9%) and 75 m (35.3%), respectively (p(ICD)=0.42 and p(ACD)=0.66). CONCLUSIONS: Treatment with aspirin did not show a difference in initial claudication distance or absolute claudication distance improvements compared with clopidogrel after a 3-month walking rehabilitation program. (J Am Heart Assoc. 2012;1:51-56.) CLINICAL TRIAL REGISTRATION: URL: http://www.ClinicalTrials.gov. Unique identifier: NCT00189618, URL: https://EudraCT.ema.europa.eu, Unique identifier: 2004-005041-35.

AIEC colonization and pathogenicity: influence of previous antibiotic treatment and preexisting inflammation.

BACKGROUND: Inflammatory bowel diseases (IBD) patients are abnormally colonized by adherent-invasive Escherichia coli (AIEC). NOD2 gene mutations impair intracellular bacterial clearance. We evaluated the impact of antibiotic treatment on AIEC colonization in wildtype (WT) and NOD2 knockout mice (NOD2KO) and the consequences on intestinal inflammation. METHODS: After 3 days of antibiotic treatment, mice were infected for 2 days with 109 CFU AIEC and sacrificed 1, 5, and 60 days later. In parallel, mice were challenged with AIEC subsequent to a dextran sodium sulfate (DSS) treatment and sacrificed 9 days later. Ileum, colon, and mesenteric tissues were sampled for AIEC quantification and evaluation of inflammation. RESULTS: Without antibiotic treatment, AIEC was not able to colonize WT and NOD2KO mice. Compared with nontreated animals, antibiotic treatment led to a significant increase in ileal and colonic colonization of AIEC in WT and/or NOD2KO mice. Persistent AIEC colonization was observed until day 5 only in NOD2KO mice, disappearing at day 60. Mesenteric translocation of AIEC was observed only in NOD2KO mice. No inflammation was observed in WT and NOD2KO mice treated with antibiotics and infected with AIEC. During DSS-induced colitis, colonization and persistence of AIEC was observed in the colon. Moreover, a dramatic increase in clinical, histological, and molecular parameters of colitis was observed in mice infected with AIEC but not with a commensal E. coli strain. CONCLUSIONS: Antibiotic treatment was necessary for AIEC colonization of the gut and mesenteric tissues and persistence of AIEC was dependent on NOD2. AIEC exacerbated a preexisting DSS-induced colitis in WT mice.

Differential diagnosis of benign and malign pancreatic masses with 18F-fluordeoxyglucose-positron emission tomography recorded with a dual-head coincidence gamma camera.

INTRODUCTION: Metabolic imaging using 18F-fluordeoxyglucose and a ring-positron emission tomography camera is an established method in the differential diagnosis of pancreatic masses. Ring-positron emission tomography cameras, however, are expensive and available in only few specialized centres. The aim of this study was to investigate how far 18F-fluordeoxyglucose scan with a conventional dual-head gamma-camera could differentiate between benign and malign pancreatic masses. MATERIAL AND METHODS: Forty-one patients (male/female: 25/16; mean age: 64.0 years; range: 41-86 years) with a pancreatic mass detected by ultrasound, computed tomography or MRI were included. In all patients 18F-fluordeoxyglucose scan was performed after overnight fasting and injection of 4 mCi 18F-fluordeoxyglucose using an ADAC Vertex MCD dual head gamma-camera (ADAC; Milpitas, California, USA), equipped with a 5/8-inch NaI-crystal. Images were acquired through a 180 degrees grade rotation in the three dimensional mode. The chosen matrix was 128 x 128 x 16, a Butterworthfilter (ADAC) was used and data were transferred into visible sinograms via Fourier-Rebinning. Coronar, sagittal and transversal slices of 3.9 mm thickness each were acquired. Focal tracer enhancement was suspicious for a malignoma and therefore regarded as positive, diffuse or no tracer uptake was suspicious for a benign process and was regarded as negative for cancer. DEFINITION OF GOLD STANDARDS: A diagnosis of cancer had to be confirmed histologically by specimens obtained by 18G-needle biopsy, surgical resection or at autopsy. A diagnosis of an inflammatory mass was considered proven, if no carcinoma could be found histologically in the surgically resected mass or at autopsy, or if there was no progression of the disease during a follow-up of at least 12 months. RESULTS: In 22 patients carcinoma was diagnosed (pancreatic cancer: n=17; endocrine tumour: n=3; carcinoma of the common bile duct: n=2). 18F-fluordeoxyglucose scan showed a focal tracer enhancement in 19 of these 22 patients (sensitivity: 86.4%). False negative results were acquired in two patients with cancer of the common bile duct and in one patient with poorly controlled insulin-dependent diabetes mellitus. In 19 patients the final diagnosis was an inflammatory pancreatic mass. 18F-fluordeoxyglucose scan showed a diffuse tracer enhancement in 15 of these 19 patients (specificity: 78.9%). False positive results were acquired in three patients whose blood tests showed signs of an acute episode of chronic pancreatitis. Positive and negative predictive values of 18F-fluordeoxyglucose scan were 82.6% and 83.3%, respectively. CONCLUSION: 18F-fluordeoxyglucose scan with a conventional dual-head gamma-camera is a highly sensitive and specific method in the differential diagnosis of benign and malign pancreatic masses.

How NOD2 mutations predispose to Crohn's disease?

NOD2 mutations are associated with the development of granulomatous inflammatory diseases, such as early-onset sarcoidosis (EOS), Blau syndrome (BS) and Crohn's disease (CD). As a pathogen-recognition molecule for muramyl dipeptide (MDP), NOD2 controls both innate and adaptive immune responses, through the regulation of cytokines, chemokines and antimicrobial peptides production. Notably, Nod2-deficient mice experienced increased susceptibility to enteric infection and to antigen-specific colitis. Furthermore, mutant mice bearing the orthologue of the major CD-associated NOD2(3020ins) allele showed increased susceptibility to DSS-induced colitis. However, many questions remain open. (i) Is antimicrobial function deficiency sufficient to initiate the development of CD? (ii) How impaired and mutant NOD2 might lead to increased adaptive immune response? (iii) How do the other disease-associated NOD2 mutations contribute to the development of chronic intestinal inflammation? Whatever the relevant mechanism(s), it provides a casual link between abnormal bacterial sensing and development of inflammatory disorders. Further work should now focus on restoring abnormal NOD2 function by modulating antimicrobial function and regulatory mechanisms of the adaptive immune system.

Effect of silver-threads-containing compression stockings on the cutaneous microcirculation: a double-blind, randomized cross-over study.

Recently silver fiber-containing compression stockings for the use in patients with chronic venous insufficiency (CVI) were introduced to the market. In order to gain some first insight into the effects of these new fabrics on the cutaneous microcirculation, a double-blind, randomized cross-over trial was performed in 10 healthy volunteers. A 3 days run-in phase preceded the (2 x10 days) treatment phases and was used to assess the reproducibility of the primary endpoint, which was the transcutaneous partial oxygen pressure (tcpO(2)) measured at a probe temperature of 44 degrees C in the perimalleolar region of the reference leg in supine and dependent leg positions. Coefficients of variation for double measured tcpO(2) values were 4.2% (3.1 SD) and 5.8% (6.0 SD) for the leg in supine and dependent position. The intra-individual comparison of the effects from both treatment phases (value end of treatment - start of treatment) resulted in a negative tcpO(2) net balance for the regular hosiery (-0.93 (2.7 SD) mmHg, supine; -1.1 (3.5 SD) mmHg, dependent) but a positive net balance for the silver fibers containing stockings (0.25 (4.0 SD) mmHg, supine; 1.7 (3.9 SD) mmHg, dependent). The inter-treatment differences were statistically significant for the leg in a dependent position. The trial provides first evidence that interweaving silver threads into regular compression stockings may result in a positive effect regarding the nutritive skin perfusion.

Increased poliovirus-specific intestinal antibody response coincides with promotion of Bifidobacterium longum-infantis and Bifidobacterium breve in infants: a randomized, double-blind, placebo-controlled trial.

To determine whether the size of the intestinal bifidobacterial population can influence the immune response to poliovirus vaccination in infants, we set up a randomized, placebo-controlled trial. From birth to 4 mo, infants were given a fermented infant formula (FIF) or a standard formula (placebo). Bifidobacteria were quantified monthly in infant stools. Antipoliovirus IgA response to Pentacoq was assessed before and 1 mo after the second vaccine injection. Thirty infants were randomized, and 20 completed the study (nine in the placebo group and 11 in the FIF group). Fecal bifidobacterial level was significantly higher with the FIF group at 4 mo of age (p=0.0498). Furthermore, B. longum/B. infantis carriage was higher at 4 mo in the FIF group (p=0.0399). Antipoliovirus IgA titers increased after Pentacoq challenge (p <0.001), and the rise was significantly higher in the FIF group (p <0.02). Antibody titers correlated with bifidobacteria, especially with B. longum/B. infantis and B. breve levels (p <0.002). Infants who harbored B. longum/B. infantis also exhibited higher levels of antipoliovirus IgAs (p <0.002). In conclusion, the present results indicate that antipoliovirus response can be triggered with a fermented formula that is able to favor intestinal bifidobacteria. Whether this effect on the immune system is achieved through the bifidogenic effect of the formula (mainly through B. longum/B. infantis and B. breve stimulation) or directly linked to compounds (i.e. peptides) produced by milk fermentation remains to be investigated.

Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin.

Three multiplex polymerase chain reactions (PCRs) targeted on Bifidobacterium and related species were designed to identify human species. The selected primers yielded amplified products of various sizes, each specific for a species. Three to four pairs were gathered in one PCR reaction and their specificity under multiplex conditions was confirmed using DNA from 26 reference strains. Using this technique on unidentified faecal strains, B. bifidum, B. longum and B. breve species were commonly recovered in infants while B. adolescentis, B. catenulatum/B. pseudocatenulatum continuum and B. longum species were predominant in adults. Thus, a single PCR can provide the assignment of a strain to one these species, reducing the number of PCR reactions and hands-on time for the identification of human isolates of bifidobacteria. Moreover, this technique is also applicable for the in situ detection of bifidobacteria in DNA extracts from human stools.

Pseudomonas grimontii sp. nov.

The vernacular name 'fluorescent Pseudomonas group 97-514' was coined for a group of 43 strains isolated from two French natural mineral waters. All these strains were gram-negative, rod-shaped and motile by means of a single polar flagellum. They produced fluorescent pigment (pyoverdin) on King B medium, catalase and cytochrome oxidase. They were capable of respiratory but not fermentative metabolism. They were not able to accumulate poly-beta-hydroxybutyrate and possessed an arginine dihydrolase system. DNA-DNA relatedness studies (S1 nuclease method) showed that the 43 strains of 'fluorescent Pseudomonas group 97-514' formed a genetically homogeneous group (DNA-DNA relatedness ranged from 70 to 100%). A total of 76 strains representing well-known or partially characterized species of the genus Pseudomonas sensu stricto had 7-56% DNA hybridization with strain CFML 97-514T. The highest DNA binding values were found with Pseudomonas veronii CIP 104663T (52%), Pseudomonas rhodesiae CIP 104664T (56%), Pseudomonas marginalis ATCC 10844T (56%), Pseudomonas gessardii CIP 105469T (53%) and Pseudomonas cedrella CIP 105541T (52%). Their unrelatedness was confirmed by deltaTm values greater than 7 degrees C. On the basis of the results of phenotypic and DNA-DNA hybridization studies, a novel Pseudomonas species, Pseudomonas grimontii sp. nov., is proposed for the 43 strains of 'fluorescent Pseudomonas group 97-514'. The type strain is strain CFML 97-514T (= CIP 106645T = ATCC BAA-140T). The G+C content of the DNA of the type strain was 58 mol%. A comparison of the complete 16S rRNA gene sequence of the type strain CFML 97-514T and the sequence of other strains of the genus Pseudomonas revealed that the novel species fell within the 'Pseudomonas fluorescens intrageneric cluster'. Members of P. grimontii grew at 4 degrees C but not at 41 degrees C. They were able to use D-xylose, alpha-L-rhamnose, alpha-aminobutyrate, meso-erythritol and itaconate as sole sources of carbon and energy and formed levan from sucrose. Strains do not possess lecithinase or Tween esterase activities. The clinical significance of P. grimontii is unknown.

[Helicobacter pylori infection--clinical aspects and indications for treatment]. / Helicobacter pylori-Infektion--Klinische Aspekte und Behandlungsindikationen.

Helicobacter pylori (H. p.) causes active chronic antrum gastritis in all infected patients. In a relatively small percentage complications of H. p.-gastritis including duodenal ulcer, gastric ulcer, giant fold gastritis, lymphocytic gastritis, autoimmune gastritis, gastric carcinoma and gastric MALT lymphoma may develop. Strongly recommended indications for eradication therapy include gastroduodenal ulcer disease, giant fold gastritis, lymphocytic gastritis, autoimmune gastritis, gastric MALT lymphoma, atrophic gastritis, corpus-predominant gastritis, post gastric cancer resection and patients who are first degree relatives of gastric cancer patients. Eradication therapy is controversial in patients with gastroesophageal reflux disease, functional dyspepsia and in patients in whom treatment with nonsteroidal antiinflammatory drugs (NSAID) or long-term treatment with proton pump inhibitors (PPI) is planned.

Pseudomonas mosselii sp. nov., a novel species isolated from clinical specimens.

Twenty-two fluorescent pseudomonad strains of clinical origin received as Pseudomonas fluorescens (10 strains), Pseudomonasputida (10 strains) and Pseudomonas sp. (2 strains), and 33 type strains of the genus Pseudomonas were studied by numerical analysis based on 280 phenotypic characters. Twelve of the 22 clinical isolates clustered within a specific group, cluster IV. The other strains clustered within groups containing well-characterized fluorescent Pseudomonas species or did not cluster. Strains belonging to cluster IV were phenotypically different from all other clusters and subclusters of fluorescent pseudomonads. DNA-DNA hybridization showed that cluster IV corresponded to a genomic group sharing 72-100% DNA relatedness. DNA-DNA hybridization values with 67 strains representing 30 species of the genus Pseudomonas sensu stricto, including six recently described species (Pseudomonas veronii, Pseudomonas rhodesiae, Pseudomonas libanensis, 'Pseudomonas orientalis', 'Pseudomonas cedrella' and Pseudomonas monteilii), were below 49%, the value found for P. monteilii. The DNA G+C content of the type strain was 63 mol%. Comparison of the 16S rRNA gene sequence of a representative strain of cluster IV (CFML 90-83T) with sequences of other strains of the genus Pseudomonas revealed that strain CFML 90-83T was part of the P. fluorescens intrageneric cluster. On the basis of phenotypic, DNA-DNA hybridization and phylogenetic analyses, a novel species, Pseudomonas mosselii sp. nov., is proposed for the 12 strains of cluster IV. The type strain is P. mosselii CFML 90-83T (= ATCC BAA-99T = CIP 105259T). The P. mosselii strains are phenotypically homogeneous and can be differentiated from other fluorescent species by several phenotypic features, including pyoverdine typing.