Guidelines for the Management of Helicobacter pylori Infection in Japan: 2009 Revised Edition

Background:  Over the past few years, the profile of Helicobacter pylori infection has changed in Japan. In particular, the relationship between H. pylori and gastric cancer has been demonstrated more clearly. Accordingly, the committee of the Japanese Society for Helicobacter Research has revised the guidelines for diagnosis and treatment of H. pylori infection in Japan.
Materials and Methods:  Four meetings of guidelines preparation committee were held from July 2007 to December 2008. In the new guidelines, recommendations for treatment have been classified into five grades according to the Minds Recommendation Grades, while the level of evidence has been classified into six grades. The Japanese national health insurance system was not taken into consideration when preparing these guidelines.
Results:  Helicobacter pylori eradication therapy achieved a Grade A recommendation, being useful for the treatment of gastric or duodenal ulcer, for the treatment and prevention of H. pylori -associated diseases such as gastric cancer, and for inhibiting the spread of H. pylori infection. Levels of evidence were determined for each disease associated with H. pylori infection. For the diagnosis of H. pylori infection, measurement of H. pylori antigen in the feces was added to the tests not requiring biopsy. One week of proton-pump inhibitor-based triple therapy (including amoxicillin and metronidazole) was recommended as second-line therapy after failure of first-line eradication therapy.
Conclusion:  The revised Japanese guidelines for H. pylori are based on scientific evidence and avoid the administrative restraints that applied to earlier versions .     

Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner

Ebolavirus (EBOV) is an enveloped, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever with mortality rates of up to 90% in humans and nonhuman primates. Previous studies suggest roles for clathrin- or caveolae-mediated endocytosis in EBOV entry; however, ebolavirus virions are long, filamentous particles that are larger than the plasma membrane invaginations that characterize clathrin- or caveolae-mediated endocytosis. The mechanism of EBOV entry remains, therefore, poorly understood. To better understand Ebolavirus entry, we carried out internalization studies with fluorescently labeled, biologically contained Ebolavirus and Ebolavirus-like particles (Ebola VLPs), both of which resemble authentic Ebolavirus in their morphology. We examined the mechanism of Ebolavirus internalization by real-time analysis of these fluorescently labeled Ebolavirus particles and found that their internalization was independent of clathrin- or caveolae-mediated endocytosis, but that they co-localized with sorting nexin (SNX) 5, a marker of macropinocytosis-specific endosomes (macropinosomes). Moreover, the internalization of Ebolavirus virions accelerated the uptake of a macropinocytosis-specific cargo, was associated with plasma membrane ruffling, and was dependent on cellular GTPases and kinases involved in macropinocytosis. A pseudotyped vesicular stomatitis virus possessing the Ebolavirus glycoprotein (GP) also co-localized with SNX5 and its internalization and infectivity were affected by macropinocytosis inhibitors. Taken together, our data suggest that Ebolavirus is internalized into cells by stimulating macropinocytosis in a GP-dependent manner. These findings provide new insights into the lifecycle of Ebolavirus and may aid in the development of therapeutics for Ebolavirus infection.     

Bermuda as an Evolutionary Life Raft for an Ancient Lineage of Endangered Lizards

Oceanic islands are well known for harboring diverse species assemblages and are frequently the basis of research on adaptive radiation and neoendemism. However, a commonly overlooked role of some islands is their function in preserving ancient lineages that have become extinct everywhere else (paleoendemism). The island archipelago of Bermuda is home to a single species of extant terrestrial vertebrate, the endemic skink Plestiodon (formerly Eumeces) longirostris. The presence of this species is surprising because Bermuda is an isolated, relatively young oceanic island approximately 1000 km from the eastern United States. Here, we apply Bayesian phylogenetic analyses using a relaxed molecular clock to demonstrate that the island of Bermuda, although no older than two million years, is home to the only extant representative of one of the earliest mainland North American Plestiodon lineages, which diverged from its closest living relatives 11.5 to 19.8 million years ago. This implies that, within a short geological time frame, mainland North American ancestors of P. longirostris colonized the recently emergent Bermuda and the entire lineage subsequently vanished from the mainland. Thus, our analyses reveal that Bermuda is an example of a “life raft” preserving millions of years of unique evolutionary history, now at the brink of extinction. Threats such as habitat destruction, littering, and non-native species have severely reduced the population size of this highly endangered lizard.     

Decay Kinetics of an Interferon Gamma Release Assay with Anti-Tuberculosis Therapy in Newly Diagnosed Tuberculosis Cases

Background

Qualitative and quantitative changes in IGRA response offer promise as biomarkers to monitor Tuberculosis (TB) drug therapy, and for the comparison of new interventions. We studied the decay kinetics of TB-specific antigen T-cell responses measured with an in-house ELISPOT assay during the course of therapy.

Methods

Newly diagnosed sputum smear positive TB cases with typical TB chest radiographs were recruited. All patients were given standard anti-TB treatment. Each subject was followed up for 6 months and treatment outcomes were documented. Blood samples were obtained for the ESAT-6 and CFP-10 (EC) ELISPOT at diagnosis, 1-, 2-, 4- and 6-months. Qualitative and quantitative reversion of the ELISPOT results were assessed with McNemar test, conditional logistic regression and mixed-effects hierarchical Poisson models.

Results

A total of 116 cases were recruited and EC ELISPOT was positive for 87% (95 of 109) at recruitment. There was a significant decrease in the proportion of EC ELISPOT positive cases over the treatment period (p<0.001). Most of the reversion occurred between the start and first month of treatment and at completion at 6 months. ESAT-6 had higher median counts compared to CFP-10 at all time points. Counts for each antigen declined significantly with therapy (p<0.001). Reverters had lower median SFUs at the start of treatment compared to non-Reverters for both antigens. Apart from the higher median counts for non-Reverters, no other risk factors for non-reversion were found.

Conclusions

TB treatment induces qualitative and quantitative reversion of a positive in-house IGRA in newly diagnosed cases of active TB disease. As this does not occur reliably in the majority of cured individuals, qualitative and quantitative reversion of an IGRA ELISPOT has limited clinical utility as a surrogate marker of treatment efficacy.     

Gene Knock-Outs of Inositol 1,4,5-Trisphosphate Receptors Types 1 and 2 Result in Perturbation of Cardiogenesis

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R1, 2, and 3) are intracellular Ca²⁺ release channels that regulate various vital processes. Although the ryanodine receptor type 2, another type of intracellular Ca²⁺ release channel, has been shown to play a role in embryonic cardiomyocytes, the functions of the IP₃Rs in cardiogenesis remain unclear.

Methodology/Principal Findings

We found that IP₃R1^−/−-IP₃R2^−/− double-mutant mice died in utero with developmental defects of the ventricular myocardium and atrioventricular (AV) canal of the heart by embryonic day (E) 11.5, even though no cardiac defect was detectable in IP₃R1^−/− or IP₃R2^−/− single-mutant mice at this developmental stage. The double-mutant phenotype resembled that of mice deficient for calcineurin/NFATc signaling, and NFATc was inactive in embryonic hearts from the double knockout-mutant mice. The double mutation of IP₃R1/R2 and pharmacologic inhibition of IP₃Rs mimicked the phenotype of the AV valve defect that result from the inhibition of calcineurin, and it could be rescued by constitutively active calcineurin.

Conclusions/Significance

Our results suggest an essential role for IP₃Rs in cardiogenesis in part through the regulation of calcineurin-NFAT signaling.     

Expanded Polyfunctional T Cell Response to Mycobacterial Antigens in TB Disease and Contraction Post-Treatment

Background

T cells producing multiple factors have been shown to be required for protection from disease progression in HIV but we have recently shown this not to be the case in TB. Subjects with active disease had a greater proportion of polyfunctional cells responding to ESAT-6/CFP-10 stimulation than their infected but non-diseased household contacts (HHC). We therefore wanted to assess this profile in subjects who had successfully completed standard TB chemotherapy.

Methods

We performed a cross-sectional study using PBMC from TB cases (pre- and post-treatment) and HHC. Samples were stimulated overnight with TB antigens (ESAT-6/CFP-10 and PPD) and their CD4+ and CD8+ T cells were assessed for production of CD107a, IFN-γ, IL-2 and TNF-α and the complexity of the responses was determined using SPICE and PESTLE software.

Results and Conclusions

We found that an increase in complexity (i.e., production of more than 1 factor simultaneously) of the T cell profile was associated with TB disease and that this was significantly reduced following TB treatment. This implies that T cells are able to respond adequately to TB antigens with active disease (at least initially) but the ability of this response to protect the host from disease progression is hampered, presumably due to immune evasion strategies by the bacteria. These findings have implications for the development of new diagnostics and vaccine strategies.     

Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1H46R-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

Background

ALS2/alsin is a guanine nucleotide exchange factor for the small GTPase Rab5 and involved in macropinocytosis-associated endosome fusion and trafficking, and neurite outgrowth. ALS2 deficiency accounts for a number of juvenile recessive motor neuron diseases (MNDs). Recently, it has been shown that ALS2 plays a role in neuroprotection against MND-associated pathological insults, such as toxicity induced by mutant Cu/Zn superoxide dismutase (SOD1). However, molecular mechanisms underlying the relationship between ALS2-associated cellular function and its neuroprotective role remain unclear.

Methodology/Principal Findings

To address this issue, we investigated the molecular and pathological basis for the phenotypic modification of mutant SOD1-expressing mice by ALS2 loss. Genetic ablation of Als2 in SOD1^H46R, but not SOD1^G93A, transgenic mice aggravated the mutant SOD1-associated disease symptoms such as body weight loss and motor dysfunction, leading to the earlier death. Light and electron microscopic examinations revealed the presence of degenerating and/or swollen spinal axons accumulating granular aggregates and autophagosome-like vesicles in early- and even pre-symptomatic SOD1^H46R mice. Further, enhanced accumulation of insoluble high molecular weight SOD1, poly-ubiquitinated proteins, and macroautophagy-associated proteins such as polyubiquitin-binding protein p62/SQSTM1 and a lipidated form of light chain 3 (LC3-II), emerged in ALS2-deficient SOD1^H46R mice. Intriguingly, ALS2 was colocalized with LC3 and p62, and partly with SOD1 on autophagosome/endosome hybrid compartments, and loss of ALS2 significantly lowered the lysosome-dependent clearance of LC3 and p62 in cultured cells.

Conclusions/Significance

Based on these observations, although molecular basis for the distinctive susceptibilities to ALS2 loss in different mutant SOD1-expressing ALS models is still elusive, disturbance of the endolysosomal system by ALS2 loss may exacerbate the SOD1^H46R-mediated neurotoxicity by accelerating the accumulation of immature vesicles and misfolded proteins in the spinal cord. We propose that ALS2 is implicated in endolysosomal trafficking through the fusion between endosomes and autophagosomes, thereby regulating endolysosomal protein degradation in vivo.     

Specific determination of threonine in biological samples by gas chromatography with electron capture detection

Threonine was oxidized into acetaldehyde at 0 degrees C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 microl sample solution and the determination limit of threonine was 1 nmol in 200 microl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.     

Establishment of a primary culture model of mouse uterine and vaginal stroma for studying in vitro estrogen effects

Effects of 17beta-estradiol (E2) on uterine and vaginal epithelial cell proliferation could be mediated by stromal cell-derived paracrine factors. To study the epithelial-stromal interactions in mice, an in vitro model of uterine and vaginal stromal cells of immature mice is essential. Therefore, we established a primary culture model of stromal cells both from uterus and vagina and examined the effect of E2 on proliferation of cultured stromal cells. We found that E2 stimulated proliferation of stromal cells from both organs in vitro, showing an increase in the number of cells and the percentage of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells. Interestingly, vaginal stromal cells responded to lower E2 than uterine stromal cells in proliferation (10(-12) M vs. 10(-8) M) and BrdU labeling (10(-14) - 10(-10) M vs. 10(-10) - 10(-6) M). To examine the effect of E2 in vivo, cells were grafted into the subrenal capsule of the host mice and grown for 2 weeks. The BrdU labeling in cultured stromal cells was increased by E2 in vivo. To examine the effect of cultured stromal cells on epithelial cell proliferation, uterine and vaginal epithelium of adult mice were separated, recombined with the cultured stromal cells, and grafted under the renal capsule of hosts for 3 weeks. Epithelial cells recombined with cultured stromal cells showed simple columnar morphology in uterine grafts and stratified and keratinized morphology in vaginal grafts under the influence of the hormonal environment of the hosts. The BrdU labeling in epithelial cells was increased by E2, suggesting that cultured stromal cells can stimulate epithelial cell proliferation. In conclusion, we established a primary culture model of uterine and vaginal stromal cells, which can be mitogenically stimulated by E2 in vitro and in vivo after being grafted under the renal capsule. This culture system will be useful for investigating the underlying molecular mechanisms of uterine and vaginal epithelial-stromal interactions.     

Divergent selection on opsins drives incipient speciation in Lake Victoria cichlids

Divergent natural selection acting on ecological traits, which also affect mate choice, is a key element of ecological speciation theory, but has not previously been demonstrated at the molecular gene level to our knowledge. Here we demonstrate parallel evolution in two cichlid genera under strong divergent selection in a gene that affects both. Strong divergent natural selection fixed opsin proteins with different predicted light absorbance properties at opposite ends of an environmental gradient. By expressing them and measuring absorbance, we show that the reciprocal fixation adapts populations to divergent light environments. The divergent evolution of the visual system coincides with divergence in male breeding coloration, consistent with incipient ecological by-product speciation.