Differential organization of the local immune response in patients with active cavitary tuberculosis or with nonprogressive tuberculoma

BACKGROUND: In 90% of all cases, Mycobacterium tuberculosis infection results in latency rather than active disease, with the pathogen being contained within granulomatous lesions at the site of primary infection. Failure of this containment leads to reactivation of postprimary tuberculosis (TB). The regional immune processes that sustain the delicate balance with persistent M. tuberculosis, however, remain unclear. METHODS: We compared activation statuses, biological functions, and interactions of host immune cells in human nonprogressive tuberculoma and active cavitary tuberculous lung tissue. RESULTS: Dissection of early granuloma formations revealed differential cellular distribution and activation statuses of distinct cell types in different regions relative to the central caseotic caverna or the tuberculoma in tuberculous lung tissue. In patients with tuberculoma with latent infection, distant parts of lung tissue exhibited strong vascularization and profound proliferative activity, indicating that continuous immune defense is required for mycobacterial containment, which is absent in cavitary tuberculous lung lesions. CONCLUSIONS: We conclude that differential regulation of the local immune response is crucial for the containment of M. tuberculosis and that a continuous antigen-specific cross talk between the host immune system and M. tuberculosis is ensured during latency. This activation requires sufficient supply of nutrients and well-coordinated structural organization, both of which are lost during reactivation of TB.     

Slowing down as an early warning signal for abrupt climate change

In the Earth's history, periods of relatively stable climate have often been interrupted by sharp transitions to a contrasting state. One explanation for such events of abrupt change is that they happened when the earth system reached a critical tipping point. However, this remains hard to prove for events in the remote past, and it is even more difficult to predict if and when we might reach a tipping point for abrupt climate change in the future. Here, we analyze eight ancient abrupt climate shifts and show that they were all preceded by a characteristic slowing down of the fluctuations starting well before the actual shift. Such slowing down, measured as increased autocorrelation, can be mathematically shown to be a hallmark of tipping points. Therefore, our results imply independent empirical evidence for the idea that past abrupt shifts were associated with the passing of critical thresholds. Because the mechanism causing slowing down is fundamentally inherent to tipping points, it follows that our way to detect slowing down might be used as a universal early warning signal for upcoming catastrophic change. Because tipping points in ecosystems and other complex systems are notoriously hard to predict in other ways, this is a promising perspective.     

Body weight is not always a good predictor of longevity in mice

There have been some observations that low body weight and a low level of some hormones (e.g. IGF-1) during the first half of life are predictors of longer life in mice. However, contradictions in the available data on the biomarkers of aging and predictors of longevity have shown that the research in these fields has become a controversial pursuit. In our study we addressed the following questions: (i) Can particular physiological parameters (body weight, food intake, estrus function, body temperature, incidence of chromosome aberrations in bone marrow cells) measured at the age of 3 and 12 months be a predictor of longevity and the rate of tumor development in five strains of mice? (ii) Can a heavy body weight at the age of 3 and 12 months be a predictor of longevity and high tumor risk in five strains of mice? Mice of five strains-CBA, SHR, SAMR, SAMP and transgenic HER-2/neu (FVB/N)-were under observation from the age of 2-3 months until natural death. Body weight and temperature, food consumption, and estrous cycle were longitudinally studied in all animals. Tumors discovered at autopsy were studied morphologically. We calculated the life span's parameters (mean, maximum, mortality rate, mortality rate doubling time) as well as their correlation with other parameters studied. The longest living CBA mice have the lowest body weight at the ages of 3 and 12 months, the lowest food consumption, body temperature, incidence of chromosome aberrations and spontaneous tumor incidence. In comparison with all other mouse strains they also have the latest disturbances in estrus function and highest body weight gain. The shortest living transgenic HER-2/neu mice have the lowest weight at the ages of 12 months, the lowest body weight gain, maximal body temperature, the most rapid disturbances in estrus function and the highest incidence of chromosome aberrations and tumor incidence in comparison to all other mouse strains. Our findings have shown that heavier body weight at the age of 12 months is a predictor of longevity in female CBA and SAMP mice but not in SHR, SAMR and HER-2/neu mice. Excessive body weight at the ages of 3 or 12 months is not a predictor of increased tumor risk in the strains studied. In general, the existence and direction of a significant correlation between body weight and life span depends upon the animals' age and genotype.     

Pyridoxal isonicotinoyl hydrazone (PIH) and its analogs as protectants against anthracycline-induced cardiotoxicity

The risk of cardiotoxicity is the main drawback of anthracycline antibiotics. However, these drugs remain among the most effective and frequently used anti cancer drugs. In this study we aimed to assess the cardioprotective effects of aroylhydrazone iron (FE) chelators: pyridoxal isonicotinoyl hydrazone (PIH) and its two analogs: salicyladehyde isonicotinoyl hydrazone (SIH) and pyridoxal o-chlorbenzoyl hydrazone (o-108). In rabbits, chronic treatment with daunorubicin (DAU) (3 mg/kg weekly for 10 weeks) induced mortality (33%) as well as left ventricular (LV) dysfunction. Co-administrations of PIH (25 mg/kg, i.p.), SIH hydrochloride [1 mg/kg, iv] as well as o-108 (10 mg/kg, i.p.), fully prevented premature deaths and most of the DAU-induced functional impairments were significantly suppressed. However, when 2- to 2.5-fold higher doses of the chelators were used, they led to rather paradoxical and mostly negative results regarding both cardioprotection and overall mortality.     

High-dose immunosuppressive therapy with autologous hematopoietic stem cell transplantation as a treatment option in multiple sclerosis

High-dose immunosuppressive therapy (HDIT) with autologous hematopoietic stem cell transplantation (auto-HSCT) is a new and promising approach to the treatment of multiple sclerosis (MS) patients because currently there are no effective treatment methods for this disease. In this article, we present results of a prospective clinical study of efficacy of HDIT + auto-HSCT in MS patients. The following treatment strategies were employed in the study: "early," "conventional," and "salvage/late" transplantation. Fifty patients with various types of MS were included in this study. No toxic deaths were reported among 50 MS patients; transplantation procedure was well-tolerated by the patients. The efficacy analysis was performed in 45 patients. Twenty-eight patients achieved an objective improvement of neurological symptoms, defined as at least 0.5-point decrease in the Expanded Disability Status Scale (EDSS) score as compared to the baseline and confirmed during 6 months, and 17 patients had disease stabilization (steady EDSS level as compared to the baseline and confirmed during 6 months). The progression-free survival at 6 years after HDIT + auto-HSCT was 72%. Magnetic resonance imaging data were available in 37 patients before transplantation showing disease activity in 43.3%. No active, new, or enlarging lesions were registered in patients without disease progression. In conclusion, HDIT + auto-HSCT suggests positive results in management of patients with different types of MS. Identification of treatment strategies based on the level of disability, namely "early," "conventional," and "salvage/late" transplantation, appears to be feasible to improve treatment outcomes.     

Transgenic alteration of Toll immune pathway in the female mosquito Aedes aegypti

Reverse genetics is a powerful tool for understanding gene functions and their interactions in the mosquito innate immunity. We took the transgenic approach, in combination with the RNA interference (RNAi) technique, to elucidate the role of mosquito REL1, a homolog of Drosophila Dorsal, in regulation of Toll immune pathway in the mosquito Aedes aegypti. By transforming the mosquitoes with DeltaREL1-A or a double-stranded RNA construct of REL1 driven by the female fat body-specific vitellogenin (Vg) promoter with the pBac[3xP3-EGFP, afm] vector, we generated two different transgenic mosquito strains, one with overexpressed AaREL1 and the second with AaREL1 knockdown. Both strains had a single copy of the respective transgene, and the expression in both transgenic mosquitoes was highly activated by blood feeding. Vg-DeltaREL1-A transgenic mosquitoes activate Toll immune pathway in the fat body by blood feeding. The overexpression of both isoforms, AaREL1-A and AaREL1-B, in Vg-DeltaREL1-A transgenic mosquitoes resulted in the concomitant activation of Aedes Sp?tzle1A and Serpin-27A, independent of septic injury. The same phenotype was observed in the mosquitoes with RNAi knockdown of an Aedes homolog to Drosophila cactus, an IkappaB inhibitor of Drosophila Toll pathway. The effect of the transgenic RNAi knockdown of AaREL1 on mosquito innate immunity was revealed by increased susceptibility to the entomopathogenic fungus Beauveria bassiana and the reduced induction of Spz1A and Serpin-27A gene expression after fungal challenge. These results have proven that AaREL1 is a key downstream regulator of Toll immune pathway in the mosquito A. aegypti.     

The cost-effectiveness of HIV preventive measures among injecting drug users in Svetlogorsk, Belarus

AIM: To undertake a cost-effectiveness analysis of a harm reduction and HIV prevention project for injecting drug users (IDUs) in Eastern Europe. Economic evaluation methods were adapted to consider the effect of an 8-month financing gap that negatively impacted on project implementation. DESIGN: Financial and economic costs of implementing the intervention were analysed retrospectively. The data were also modelled to estimate the costs of a fully functioning project. Estimates of the intervention impact on sexual and drug injecting behaviour were obtained from existing pre- and post-intervention behavioural surveys of IDUs. A dynamic mathematical model was used to translate these changes into estimates of HIV infections averted among IDUs and their sexual partners. Projections of the potential effect of the shortfall in funding on the impact and cost-effectiveness of the intervention were made. SETTING: Svetlogorsk, Belarus, where in 1997 the IDU HIV prevalence was 74%. FINDINGS: The intervention averted 176 HIV infections (95% CI 60-270) with a cost-effectiveness of 359 dollars per HIV infection averted (95% CI 234-1054 dollars). Without the 2311 dollars reduction (7%) in financing, the estimated cost-effectiveness ratio of the project would have been 11% lower. The costing methods used to measure donated mass media can substantially influence cost and cost-effectiveness estimates. CONCLUSIONS: Harm reduction activities among IDUs can be cost-effective, even when IDU HIV prevalence and incidence is high. Relatively small shortfalls in funding reduce impact and cost-effectiveness. Increased and consistent allocation of resources to harm reduction projects could significantly reduce the pace of the HIV epidemic in Eastern Europe.     

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.Complexities of natural biological systems make it difficult to understand and define precisely the roles of individual genes and their integrated functions. The use of model organisms with a relatively small number of genes enables the isolation of core biological processes from their complex regulatory networks for extensive characterization. However, even the simplest natural microbes contain many genes of unknown function, as well as genes that can be singly or simultaneously deleted without any noticeable effect on growth rate in a laboratory setting (Hutchison et al. 1999; Glass et al. 2006; Posfai et al. 2006). Ill-defined genes and those mediating functional redundancies both compound the challenge of understanding even the simplest life forms.Toward generating a minimal cell where every gene is essential for the axenic viability of the organism, we are pursuing strategies to reduce the 1-Mb genome of Mycoplasma mycoides JCVI-syn1.0 (Gibson et al. 2010). Because we can (1) introduce this genome into yeast and maintain it as a plasmid (Benders et al. 2010; Karas et al. 2013a); and (2) “transplant” the genome from yeast into mycoplasma recipient cells (Lartigue et al. 2009), genetic tools in yeast are available for reducing this bacterial genome. Several systems offer advanced tools for bacterial genome engineering. Here we further exploit distinctive features of yeast for this purpose.Methods for serially replacing genomic regions with selectable markers are limited by the number of available markers. One effective approach is to reuse the same marker after precise and scarless marker excision (Storici et al. 2001). We have previously used a self-excising marker (Noskov et al. 2010) six times in yeast to generate a JCVI-syn1.0 genome lacking all six restriction systems (JCVI-syn1.0 ∆1-6) (Karas et al. 2013a). Despite the advantages of scarless engineering, sequential procedures are time-consuming. When applied to poorly characterized genes with the potential to interact with other genes, some paths for multigene knockout may lead to dead ends that result from synergistic mutant phenotypes. When a dead end is reached, sequentially returning to a previous genome in an effort to find a detour to a viable higher-order multimutant may be prohibitively time-consuming.An alternative approach to multigene engineering, available in yeast, is to prepare a set of single mutants and combine the deletions into a single strain via cycles of mating and meiotic recombination (Fig. 1A; Pinel et al. 2011; Suzuki et al. 2011, 2012). With a green fluorescent protein (GFP) reporter gene inserted in each deletion locus, the enrichment of higher-order yeast deletion strains in the meiotic population can be accomplished using flow cytometry. Here we apply this method to the JCVI-syn1.0 ∆1-6 exogenous, bacterial genome harbored in yeast to nonsequentially assemble deletions for genes predicted to be individually deletable based on biological knowledge or transposon-mediated disruption data. The functional identification of simultaneously deletable regions is expected to accelerate the effort to construct a minimal genome.Open in a separate windowFigure 1.Progressive clustering of deleted genomic segments. (A) Scheme of the method. Light blue oval represents a bacterial cell. Black ring or horizontal line denotes a bacterial genome, with the orange box indicating the yeast vector used as a site for linearization and recircularization. Gray shape denotes a yeast cell. Green dot in the genome indicates a deletion replaced with a GFP marker. (B) Map of deleted regions. Orange box indicates the yeast vector sequence used for genome linearization and recircularization. Green boxes indicate regions deleted in multimutant mycoplasma strains. Blue boxes denote restriction modification (RM) systems that are also deleted in the strains. (C) Pulsed-gel electrophoresis result for deleted genomes. The starting strain was the JCVI-syn1.0 ∆1–6 strain (1062 kb). Two strains were analyzed for each design of simultaneous deletion (962 kb for eight-deletion or 974 kb for seven-deletion genome). Ladder is a set of yeast chromosomes (New England BioLabs). (D) GFP-RFP ratio sorting result. Standard sorting was compared with sorting based on a GFP-RFP ratio (Methods).     

Cadmium specific proteomic responses of a highly resistant Pseudomonas aeruginosa san ai

Pseudomonas aeruginosa san ai is a promising candidate for bioremediation of cadmium pollution, as it resists a high concentration of up to 7.2 mM of cadmium. Leaving biomass of P. aeruginosa san ai exposed to cadmium has a large biosorption potential, implying its capacity to extract heavy metal from contaminated medium. In the present study, we investigated tolerance and accumulation of cadmium on protein level by shotgun proteomics approach based on liquid chromatography and tandem mass spectrometry coupled with bioinformatics to identify proteins. Size exclusion chromatography was used for protein prefractionation to preserve native forms of metalloproteins and protein complexes. Using this approach a total of 60 proteins were observed as up-regulated in cadmium-amended culture. Almost a third of the total numbers of up-regulated were metalloproteins. Particularly interesting are denitrification proteins which are over expressed but not active, suggesting their protective role in conditions of heavy metal exposure. P. aeruginosa san ai developed a complex mechanism to adapt to cadmium, based on: extracellular biosorption, bioaccumulation, the formation of biofilm, controlled siderophore production, enhanced respiration and modified protein profile. An increased abundance of proteins involved in: cell energy metabolism, including denitrification proteins; amino acid metabolism; cell motility and posttranslational modifications, primarily based on thiol-disulfide exchange, were observed. Enhanced oxygen consumption of biomass in cadmium-amended culture versus control was found. Our results signify that P. aeruginosa san ai is naturally well equipped to overcome and survive high doses of cadmium and, as such, has a great potential for application in bioremediation of cadmium polluted sites.

When exposed to cadmium a highly resistant strain P. aeruginosa san ai responds by an increased metalloprotein expression (particularly denitrification proteins), an enhanced respiration, and a pronounced thiol-disulfide protein modifications.