Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.     

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Adipose Tissue from Obese Rats

Objective

Obesity is associated with low-grade chronic inflammation. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery would reduce activation of the NLRP3 inflammasome in metabolically active adipose tissue (AT) of obese rats, and this change would be related to decreases in body weight and improved glycemic control.

Methods

Omental, mesenteric and subcutaneous fat depots were collected from Sprague-Dawley rats: Sham control and RYGB; 90-days after surgery. NLRP3, caspase–1, apoptosis-associated speck-like protein (ASC), IL–1β, IL–18, IL–6 and MCP–1 gene and protein expression were quantified. Glucose metabolism was assessed by oral glucose tolerance test (OGTT).

Results

Compared to Sham surgery controls, RYGB surgery decreased IL–6, MCP–1, NLRP3, IL–18, caspase–1 and ASC in omental fat, and decreased IL–6, MCP1, IL–1β, IL–18, caspase–1 and ASC gene expression in mesenteric fat. We observed differential gene expression between visceral and subcutaneous fat for IL–6 and IL–1β, both being downregulated by RYGB in visceral, and upregulated in subcutaneous depots. These changes in gene expression were accompanied by a decrease in NLRP3, ASC, IL–18, caspase–1 and IL–1β protein expression in omental tissue. We found a positive correlation between caspase–1, ASC, MCP–1, IL–18 and IL–6 gene expression following surgery and glucose AUC response in omental fat, while the change in glucose AUC response correlated with caspase–1 gene expression in subcutaneous fat.

Conclusion

This study demonstrates that bariatric surgery reverses inflammation in visceral adipose tissue by suppressing NLRP3 inflammasome activation. These are the first data to implicate the NLRP3 inflammasome in diabetes remission after RYGB surgery.     

Cardiovascular Patterning as Determined by Hemodynamic Forces and Blood Vessel Genetics

Background

Vascular patterning depends on coordinated timing of arteriovenous specification of endothelial cells and the concomitant hemodynamic forces supplied by the onset of cardiac function. Using a combination of 3D imaging by OPT and embryo registration techniques, we sought to identify structural differences between three different mouse models of cardiovascular perturbation.

Results

Endoglin mutant mice shared a high degree of similarity to Mlc2a mutant mice, which have been shown to have a primary developmental heart defect causing secondary vessel remodeling failures. Dll4 mutant mice, which have well-characterized arterial blood vessel specification defects, showed distinct differences in vascular patterning when compared to the disruptions seen in Mlc2a ^-/- and Eng ^-/- models. While Mlc2a ^-/- and Eng ^-/- embryos exhibited significantly larger atria than wild-type, Dll4 ^-/- embryos had significantly smaller hearts than wild-type, but this quantitative volume decrease was not limited to the developing atrium. Dll4 ^-/- embryos also had atretic dorsal aortae and smaller trunks, suggesting that the cardiac abnormalities were secondary to primary arterial blood vessel specification defects.

Conclusions

The similarities in Eng ^-/- and Mlc2a ^-/- embryos suggest that Eng ^-/- mice may suffer from a primary heart developmental defect and secondary defects in vessel patterning, while defects in Dll4 ^-/- embryos are consistent with primary defects in vessel patterning.     

The Interplay of Habitat and Seed Type on Scatterhoarding Behavior in a Fragmented Afromontane Forest Landscape

Scatterhoarding by rodents, whereby seeds are collected and stored for later consumption, can result in seed dispersal. Seeds may be covered in litter on the forest floor (cached) or buried. This is particularly so in the Neotropics for large, nutritious seeds, and where primary dispersers are rare or missing. In African forests, contemporary anthropogenic pressures such as hunting, forest degradation, and fragmentation are contributing toward major declines in large frugivores, yet the potential for scatterhoarding to mitigate this loss is largely unknown. In this study, we used thread‐marked seed to explore the balance between seed predation and dispersal by rodents in Afromontane forest. We studied two tree species in three habitats: (1) continuous forest; (2) continuous forest edge, and (3) small, degraded riparian forest patches. We found that seed removal rates were high and almost the same in all three habitats for both tree species, but that the predation/dispersal balance differed among habitats. In continuous forest, more seeds of each species were scatterhoarded than depredated, and rates of scatterhoarding differed between the two species. In all habitats, burying seeds up to 2 cm belowground was more common than caching. Distances seeds were moved was approximately five times greater in continuous forest than in forest edge or riparian patches. We found strong evidence to suggest that the African pouched rat, Cricetomys sp. nov was responsible for the scatterhoarding.     

Detection of Botrytis cinerea by loop-mediated isothermal amplification

Aims: To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop‐mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. Methods and Results: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross‐reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real‐time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. Conclusions: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. Significance and Impact of the Study: The LAMP method combines the sensitivity and specificity of nucleic acid‐based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on‐site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.     

Environmental Management for Malaria Control: Knowledge and Practices in Mvomero,Tanzania

Environmental conditions play an important role in the transmission of malaria; therefore, regulating these conditions can help to reduce disease burden. Environmental management practices for disease control can be implemented at the community level to complement other malaria control methods. This study assesses current knowledge and practices related to mosquito ecology and environmental management for malaria control in a rural, agricultural region of Tanzania. Household surveys were conducted with 408 randomly selected respondents from 10 villages and qualitative data were collected through focus group discussions and in-depth interviews. Results show that respondents are well aware of the links between mosquitoes, the environment, and malaria. Most respondents stated that cleaning the environment around the home, clearing vegetation around the home, or draining stagnant water can reduce mosquito populations, and 63% of respondents reported performing at least one of these techniques to protect themselves from malaria. It is clear that many respondents believe that these environmental management practices are effective malaria control methods, but the actual efficacy of these techniques for controlling populations of vectors or reducing malaria prevalence in the varying ecological habitats in Mvomero is unknown. Further research should be conducted to determine the effects of different environmental management practices on both mosquito populations and malaria transmission in this region, and increased participation in effective techniques should be promoted.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle α‐actin gene

Smooth muscle α actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells (vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full‐length SMA promoter and intronic sequences. We determined that the founders and F1 progeny of five independent lines contain 1–3 copies of the mCherry‐substituted BAC vector. Furthermore, we characterized the expression of SMA‐mCherry in relation to endogenous SMA in the embryo and in adult tissues, and found that the transgenic reporter in each line recapitulated endogenous SMA expression at all time points. We were also able to isolate SMA expressing cells from embryonic tissues using fluorescence‐activated cell sorting (FACS). We demonstrated that this marker can be combined with other vital fluorescent reporters and it can be used for live imaging of embryonic cardiodynamics. Therefore, these transgenic mice will be useful for isolating live SMA‐expressing cells via FACS and for studying the emergence, behavior, and regulation of SMA‐expressing cells, including vascular smooth muscle cells and pericytes throughout embryonic and postnatal development. genesis 48:457–463, 2010. © 2010 Wiley‐Liss, Inc.     

Microbial community biofabrics in a geothermal mine adit

Speleothems such as stalactites and stalagmites are usually considered to be mineralogical in composition and origin; however, microorganisms have been implicated in the development of some speleothems. We have identified and characterized the biological and mineralogical composition of mat-like biofabrics in two novel kinds of speleothems from a 50 degrees C geothermal mine adit near Glenwood Springs, CO. One type of structure consists of 2- to 3-cm-long, 3- to 4-mm-wide, leather-like, hollow, soda straw stalactites. Light and electron microscopy indicated that the stalactites are composed of a mineralized biofabric with several cell morphotypes in a laminated form, with gypsum and sulfur as the dominant mineral components. A small-subunit rRNA gene phylogenetic community analysis along the stalactite length yielded a diverse gradient of organisms, with a relatively simple suite of main constituents: Thermus spp., crenarchaeotes, Chloroflexi, and Gammaproteobacteria. PCR analysis also detected putative crenarchaeal ammonia monooxygenase subunit A (amoA) genes in this community, the majority related to sequences from other geothermal systems. The second type of speleothem, dumpling-like rafts floating on a 50 degrees C pool on the floor of the adit, showed a mat-like fabric of evidently living organisms on the outside of the dumpling, with a multimineral, amorphous, gypsum-based internal composition. These two novel types of biofabrics are examples of the complex roles that microbes can play in mineralization, weathering, and deposition processes in karst environments.