HIV Viremia and T-Cell Activation Differentially Affect the Performance of Glomerular Filtration Rate Equations Based on Creatinine and Cystatin C

Background

Serum creatinine and cystatin C are used as markers of glomerular filtration rate (GFR). The performance of these GFR markers relative to exogenously measured GFR (mGFR) in HIV-positive individuals is not well established.

Methods

We assessed the performance of the chronic kidney disease epidemiology collaboration equations based on serum concentrations of creatinine (eGFR_cr), cystatin C (eGFR_cys) and both biomarkers combined (eGFR_cr-cys) in 187 HIV-positive and 98 HIV-negative participants. Measured GFR was calculated by plasma iohexol clearance. Bias and accuracy were defined as the difference between eGFR and mGFR and the percentage of eGFR observations within 30% of mGFR, respectively. Activated CD4 and CD8 T-cells (CD38+ HLA-DR+) were measured by flow cytometry.

Results

The median mGFR was >100 ml/min/1.73 m² in both groups. All equations tended to be less accurate in HIV-positive than in HIV-negative subjects, with eGFR_cr-cys being the most accurate overall. In the HIV-positive group, eGFR_cys was significantly less accurate and more biased than eGFR_cr and eGFR_{cr_cys}. Additionally eGFR_cys bias and accuracy were strongly associated with use of antiretroviral therapy, HIV RNA suppression, and percentages of activated CD4 or CD8 T-cells. Hepatitis C seropositivity was associated with larger eGFR_cys bias in both HIV-positive and HIV-negative groups. In contrast, eGFR_cr accuracy and bias were not associated with HIV-related factors, T-cell activation, or hepatitis C.

Conclusions

The performance of eGFR_cys relative to mGFR was strongly correlated with HIV treatment factors and markers of T-cell activation, which may limit its usefulness as a GFR marker in this population.     

Fluxomics with Ratiometric Metabolite Dyes

Today''s major excitement in biology centers on signaling: How can a cell or organism measure the myriad of environmental cues, integrate it, and acclimate to the new conditions? Hormonal signals and second messengers are in the focus of most of these studies, e.g., regulation of glucose transporter GLUT4 cycling by insulin, or regulation of plant growth by auxin or brassinosteroids.^1–3 In comparison, we generally assume that we know almost everything about basic metabolism since it has been studied for many decades; for example we know since the early 80s that allosteric regulation by fructose-2,6-bisphophate plays an important role in regulating glycolysis in plants and animals.⁴ This may be the reason why studies of metabolism appear to be a bit out of fashion. But if we look to other organisms such as E. coli or yeast, we rapidly realize that metabolism is controlled by complex interconnected signaling networks, and that we understand little of these signaling networks in humans and plants.^5,6 As it turns out, the cell registers many metabolites, and flux through the pathways is regulated using complex signaling networks that involve calcium as well as hormones.Key Words: flux, fluxome, glucose, glutamate, phosphate, sucrose, fluorescence resonance energy transfer, biosensorOne of the reasons for the fable for hormones lies in the simple fact that it is easier to observe macroscopic changes, such as changes in the architecture of a plant than to determine metabolite levels, but also here new tools are urgently needed that allow quantification of these small molecules. Visualization of starch levels provided a significant advance, and in combination with mutant screens allowed to identify fundamental components of starch metabolism.^7–9 The biggest advance for the signaling field was the development of advanced chemical and genetically encoded calcium dyes.^10–12 No such dyes are available for hormones or metabolites, as soon as we try to determine levels of metabolites (or signaling molecules), we run into the issues of compartmentation and cellular differences in tissues. Today, the same enzymatic assays used decades ago are still widely used to determine metabolite levels. Although significant advances in chromatography and mass spectrometry based metabolite analysis have moved the study of metabolism to ‘omics’ era, compartmentalization of metabolism still presents a major challenge. Especially the large vacuoles of plant cells are a major obstacle, since even fractionation studies suffer from contamination. Moreover, with the current set of tools it is not possible to determine the dynamic changes in metabolite levels in different subcellular compartments in real time in vivo. Radiotracers have helped a lot to identify and quantify intermediates and to assemble pathways, originally using pulse labeling followed by paper chromatography. Today ¹³C-labeling is used together with mass spectrometry to obtain insights into metabolic flux control.¹³ This tool set for the first time enabled the comparison of mutants and study regulatory networks involved in sugar signaling. While significant, advances in radiotracer experiments do not provide cellular or subcellular information and only limited temporal resolution, they do provide efficient means for studying metabolite fluxes through complex and/or not well-defined pathways. Thus there is a clear need for metabolite specific dyes that can be targeted to subcellular compartments and that would enable flux measurements in response to environmental cues helping to push metabolic research back into the focus of signaling-related biology.In 2002, we developed the first prototype “metabolic dye” FRET sensor for maltose.^14,15 A similar glucose sensor was recently employed for measuring tracer-independent transport of glucose across the ER membrane of liver cells.¹⁶ After resolving some issues such as low signal-to-noise and gene silencing in plants, we are now able to compare glucose levels between cells in an intact root in real time.¹⁷ The parallel development of sucrose and phosphate sensors complements the set of tools, in future experiments providing a comparison of sucrose, phosphate and glucose fluxes in intact tissues with both temporal (below seconds) and spatial resolution (cellular and subcellular).^18,19The first experiments already led to a big surprise: glucose supplied to the root is rapidly taken up and is rapidly metabolized.¹⁷ Roots expressing the highest affinity sensor FLIPglu170n responded to glucose perfusion suggesting that the steady state glucose level in the root is less than 100 nM, the estimated detection limit for this sensor in these first experiments. The first experiments were limited by the mixing kinetics in the bath used for perfusion, while improvement of the chamber now allow for faster for glucose exchange. We estimate that glucose levels fall from a steady state level of approximately 5 mM in the cytosol when perfused with 5 mM glucose to below 100 nM in about three minutes. For the sensor with an affinity of 600 µM the rate of glucose accumulation, which is composed of the various rates that affect the steady state in the cytosol such as metabolism, compartmentation and transport across the plasma membrane, is in the range of 527 ± 77 µM glucose/min and that for glucose removal is 317 ± 37 (Fig. 1; Chaudhuri B, Frommer WB, unpublished). Questions that arise are: Which transport systems drive uptake? How much does the vacuole contribute to the observed flux and steady state levels? Is the capacity of hexokinase at levels below its K_m still sufficient to phosphorylate glucose efficient enough to pull glucose below 100 nM or does hexokinase have different properties in vivo compared to what we know from the purified enzyme? Are there different transporters and enzymes contributing to flux in the low (1–10 mM) and the ultrahigh affinity (low µM) phases? Are there spatial differences in the root? Why do roots take up glucose so efficiently in the first place? The combination of the sensors with information from the expression-LEDs from Birnbaum and Benfey²⁰ and specific knock-out mutants should help answering some of these questions.Open in a separate windowFigure 1Quantitative analysis of glucose flux from an Arabidopsis root expressing FLIPglu-600µΔ13, a FRET sensor for glucose with an affinity of 600 µM. The root of a 10 day-old seedling was placed into a perfusion chamber and perfused with hydroponic medium with or without 5 mM glucose. eCFP was excited and emission was recorded for eCFP and eYFP every 10 seconds (essentially as decsribed in ref. 17). The emission intensities for a region-of-interest were averaged and the emission ratio was determined at the two wavelengths for each image of a time series and plotted on the Y-axis against time on the X-axis. Addition of glucose is indicated.Another big surprise is the dramatic gradient of glucose across the plasma membrane, which has important implications for our understanding of transport processes across the plasma membrane as well as the intracellular membranes.¹⁷ Information about the gradients is relevant in the context of apo- and symplasmic unloading routes in roots²¹ and the contribution of proton-coupled transporters in cellular export.²² It will thus be interesting to follow the extracellular levels using surface-anchored sensors. Now that besides high sensitivity glucose FLIPs¹⁷ we also generated nanosensors for sucrose¹⁹ and phosphate,¹⁸ complementing the similar tool sets for calcium²³ and pH,²⁴ it is possible to compare multiple parameters and to follow flux at different levels and to calibrate against other influences.The improvements of the signal-to-noise ratio of the FRET-based metabolite sensors²⁵ makes the FLIPs a standard tool for every lab interested in measuring ion-, sugar- or amino acid flux in living cells. Since the nanosensors are genetically encoded, they can be used to characterize intracellular fluxes^16,26 in any organism for which transformation protocols have been established. The existing sets of sensors are simple to use, constructs are available through Addgene and Arabidopsis lines from the Arabidopsis Stock Center. Detailed instructions for imaging can be found at: http://carnegiedpb.stanford.edu/research/frommer/research_frommer_protocols.php. These tools will hopefully become a standard system not only for physiological analyses, but in addition provide a new way for high throughput fluxomics studies.     

MICA polymorphism in South American Indians

We have studied the MICA alleles of 196 unrelated subjects from three South American Indian tribes (Toba, Wichi and Terena). They are members of isolated tribes located in the Gran Chaco area in northeastern Argentina and in Mato Grosso do Sul in South Central Brazil. Of 55 previously known alleles, nine were observed in South American Indians, compared with 16 that were found in North American Caucasians, suggesting a more restricted allelic distribution of MICA in these tribes. In South American Indians, MICA*00201 was the most frequent allele, with a gene frequency of 33% in Toba, 47% in Wichi and 44% in Terena. MICA*00201, MICA*027 (external domain sequence like MICA*008/TM allele A5) and MICA*010 accounted for more than 90% of all the MICA genes in South American Indians. In North American Caucasians, MICA*00801 (*008/A5.1) accounted for 42% of the genes and was the most common allele. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of HLA-B in the South American Indian populations. Phylogenetic trees constructed using gene frequencies of the transmembrane short tandem repeats in the populations reported here, and in other populations taken from published reports, suggest that South American Indians are more closely related to Asians than to Europeans.     

Immunogenicity and protective efficacy of three DNA vaccines encoding pre-erythrocytic- and erythrocytic-stage antigens of Plasmodium cynomolgi in rhesus monkeys

Although several malaria vaccine candidate antigens have been identified, the most suitable methods for their delivery are still being investigated. In this regard, direct immunization with DNA encoding these vaccine target antigens is an attractive alternative. Here, we have investigated the immune responses to DNA immunization with three major vaccine target antigens: the apical membrane antigen-1 and the 19-kDa C-terminal fragment of merozoite surface protein-1 from the erythrocytic stage, and the thrombospondin-related adhesive protein from the pre-erythrocytic stage of Plasmodium cynomolgi in rhesus monkeys. Antigen-specific antibodies were developed in all the immunized monkeys and peripheral blood mononuclear cells from all immunized monkeys proliferated to different extents upon in vitro stimulation with the corresponding recombinant proteins. The immunized monkeys were challenged with P. cynomolgi sporozoites. All of the immunized animals developed infection but although there was no significant difference between the control and vaccinated animals in terms of pre-patent period, total duration of patency and primary peak parasitemia, the vaccinated animals had significantly lower secondary peak parasitemia than the control animals.     

In silico detection of binding mode of J-superfamily conotoxin pl14a with Kv1.6 channel

A novel conotoxin pl14a containing 25 amino acid residues with an amidated C-terminus from vermivorous cone snail, Conus planorbis belongs to J-conotoxin superfamily and this is the first conotoxin, which inhibits both nicotinic acetylcholine receptor subtypes and Kv1.6 channel. We have attempted through bioinformatics approaches to elucidate the extent of specificity of pl14a towards Kv1 channel subtypes (Kv1.1-Kv1.6). Our work provides rationale for the relatively high specificity and binding mode of pl14a to Kv1.6 channel. The pl14a peptide contains two types of structural elements, namely the putative dyad (Lys18 and Tyr19) and basic residue ring constituted of arginine residues. We have carried out in silico docking studies so as to assess the contribution of one or combination of both structural elements of pl14a in blocking of Kv1.6 channel. For this purpose, we have built by homology modelling, the theoretical 3D structure of Kv1.6 channel based on the available crystal structure of mammalian shaker Kv1.2 channel. Docking studies suggest that positively charged residues ring may be involved in the blocking mechanism of Kv1.6 channel. The models suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kappaM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. The newly detected pharmacophore for pl14a interacting with Kv1.6 channel provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively-charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profiles against subtypes of the potassium channels for these conopeptides.     

IL-6 promotes head and neck tumor metastasis by inducing epithelial-mesenchymal transition via the JAK-STAT3-SNAIL signaling pathway

Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions and cell polarity, resulting in the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process in head and neck cancers are poorly understood. Increasing evidence suggests that tumor microenvironment plays an important role in promoting EMT in tumor cells. We have previously shown that head and neck tumors exhibit significantly higher Bcl-2 expression in tumor-associated endothelial cells and overexpression of Bcl-2 alone in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a severe combined immunodeficient (SCID) mouse model. In this study, we show that endothelial cells expressing Bcl-2 (EC-Bcl-2), when cocultured with head and neck tumor cells (CAL27), significantly enhance EMT-related changes in tumor cells predominantly by the secretion of IL-6. Treatment with recombinant IL-6 or stable IL-6 overexpression in CAL27 cells or immortalized oral epithelial cells (IOE) significantly induced the expression of mesenchymal marker, vimentin, while repressing E-cadherin expression via the JAK/STAT3/Snail signaling pathway. These EMT-related changes were further associated with enhanced tumor and IOE cell scattering and motility. STAT3 knockdown significantly reversed IL-6-mediated tumor and IOE cell motility by inhibiting FAK activation. Furthermore, tumor cells overexpressing IL-6 showed marked increase in lymph node and lung metastasis in a SCID mouse xenograft model. Taken together, these results show a novel function for IL-6 in mediating EMT in head and neck tumor cells and increasing their metastatic potential.     

The assembly of individual chaplin peptides from Streptomyces coelicolor into functional amyloid fibrils

The self-association of proteins into amyloid fibrils offers an alternative to the natively folded state of many polypeptides. Although commonly associated with disease, amyloid fibrils represent the natural functional state of some proteins, such as the chaplins from the soil-dwelling bacterium Streptomyces coelicolor, which coat the aerial mycelium and spores rendering them hydrophobic. We have undertaken a biophysical characterisation of the five short chaplin peptides ChpD-H to probe the mechanism by which these peptides self-assemble in solution to form fibrils. Each of the five chaplin peptides produced synthetically or isolated from the cell wall is individually surface-active and capable of forming fibrils under a range of solution conditions in vitro. These fibrils contain a highly similar cross-β core structure and a secondary structure that resembles fibrils formed in vivo on the spore and mycelium surface. They can also restore the growth of aerial hyphae to a chaplin mutant strain. We show that cysteine residues are not required for fibril formation in vitro and propose a role for the cysteine residues conserved in four of the five short chaplin peptides.     

VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.