A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals

The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.     

Mannose-binding lectin does not explain the course and outcome of pregnancy in rheumatoid arthritis

Introduction  

Rheumatoid arthritis (RA) improves during pregnancy and flares after delivery. It has been hypothesized that high levels of the complement factor mannose-binding lectin (MBL) are associated with a favourable disease course of RA by facilitating the clearance of pathogenic immunoglobulin G (IgG) lacking galactose sugar moieties. During pregnancy, increased galactosylation of IgG and simultaneously increased MBL levels can be observed, with the latter being strictly related to maternal MBL genotypes. Therefore, increased MBL levels in concert with increased IgG galactosylation may be associated with pregnancy-induced improvement of RA. The objective of this study was to investigate whether MBL genotypes are associated with changes in RA disease activity and with changes in IgG galactosylation during pregnancy and in the postpartum period. We also studied the association between MBL genotypes and pregnancy outcomes in RA.     

Multicentre Double-Blind Placebo-Controlled Food Challenge Study in Children Sensitised to Cashew Nut

Background

Few studies with a limited number of patients have provided indications that cashew-allergic patients may experience severe allergic reactions to minimal amounts of cashew nut. The objectives of this multicentre study were to assess the clinical relevance of cashew nut sensitisation, to study the clinical reaction patterns in double-blind placebo-controlled food challenge tests and to establish the amount of cashew nuts that can elicit an allergic reaction.

Methods and Findings

A total of 179 children were included (median age 9.0 years; range 2–17 years) with cashew nut sensitisation and a clinical history of reactions to cashew nuts or unknown exposure. Sensitised children who could tolerate cashew nuts were excluded. The study included three clinical visits and a telephone consultation. During the first visit, the medical history was evaluated, physical examinations were conducted, blood samples were drawn and skin prick tests were performed. The children underwent a double-blind placebo-controlled food challenge test with cashew nut during the second and third visits. The study showed that 137 (76.5%) of the sensitised children suspected of allergy to cashew nut had a positive double-blind placebo-controlled food challenge test, with 46% (63) manifesting subjective symptoms to the lowest dose of 1 mg cashew nut protein and 11% (15) developing objective symptoms to the lowest dose. Children most frequently had gastro-intestinal symptoms, followed by oral allergy and skin symptoms. A total of 36% (49/137) of the children experienced an anaphylactic reaction and 6% (8/137) of the children were treated with epinephrine.

Conclusion

This prospective study demonstrated a strikingly high percentage of clinical reactions to cashew nut in this third line population. Severe allergic reactions, including anaphylaxis requiring epinephrine, were observed. These reactions were to minimal amounts of cashew nut, demonstrated the high potency of this allergens.

Trial Registration

www.ncbi.nlm.nih.gov/pubmed NTR3572     

NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri

It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP⁺ with reduced ferredoxin (Fd_red) and the endergonic reduction of NADP⁺ with NADH in a reversible reaction: Fd_red²⁻ + NADH + 2 NADP⁺ + H⁺ = Fd_ox + NAD⁺ + 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H₂ (reaction 1) and in deriving a large (30%) portion of its cell carbon from CO₂. Both the energy metabolism and the pathways of biosynthesis have therefore been the subject of many investigations (for relevant literature, see references 12 and 27). (1)During growth of C. kluyveri on ethanol and acetate, approximately five ethanol and four acetate molecules are converted to three butyrate molecules and one caproate molecule (reaction 1a), and one ethanol molecule is oxidized to one acetate⁻, one H⁺, and two H₂ (reaction 1b) molecules (23, 31). How exergonic reaction 1a is coupled with endergonic reaction 1b and with ATP synthesis from ADP and P_i (ΔG^o′ = +32 kJ/mol) has remained unclear for many years. (1a) (1b)We recently showed (12) that, in Clostridium kluyveri, the exergonic reduction of crotonyl-coenzyme A (crotonyl-CoA) (E_o′ = −10 mV) with NADH (E_o′ = −320 mV) involved in reaction 1a is coupled with the endergonic reduction of ferredoxin (Fd_ox) (E_o′ = −420 mV) with NADH (E_o′ = −320 mV) involved in reaction 1b via the recently proposed mechanism of flavin-based electron bifurcation (7). The coupling reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase-EtfAB complex (reaction 2) (12): (2)The reduced ferredoxin (Fd_red²⁻) is assumed to be used for rereduction of NAD⁺ via a membrane-associated, proton-translocating ferredoxin:NAD oxidoreductase (RnfABCDEG) (reaction 3), and the proton motive force thus generated is assumed to drive the phosphorylation of ADP via a membrane-associated F₁F₀ ATP synthetase (reaction 4): (3) (4)The novel coupling mechanism represented by reactions 2 and 3 allowed for the first time the possibility of formulating a metabolic scheme for the ethanol-acetate fermentation that could account for the observed fermentation products and growth yields and thus for the observed ATP gains (27). One issue, however, remained open, namely, why the formation of butyrate from ethanol and acetate in the fermentation involves both an NADP⁺- and an NAD⁺-specific β-hydroxybutyryl-CoA dehydrogenase (16), considering that, in the oxidative part of the fermentation (ethanol oxidation to acetyl-CoA), only NADH is generated (8, 9, 13).The presence of a reduced ferredoxin:NADP⁺ oxidoreductase was proposed based on results of enzymatic studies performed 40 years ago. Cell extracts of Clostridium kluyveri were found to catalyze the formation of H₂ from NADPH in a ferredoxin- and NAD⁺-dependent reaction (34). The results were interpreted to indicate that C. kluyveri contains a ferredoxin-dependent hydrogenase and an NADPH:ferredoxin oxidoreductase with transhydrogenase activity. H₂ formation from NADPH was strictly dependent on the presence of NAD⁺ and was inhibited by NADH, inhibition being competitive with the presence of NAD⁺, indicating that ferredoxin reduction with NADPH is under the allosteric control of the NAD⁺/NADH couple. The cell extracts also catalyzed the NADH-dependent reduction of NADP⁺ with reduced ferredoxin (21, 34). Purification of the enzyme catalyzing these reactions was not achieved, and no function in the energy metabolism of C. kluyveri was assigned.In this communication, we report on the properties of the recombinant enzyme that catalyzes the NAD⁺-dependent reduction of ferredoxin with NADPH and the NADH-dependent reduction of NADP⁺ with reduced ferredoxin and show that the cytoplasmic heterodimeric enzyme couples the exergonic reduction of NADP⁺ with reduced ferredoxin with the endergonic reduction of NADP⁺ with NADH in a fully reversible reaction. The transhydrogenation reaction is endergonic, because in vivo the NADH/NAD⁺ ratio is generally near 0.3 and the NADPH/NADP⁺ ratio is generally above 1 (2, 30). (5)NADP⁺ reduction is most probably the physiological function of the enzyme, which is why we chose the abbreviation NfnAB (for NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase).     

Identification of novel virulence-associated loci in uropathogenic Escherichia coli by suppression subtractive hybridization

To identify novel virulence-associated genes in uropathogenic Escherichia coli (UPEC) strains, a suppression subtractive hybridization strategy was applied to genomic DNA of four clinical UPEC isolates from patients suffering from cystitis or pyelonephritis. The genomic DNA of four isolates (tester strains) was subtracted from the DNA of two different driver strains, the well characterized UPEC strain CFT073 and the non-pathogenic E. coli K-12 strain MG1655. We determined the sequence of 172 tester strain-specific DNA fragments, 86 of which revealed only low or no homology to nucleotide sequences of public databases. We further determined the virulence association of the 86 novel DNA fragments using each DNA fragment as a probe in Southern hybridizations of a reference strain collection consisting of 60 extraintestinal pathogenic E. coli isolates, and 40 non-virulent E. coli strains from stool samples. From this, 19 novel DNA fragments were demonstrated to be significantly associated with virulent strains and thus may represent new virulence traits. Our results support the idea of a considerable genetic variability among UPEC strains and suggest that novel genomic determinants might contribute to virulence of UPEC.     

The structure and dynamics of tandem WW domains in a negative regulator of notch signaling, Suppressor of deltex

WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.     

Snow-induced changes in dwarf birch chemistry increase moth larval growth rate and level of herbivory

Changes in snow cover might influence arctic ecosystems to the same extent as increased temperatures. Although the duration of snow cover is generally expected to decrease in the future as a result of global warming, the amounts of snow might increase in arctic areas where much of the elevated precipitation will fall as snow. We examined the effects of an increased snow cover, as a result of a snow fence treatment, on soil nitrogen mineralization, plant phenology, plant chemistry (nitrogen and potential defense compounds), the level of invertebrate herbivory, and performance of invertebrate herbivores in an arctic ecosystem, using dwarf birch (Betula nana) and the autumnal moth (Epirrita autumnata) as study organisms. An enhanced and prolonged snow cover increased the level of herbivory on dwarf birch leaves. Larvae feeding on plants that had experienced enhanced snow cover grew faster and pupated earlier than larvae fed with plant material from control plots, indicating that plants from enhanced snow-lie plots produce higher-quality food to herbivores. The increased larval growth rate was strongly correlated with higher leaf nitrogen concentration in plants subjected to snow manipulation, and also to certain phenolic acids. Snow manipulation did not change net nitrogen mineralization rates in the soil or total carbon concentration in leaves, but it altered the within-season fluctuating pattern of leaf phenolic compounds. This study demonstrates a positive relationship between increased snow cover and level of herbivory on deciduous shrubs, thus proposing a negative feedback on the climate-induced dwarf shrub expansion in arctic areas.     

Evidence for a Relationship between VEGF and BMI Independent of Insulin Sensitivity by Glucose Clamp Procedure in a Homogenous Group Healthy Young Men

Background

This is the first study to experimentally explore the direct relationship between circulating VEGF levels and body mass index (BMI) as well as to unravel the role of insulin sensitivity in this context under standardized glucose clamp conditions as the methodical gold-standard. In order to control for known influencing factors such as gender, medication, and arterial hypertension, we examined a highly homogeneous group of young male subjects. Moreover, to encompass also subjects beyond the normal BMI range, low weight and obese participants were additionally included and stress hormones as a main regulator of VEGF were assessed.

Methodology/Principal Findings

Under euglycemic clamp conditions, VEGF was measured in 15 normal weight (BMI 20–25 kg/m²), 15 low weight (BMI<20 kg/m²), and 15 obese (BMI>30 kg/m²) male subjects aged 18–30 years and the insulin sensitivity index (ISI) was calculated. Since stress axis activation promotes VEGF secretion, concentrations of ACTH, cortisol, and catecholamines were monitored. Despite of comparable ACTH (P = 0.145), cortisol (P = 0.840), and norepinephrine (P = 0.065) levels, VEGF concentrations differed significantly between BMI-groups (P = 0.008) with higher concentrations in obese subjects as compared to normal weight (P = 0.061) and low weight subjects (P = 0.002). Pearson''s correlation analysis revealed a positive relationship between BMI and VEGF levels (r = 0.407; P = 0.010) but no correlation of VEGF with ISI (r = 0.224; P = 0.175).

Conclusions/Significance

Our data demonstrate a positive correlation between concentrations of circulating VEGF levels and BMI in healthy male subjects under highly controlled conditions. This relationship which is apparently disconnected from insulin sensitivity may be part of some pathogenetic mechanisms underlying obesity and type 2 diabetes.     

HIV Infection Is Associated with a Preferential Decline in Less-Differentiated CD56dim CD16+ NK Cells

HIV-1 infection is characterized by loss of CD56^dim CD16⁺ NK cells and increased terminal differentiation on various lymphocyte subsets. We identified a decrease of CD57⁻ and CD57^dim cells but not of CD57^bright cells on CD56^dim CD16⁺ NK cells in chronic HIV infection. Increasing CD57 expression was strongly associated with increasing frequencies of killer immunoglobulin-like receptors (KIRs) and granzyme B-expressing cells but decreasing percentages of cells expressing CD27⁺, HLA-DR⁺, Ki-67⁺, and CD107a. Our data indicate that HIV leads to a decline of less-differentiated cells and suggest that CD57 is a useful marker for terminal differentiation on NK cells.NK cells are effector cells of innate immunity which are pivotal as first-line defense against viral infections, such as HIV infection (14). Large genotypic studies demonstrated a delayed onset of AIDS in HIV-seropositive individuals carrying the activating receptor KIR3DS1 and/or alleles of the inhibiting receptor KIR3DL1 in conjunction with HLA-Bw4-80I (18, 19). Development of NK cells mainly takes place in the bone marrow, from which mature NK cells move out to reside and circulate in peripheral sites (13). Mature NK cells are characterized by granules which harbor granzymes and perforin. These NK cells are fully armed, “ready-to-go” effector cells (17).A number of NK cell abnormalities have been reported in HIV infection (9), including high activation status (2, 10), increased turnover (16), differential expression of activating and inhibitory receptors (20), impaired interaction with dendritic cells (12), and loss of CD56^dim CD16⁺ NK cells (23). CD56^dim CD16⁺ NK cells represent the largest NK cell subset in peripheral blood in healthy individuals. The expression of killer immunoglobulin-like receptors (KIRs) and CD57 are predominant features of this subpopulation (8, 15). CD57 expression on NK cells has been previously associated with replicative senescence on T and NK cells (4), raising the question of how HIV-1 infection alters CD57 expression on CD56^dim CD16⁺ NK cells.To the best of our knowledge, no one has addressed the phenotypic and functional properties of CD56^dim CD16⁺ NK cells that are preferentially lost during HIV infection. Here, we provide evidence that increasing CD57 expression indicates terminal differentiation in healthy individuals, as well in as HIV-infected subjects. We furthermore show that HIV infection is associated with preferential loss of less-differentiated cells, which are characterized by high activation status and turnover.In this study, blood samples from 37 HIV-seropositive individuals and 15 healthy subjects were analyzed; all HIV-infected patients were either antiretroviral therapy naïve or untreated for more than one year. The HIV-positive study cohort comprised 10 patients with a viral load of less than 2,000 copies/ml, 14 patients with a viral load ranging from 2,000/ml to 20,000 copies/ml, and 13 patients with a viral load above 20,000 copies/ml. CD4 T cell counts ranged from 180/μl to 1,355/μl, the average being 457.3/μl.The study was approved by the local ethics commission (Ethikkommission der Medizinischen Hochschule Hannover, Votum No. 3150), and all study participants gave informed written consent for their participation.Flow cytometric analysis was performed on cryopreserved peripheral blood mononuclear cells (PBMCs) as previously described (21, 22). A list of monoclonal antibodies employed in this study is available upon request. For intracellular analysis of granzyme B, perforin, and Ki-67, we used a fixation and permeabilization kit (Invitrogen). At least 1 million events were acquired for each sample, using either a FACSAria or LSR II flow cytometer (BD Biosciences). Data were analyzed with FlowJo (TreeStar). Lymphocytes were defined by forward and side scatter. CD3⁺, CD14⁺, CD19⁺, dead cells, and cell aggregates were removed from analysis based on peridinin chlorophyll protein and Viaprobe staining and gating on a plot of forward-scatter area versus forward-scatter height (Fig. ​(Fig.1A).1A). NK cells and their distinctive subpopulations were defined based on their CD56 and/or CD16 expression. Fluorescence-minus-one (FMO) staining was used to determine threshold values for the expression of specific markers.Open in a separate windowFIG. 1.HIV infection is associated with loss of CD57⁻ and CD57^dim but not CD57^bright CD56^dim CD16⁺ NK cells. (A) Representative gating scheme for identification of NK cells. NK cells were defined as CD3⁻ CD14⁻ CD19⁻ lymphocytes expressing either CD56 or CD16 or both. We divided CD56^dim CD16⁺ NK cells into three subsets based on their level of CD57 expression: CD57⁻, CD57^dim, and CD57^bright cells. Numbers on FACS plots indicate frequency of gated population. SSC-A, side scatter area; FSC-A, forward scatter area; FSC-W, forward scatter width. (B) Comparison of percentages of the CD57⁻, CD57^dim, and CD57^bright subpopulations in control subjects (n = 14) and HIV-seropositive individuals (n = 34) on CD56^dim CD16⁺ NK cells. ns, not significant (P > 0.05); **, P < 0.01; ***, P < 0.001. (C) Frequencies of CD57⁻, CD57^dim, and CD57^bright expressing CD56^dim CD16⁺ NK cells in relation to total NK cells in control subjects (n = 14) and HIV-seropositive individuals (n = 34). (D) Mean frequency of CD56^dim CD16⁺ NK cells in 14 control individuals and in 34 HIV-infected people and the distribution of CD57⁻, CD57^dim, and CD57^bright cells within CD56^dim CD16⁺ NK cells is shown. (E) Relationship between percentage of CD57^dim CD56^dim CD16⁺NK cells and percentage of CD56^neg CD16⁺ NK cells on total NK cells. Horizontal bars in dot plots show the means.NK cells as defined above were sorted from cryopreserved PBMCs on a FACSAria (purities ranged from 91% to 99%). An amount of 10⁵ NK cells was plated per well and stimulated with 10 ng/ml interleukin-15 (IL-15), 100 ng/ml IL-12, and 5 × 10⁴ K562 cells. A CD107a degranulation assay was performed as described previously (1, 12). GraphPad Prism (version 5.0) software was used for statistical evaluation of data. Correlation analysis was performed using the Pearson test. The unpaired t test was performed when two groups were compared, and all t tests were two tailed. Comparison of more than two groups was performed using one-way analysis of variance followed by Tukey''s post-hoc test. P values of less than 0.05 were considered significant.We found that CD57 on NK cells was predominantly expressed on the CD56^dim CD16⁺ population (Fig. ​(Fig.1A).1A). The expression patterns of CD57 allowed us to differentiate between three subfractions within CD56^dim CD16⁺ NK cells, namely, CD57⁻, CD57^dim, and CD57^bright cells. The frequency of the CD57^bright subpopulation on CD56^dim CD16⁺ NK cells was increased compared to the frequency of the CD57^dim subpopulation on CD56^dim CD16⁺ NK cells in HIV-seropositive patients but not in HIV-seronegative control subjects (Fig. ​(Fig.1B).1B). This relative increase was associated with substantial reductions of the CD57⁻ CD56^dim and the CD57^dim CD56^dim NK cell subpopulations of total NK cells in our HIV-seropositive cohort compared to these subpopulations in healthy control subjects (means, 36.6% versus 24.8% [P = 0.0002] and 22.4% versus 15.4% [P = 0.0001]), but the frequencies of CD57^bright CD56^dim NK cells within total NK cells were similar between HIV-infected patients and HIV-seronegative individuals (Fig. ​(Fig.1C).1C). In accordance with previously published data (3, 23), we could confirm that there is a relative loss of CD56^dim CD16⁺ NK cells in HIV infection (mean, 84.3% versus 67.0%, P = 0.0004) (Fig. ​(Fig.1D).1D). Our data indicate that this loss is predominantly due to decreased numbers of CD57⁻ CD56^dim and CD57^dim CD56^dim NK cells, leading to a relative overrepresentation of CD57^bright cells within CD56^dim CD16⁺ NK cells in HIV infection (Fig. ​(Fig.1C).1C). There was no significant correlation between the relative loss of CD57⁻ and CD57^dim NK cells and absolute numbers of CD56^dim CD16⁺ NK cells, but there was a significant inverse correlation between loss of CD57^dim NK cells and increasing percentages of CD56⁻ CD16⁺ cells (Pearson r = −0.54, P = 0.001) (Fig. ​(Fig.1E1E).To determine whether the relative decrease of CD57⁻ and CD57^dim NK cells was associated with parameters of HIV disease progression, we performed correlation analysis of the percentages of CD57⁻ or CD57^dim cells with viral load and CD4 T cell counts. We found no such correlations (Pearson r < 0.2 and P > 0.05 for all) (data not shown). A recent cross-sectional and longitudinal study demonstrated that changes in the NK cell compartment, as shown by down-modulation of Siglec-7 on CD56^dim NK cells, are associated with HIV viremia (5). The longitudinal data in the study indicated that the full restoration of NK cell pathologies required 24 months of antiviral treatment. This suggests that alterations in the NK cell compartment can be driven by HIV viral load but that these changes seem to require a significant amount of time.We next investigated the phenotypic and functional properties of the CD57⁻, CD57^dim, and CD57^bright subpopulations on CD56^dim CD16⁺ NK cells. For KIR2DL2/DL3/DS2, we detected increasing prevalences of KIR-expressing NK cells with increasing expression of CD57 in both healthy control subjects and HIV-infected blood donors (Fig. ​(Fig.2A).2A). As for KIR3DS1/DL1, we found an increase of KIR⁺-expressing NK cells between CD57⁻ and CD57^bright cells in control individuals and significant differences in percentages of KIR3DS1/DL1-expressing NK cells between CD57⁻ and CD57^dim, as well as between CD57⁻ and CD57^bright, NK cells in our HIV-positive cohort (Fig. ​(Fig.2A).2A). These results suggest that increasing CD57 expression is associated with higher numbers of KIR-expressing NK cells in control subjects and HIV-infected subjects.Open in a separate windowFIG. 2.Phenotypic characterization of the CD57⁻, CD57^dim, and CD57^bright subpopulations of CD56^dim CD16⁺ NK cells. Representative flow cytometry plots for one control and one HIV-infected subject and summary data for all individuals whose PBMCs were analyzed are shown. CD57⁻, CD57^dim, and CD57^bright NK cells are concatenated to visualize them in a single dot plot. Numbers in contour plots indicate percentages of gated events of the respective subset. (A) Percentages of KIR2DL2/DL3/DS2 and KIR3DS1/DL1-expressing CD57⁻, CD57^dim, and CD57^bright cells were analyzed in control individuals (n = 15) and HIV-infected subjects (n = 37). (B) Numbers of HLA-DR-expressing and CD27-expressing CD57⁻, CD57^dim, and CD57^bright cells in control individuals'' (n = 15) and HIV-infected subjects'' (n = 37) PBMCs were analyzed. Horizontal bars in dot plots show the means. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.We next addressed the question of whether increasing CD57 expression is linked to differential phenotypic properties of NK cells and analyzed the HLA-DR and CD27 expression of the CD57⁻, CD57^dim, and CD57^bright subpopulations on CD56^dim CD16⁺ NK cells. A significantly higher fraction of NK cells expressed HLA-DR in the CD57⁻ than in the CD57^bright subset in both healthy control individuals and HIV-infected subjects (Fig. ​(Fig.2B).2B). A considerably higher portion of NK cells was positive for HLA-DR in HIV-infected individuals than in control subjects (means, 3.2% versus 13.2% [P < 0.0001], 1.8% versus 10.4% [P = 0.001], and 0.9% versus 6.5% [P = 0.005] for CD57⁻, CD57^dim, and CD57^bright subpopulations, respectively). We furthermore detected marked differences in frequencies of cells expressing CD27, a member of the tumor necrosis factor (TNF) receptor family (24). CD57⁻ NK cells displayed the highest percentages of CD27⁺ cells, whereas CD57^bright cells were almost all negative for CD27, in both control individuals and HIV-seropositive subjects (Fig. ​(Fig.2B).2B). We thus show that increasing expression of CD57 is associated with differential activation status and differential phenotype.Next, we sought to determine whether CD57 is linked to differential functional phenotypes by assessing the intracellular expression of granzyme B, perforin, and Ki-67. The frequencies of perforin-expressing NK cells did not vary within the different CD57 subsets of CD56^dim CD16⁺ NK cells (Fig. ​(Fig.3A).3A). However, we found that CD57^bright cells displayed the highest frequencies of granzyme B⁺ in both control and HIV-seropositive subjects, whereas CD57⁻ cells exhibited the lowest percentages for granzyme B⁺ cells (Fig. ​(Fig.3A).3A). Conversely, when we studied the expression of Ki-67, we identified the opposite trend: less than 5% of CD57^bright cells in control individuals and less than 10% of CD57^bright cells in HIV-infected study subjects expressed Ki-67 (Fig. ​(Fig.3B).3B). The highest numbers of Ki-67⁺ cells were found in the CD57⁻ population.Open in a separate windowFIG. 3.Functional characterization of CD57⁻, CD57^dim, and CD57^bright cells within the CD56^dim CD16⁺ NK cell population. (A) Representative staining results for granzyme B and perforin and summary data for control (n = 14) and HIV-seropositive subjects (n = 36). Numbers in the concatenated contour plots indicate percentages of gated events of the respective subset. B cells were defined as the negative control for granzyme and perforin staining. (B) Percentages of Ki-67⁺ and CD107a⁺ cells on CD57⁻, CD57^dim, and CD57^bright cells within the CD56^dim NK cell population in control (n = 14 and n = 9, respectively) and HIV-seropositive (n = 36 and n = 21, respectively) subjects'' PBMCs were analyzed. Horizontal bars in dot plots show the means. NC, negative control; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.We also assessed the presence of the degranulation marker CD107a on CD57⁻, CD57^dim, and CD57^bright subpopulations of CD56^dim CD16⁺ NK cells after stimulation with IL-12 and IL-15 and exposure to K562 cells. Similarly to what we had observed for Ki-67 expression, CD57⁻ cells were the most efficient at degranulation when compared with CD57^dim and CD57^bright cells in HIV-infected individuals. Comparison to healthy controls revealed that there was a higher expression of CD107a in HIV-seropositive subjects for each CD57 subset. However, the most effective degranulation occurred in the CD57⁻ and CD57^dim subsets, which are preferentially depleted in HIV infection.We focused our analysis on CD56^dim CD16⁺ NK cells because they constitute the largest NK cell subset in peripheral blood, they are the major NK cell subset expressing CD57 and KIRs, and they are the most prominent subpopulation for cytolytic activity. CD56^dim CD16⁺ cells but not CD56^bright CD16⁻ NK cells were reported to be decreased in HIV-infected subjects (23), which we could confirm in our experiments (data not shown). We did not find CD57 on CD56^bright CD16⁻ NK cells either in healthy or in HIV-infected individuals. CD57 has been described as a marker for replicative senescence, and its expression has been associated with shorter telomeres and diminished proliferative capacities on T and NK cells (4). The presence of this marker on CD56^dim CD16⁺ but not on CD56^bright CD16⁺ NK cells might explain why the latter subset was shown to proliferate more efficiently upon cytokine stimulation (6). We demonstrated that increasing CD57 expression on NK cells was associated with lower numbers of CD27-expressing cells, a marker which is mainly expressed by CD56^bright CD16⁻ NK cells (24). CD56^bright CD16⁻ cells were suggested to be early NK cells, which differentiate from CD34^dim CD45RA⁺ hematopoietic precursor cells with high expression of integrin α₄β₇ (11). These cells can furthermore give rise to CD56^dim CD16⁺ NK cells (7). Our data support this hypothesis, as we show that CD57 can be found on CD56^dim CD16⁺ NK cells but not on CD56^bright NK cells, whereas the opposite is observed for CD27.We demonstrate that differential CD57 expression is associated with distinct functional characteristics. We show for the first time that increasing expression of CD57 on CD56^dim CD16⁺ NK cells is associated with increasing prevalence of KIR⁺ and granzyme B⁺ cells. These cells appear to be more mature and differentiated in terms of KIR and granzyme B expression but less functionally active, as shown by decreased expression of Ki-67 and CD107a. We therefore propose that CD57 is not only a marker for replicative senescence but, in addition, a marker for terminal differentiation on NK cells, which is characterized by increased expression of KIR and higher granzyme B content and “counterbalanced” by decreased degranulation (CD107a) and decreased proliferation (Ki-67).Notably, we observed consistently higher frequencies of granzyme B⁺ cells in all three subsets within CD56^dim CD16⁺ NK cells from HIV-seropositive individuals than in healthy control subjects (means, 52.9% versus 78.7% [P < 0.0001], 65.3% versus 89.6% [P < 0.0001], and 76.5% versus 95.0% [P < 0.0001]for CD57⁻, CD57^dim, and CD57^bright subpopulations, respectively) (Fig. ​(Fig.1C).1C). Furthermore, HIV infection was associated with higher numbers of Ki-67-expressing NK cells (means, 8.4% versus 16.1% [P = 0.0005], 5.3% versus 11.6% [P = 0.0016], and 4.1% versus 6.2% [P = 0.04]) (Fig. ​(Fig.1C).1C). These changes, including the strong increase in HLA-DR-expressing NK cells, probably reflect the systemic immune activation in HIV-infected individuals.In summary, these findings support a view of a differential regulation of NK function and are in concordance with maturation of NK cells with high expression of CD57 on NK cells with a more terminally differentiated phenotype. Our data indicate that high turnover; activation status; and active degranulation as characterized by the expression of Ki-67, HLA-DR, and CD107a are mainly features of CD57⁻ and much less of CD57^dim NK cells. HIV infection is associated with increased activation, proliferation, and cytotoxicity during “early” stages of CD56^dim CD16⁺ NK cell differentiation compared to their occurrence in healthy controls, but those are the very cells that are significantly decreased in chronic HIV infection. A loss of these functionally more active NK cells may be a yet-unappreciated factor in overall NK cell pathology and a further possible explanation for the impairment of NK cells in their contribution to viral control in HIV infection.     

Community structure and diversity of tropical forest mammals: data from a global camera trap network

Terrestrial mammals are a key component of tropical forest communities as indicators of ecosystem health and providers of important ecosystem services. However, there is little quantitative information about how they change with local, regional and global threats. In this paper, the first standardized pantropical forest terrestrial mammal community study, we examine several aspects of terrestrial mammal species and community diversity (species richness, species diversity, evenness, dominance, functional diversity and community structure) at seven sites around the globe using a single standardized camera trapping methodology approach. The sites-located in Uganda, Tanzania, Indonesia, Lao PDR, Suriname, Brazil and Costa Rica-are surrounded by different landscape configurations, from continuous forests to highly fragmented forests. We obtained more than 51 000 images and detected 105 species of mammals with a total sampling effort of 12 687 camera trap days. We find that mammal communities from highly fragmented sites have lower species richness, species diversity, functional diversity and higher dominance when compared with sites in partially fragmented and continuous forest. We emphasize the importance of standardized camera trapping approaches for obtaining baselines for monitoring forest mammal communities so as to adequately understand the effect of global, regional and local threats and appropriately inform conservation actions.