Single Human Donor Research Articles

Structural disruption of Ntox15 nuclease effector domains by immunity proteins protects against type VI secretion system intoxication in Bacteroidales.

Bacteroidales use type VI secretion systems (T6SS) to competitively colonize and persist in the colon. We identify a horizontally transferred T6SS with Ntox15 family nuclease effector (Tde1) that mediates interbacterial antagonism among Bacteroidales, including several derived from a single human donor. Expression of cognate (Tdi1) or orphan immunity proteins in acquired interbacterial defense systems protects against Tde1-dependent attack. We find that immunity protein interaction induces a large effector conformational change in Tde nucleases, disrupting the active site and altering the DNA-binding site. Crystallographic snapshots of isolated Tde1, the Tde1/Tdi1 complex, and homologs from Phocaeicola vulgatus (Tde2/Tdi2) illustrate a conserved mechanism of immunity inserting into the central core of Tde, splitting the nuclease fold into two subdomains. The Tde/Tdi interface and immunity mechanism are distinct from all other polymorphic toxin-immunity interactions of known structure. Bacteroidales abundance has been linked to inflammatory bowel disease activity in prior studies, and we demonstrate that Tde and T6SS structural genes are each enriched in fecal metagenomes from ulcerative colitis subjects. Genetically mobile Tde1-encoding T6SS in Bacteroidales mediate competitive growth and may be involved in inflammatory bowel disease. Broad immunity is conferred by Tdi1 homologs through a fold-disrupting mechanism unique among polymorphic effector-immunity pairs of known structure. IMPORTANCE Bacteroidales are related to inflammatory bowel disease severity and progression. We identify type VI secretion system (T6SS) nuclease effectors (Tde) which are enriched in ulcerative colitis and horizontally transferred on mobile genetic elements. Tde-encoding T6SSs mediate interbacterial competition. Orphan and cognate immunity proteins (Tdi) prevent intoxication by multiple Tde through a new mechanism among polymorphic toxin systems. Tdi inserts into the effector central core, splitting Ntox15 into two subdomains and disrupting the active site. This mechanism may allow for evolutionary diversification of the Tde/Tdi interface as observed in colicin nuclease-immunity interactions, promoting broad neutralization of Tde by orphan Tdi. Tde-dependent T6SS interbacterial antagonism may contribute to Bacteroidales diversity in the context of ulcerative colitis.

High-throughput, single-microbe genomics with strain resolution, applied to a human gut microbiome

Characterizing complex microbial communities with single-cell resolution has been a long-standing goal of microbiology. We present Microbe-seq, a high-throughput method that yields the genomes of individual microbes from complex microbial communities. We encapsulate individual microbes in droplets with microfluidics and liberate their DNA, which we then amplify, tag with droplet-specific barcodes, and sequence. We explore the human gut microbiome, sequencing more than 20,000 microbial single-amplified genomes (SAGs) from a single human donor and coassembling genomes of almost 100 bacterial species, including several with multiple subspecies strains. We use these genomes to probe microbial interactions, reconstructing the horizontal gene transfer (HGT) network and observing HGT between 92 species pairs; we also identify a significant in vivo host-phage association between crAssphage and one strain of Bacteroides vulgatus . Microbe-seq contributes high-throughput culture-free capabilities to investigate genomic blueprints of complex microbial communities with single-microbe resolution.

Analysis in a murine model points to IgG responses against the 34k2 salivary proteins from Aedes albopictus and Aedes aegypti as novel promising candidate markers of host exposure to Aedes mosquitoes.

BackgroundAedes mosquitoes are vectors of arboviral diseases of great relevance for public health. The recent outbreaks of dengue, Zika, chikungunya and the rapid worldwide spreading of Aedes albopictus emphasize the need for improvement of vector surveillance and control. Host antibody response to mosquito salivary antigens is emerging as a relevant additional tool to directly assess vector-host contact, monitor efficacy of control interventions and evaluate risk of arboviral transmission.Methodology/principal findingsGroups of four BALB/c mice were immunized by exposure to bites of either Aedes albopictus or Aedes aegypti. The 34k2 salivary proteins from Ae. albopictus (al34k2) and Ae. aegypti (ae34k2) were expressed in recombinant form and Ae. albopictus salivary peptides were designed through B-cell epitopes prediction software. IgG responses to salivary gland extracts, peptides, al34k2 and ae34k2 were measured in exposed mice. Both al34k2 and ae34k2, with some individual and antigen-specific variation, elicited a clearly detectable antibody response in immunized mice. Remarkably, the two orthologous proteins showed very low level of immune cross-reactivity, suggesting they may eventually be developed as species-specific markers of host exposure. The al34k2 immunogenicity and the limited immune cross-reactivity to ae34k2 were confirmed in a single human donor hyperimmune to Ae. albopictus saliva.Conclusions/significanceOur study shows that exposure to bites of Ae. albopictus or Ae. aegypti evokes in mice species-specific IgG responses to al34k2 or ae34k2, respectively. Deeper understanding of duration of antibody response and validation in natural conditions of human exposure to Aedes mosquitoes are certainly needed. However, our findings point to the al34k2 salivary protein as a promising potential candidate for the development of immunoassays to evaluate human exposure to Ae. albopictus. This would be a step forward in the establishment of a serological toolbox for the simultaneous assessment of human exposure to Aedes vectors and the pathogens they transmit.

Molecular characterization of human anti-hinge antibodies derived from single-cell cloning of normal human B cells

Anti-hinge antibodies (AHAs) are an autoantibody subclass that, following proteolytic cleavage, recognize cryptic epitopes exposed in the hinge regions of immunoglobulins (Igs) and do not bind to the intact Ig counterpart. AHAs have been postulated to exacerbate chronic inflammatory disorders such as inflammatory bowel disease and rheumatoid arthritis. On the other hand, AHAs may protect against invasive microbial pathogens and cancer. However, despite more than 50 years of study, the origin and specific B cell compartments that express AHAs remain elusive. Recent research on serum AHAs suggests that they arise during an active immune response, in contrast to previous proposals that they derive from the preexisting immune repertoire in the absence of antigenic stimuli. We report here the isolation and characterization of AHAs from memory B cells, although anti-hinge-reactive B cells were also detected in the naive B cell compartment. IgG AHAs cloned from a single human donor exhibited restricted specificity for protease-cleaved F(ab')2 fragments and did not bind the intact IgG counterpart. The cloned IgG-specific AHA-variable regions were mutated from germ line-derived sequences and displayed a high sequence variability, confirming that these AHAs underwent class-switch recombination and somatic hypermutation. Consistent with previous studies of serum AHAs, several of these clones recognized a linear, peptide-like epitope, but one clone was unique in recognizing a conformational epitope. All cloned AHAs could restore immune effector functions to proteolytically generated F(ab')2 fragments. Our results confirm that a diverse set of epitope-specific AHAs can be isolated from a single human donor.

Graft preparation for hemi-Descemet membrane endothelial keratoplasty (hemi-DMEK)

Background/aimsThere is a substantial shortage of donor tissue for endothelial keratoplasty worldwide. Using two endothelial grafts of one donor cornea may potentially increase the pool of suitable donor tissue for...

Single Cell Genomics of Uncultured, Health-Associated Tannerella BU063 (Oral Taxon 286) and Comparison to the Closely Related Pathogen Tannerella forsythia

The uncultivated bacterium Tannerella BU063 (oral taxon 286) is the closest relative to the periodontal pathogen Tannerella forsythia, but is not disease-associated itself. Using a single cell genomics approach, we isolated 12 individual BU063 cells by flow cytometry, and we amplified and sequenced their genomes. Comparative analyses of the assembled genomic scaffolds and their gene contents allowed us to study the diversity of this taxon within the oral community of a single human donor that provided the sample. Eight different BU063 genotypes were represented, all about 5% divergent at the nucleotide level. There were 2 pairs of cells and one group of three that were more highly identical, and may represent clonal populations. We did pooled assemblies on the nearly identical genomes to increase the assembled genomic coverage. The presence of a set of 66 “core” housekeeping genes showed that two of the single cell assemblies and the assembly derived from the three putatively identical cells were essentially complete. As expected, the genome of BU063 is more similar to Tannerella forsythia than any other known genome, although there are significant differences, including a 44% difference in gene content, changes in metabolic pathways, loss of synteny, and an 8–9% difference in GC content. Several identified virulence genes of T. forsythia are not found in BU063 including karilysin, prtH, and bspA. The absence of these genes may explain the lack of periodontal pathogenesis by this species and provides a new foundation to further understand the genome evolution and mechanisms of bacterial-host interaction in closely related oral microbes with different pathogenicity potential.

Structure and Dynamics of Lens Lipid Membranes Derived from a Single Porcine Donor: High Field EPR Study

To obtain correct EPR line shapes, spin-lattice relaxation times, and bimolecular collision rates between spin labels and oxygen, samples must be thoroughly deoxygenated or equilibrated with a controlled oxygen partial pressure. These measurements are conveniently carried out using a gas permeable plastic sample tube of small diameter that fits in a loop-gap resonator. Flow of gas over the tube allows easy deoxygenation or controlled oxygenation of the sample. In the initial design of the W-band (94 GHz) loop-gap resonator, samples were equilibrated with gas at room temperature outside the resonator, transferred to a quartz capillary, and positioned in the resonator. In a recently designed W-band loop-gap resonator, the sample is positioned in a gas-permeable Teflon tubing inside the resonator, which allows measurements of extremely small volume (∼30 nL) of sample. We used this new design to measure properties of lens lipid membranes derived from total lipids extracted from both lenses (single donor) of a 2-year-old porcine cortex and nucleus. Detailed profiles of membrane fluidity and oxygen transport parameter were obtained from saturation recovery EPR. Analysis of conventional spectra using the microscopic-order macroscopic-disorder (MOMD) model provided rotational diffusion coefficients (R(perpendicular) and R(parallel)) and order parameters. Three different types of motion of lipid spin labels n-PC, T-PC, and CSL (ASL) with, respectively, nitroxide z-axis, x-axis, and y-axis parallel to the bilayer normal, are discussed. Results demonstrate that EPR at W-band has the potential to be a powerful tool for studying samples of small volume, ∼30 nL, obtained from eye lenses of a single human donor.

One-step grafting procedure using artificial dermis and split-thickness skin in burn patients

The study aimed to achieve a one-step grafting procedure using artificial dermis and split-thickness skin. We performed simultaneous grafting of artificial dermis and skin in two severely burned patients. Artificial dermis was treated with fresh autogenous platelet-derived wound-healing factors (PDWHF), cryopreserved allogeneic cultured endothelial cells, and fibroblasts. Dermal microvascular endothelial cells and fibroblasts were obtained from a single human donor’s skin. The cultured cells were cryopreserved until use in grafting. The PDWHF was prepared from autogenous blood from each patient prior to the surgery. In two patients, the artificial dermis treated with this method was grafted to a full-thickness burn wound. Immediately after artificial dermis grafting, meshed split-thickness skin was grafted. In each case, the skin graft took well, and the skin texture was acceptable. Histological examination revealed that bovine collagen tissue remained in the dermis after surgery, indicating the success of the simultaneous grafting of the artificial dermis and the skin. The present study indicates that one-step grafting of artificial dermis and split-skin is possible when the artificial dermis is treated with PDWHF and cultured endothelial cells and fibroblasts. Level of Evidence: Level V, therapeutic study.

Pancreatic β-cell function relates positively to HDL functionality in well-controlled Type 2 diabetes mellitus

BackgroundHigh density lipoproteins (HDLs) have been implicated in glucose homeostasis. Among subjects with normal fasting glucose (NFG), impaired fasting glucose (IFG) and Type 2 diabetes mellitus (T2DM) we tested whether pancreatic β-cell function relates to HDL functionality, as determined by HDL anti-oxidative capacity and cellular cholesterol efflux to plasma. Subjects and methodsHDL anti-oxidative capacity (inhibition of LDL oxidation in vitro), cellular cholesterol efflux (the ability of plasma to stimulate cholesterol efflux out of cultured fibroblasts obtained from a single human donor), glucose and insulin were determined in fasting plasma samples from 37 subjects with NFG, 36 with IFG and 22 with T2DM (no glucose lowering drug or insulin treatment; HbA1c 6.0±1.0%). Homeostasis model assessment was used to estimate pancreatic β-cell function (HOMA-β) and insulin resistance (HOMAir). ResultsHOMA-β was lowest, whereas HOMAir was highest in T2DM (P<0.01 and P<0.001 vs. NFG). HDL anti-oxidative capacity and cellular cholesterol efflux did not differ significantly according to glucose tolerance category. In univariate analysis and after controlling for HOMAir both HDL anti-oxidative capacity (P<0.05) and cellular cholesterol efflux (P<0.01) were positively correlated with HOMA-β in T2DM, but not in NFG and IFG. In age-, sex- and HOMAir-adjusted analyses, T2DM status interacted positively with HDL anti-oxidative capacity (P=0.001) and cellular cholesterol efflux (P=0.042) on HOMA-β. HbA1c interacted similarly with HDL functionality measures on HOMA-β. ConclusionsPancreatic β-cell function relates to pathophysiologically relevant measures of HDL function in T2DM, but not in NFG and IFG. Better HDL functionality may contribute to maintenance of β-cell function in subjects with well-controlled T2DM.

A strategy for the generation of specific human antibodies by directed evolution and phage display

This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

Comparative Study of Different Biological Glues in an Experimental Model of Surgical Bleeding in Anesthetized Rats: Platelet-Rich and -Poor Plasma-Based Glue with and without Aprotinin versus Commercial Fibrinogen-Based Glue

The use of fibrin glue in cardiovascular surgery has been associated with decreased operative time, effective control of localized bleeding, and reduced postoperative blood loss. All preparations of fibrin glue mimic the final common pathway of the coagulation cascade in which fibrinogen is converted to fibrin in the presence of thrombin and calcium. The goal of the study was to compare five different types of fibrin glue, with or without aprotinin, on a surgical bleeding model in the rat. In 70 anesthetized Wistar rats, after laparotomy, a 3 cm liver incision was performed. After randomization, seven groups were studied. In the first group, Biocol was used as a pinpoint application to the bleeding site. Four groups received a fibrin glue obtained from a single human donor plasma using Cell Saver V (Haemonetics). The sealant was applied as a two-component system. The first component of the glue was either platelet-rich-plasma (PRP) or platelet-poor-plasma (PPP). The second component consisted of a mixture of 0.5 ml CaCl 10% with 1000 U of human thrombin, with or without 400KUI of aprotinin (AP). The last two groups, control and aprotinin were treated using saline solution or topical aprotinin respectively. Hemoglobin and hematocrit were measured before surgery and 30 min after application of the glue. The decrease in hemoglobin (Hb) and hematocrit (Hct) was the primary efficacy variable. Before surgery, there was no difference regarding Hb and Hct values between groups. Thirty min after the application of the glue, the decrease in hemoglobin expressed as percent of the control values is only significantly lower in the Biocol group when compared to control. No significant difference was observed with the other groups in comparison to control. The commercial fibrin glue (Biocol) is more efficient than other preparations. This efficacy is likely due to a higher fibrinogen concentration.

Is There a Possibility of a Bioartificial Pancreas?

The use of pancreatic living tissue, namely the transplantation of the pancreas or of isolated islets, for the cure of diabetes mellitus is extremely attractive. It is the only way to obtain in type 1 diabetic patients a perfect normalization of glycosylated hemoglobin, a marker of metabolic control. However, there are two major obstacles which limit this approach: The immune rejection of the transplanted tissue and the scarcity of pancreas donors in the face of a common disease. These two obstacles explain that a relatively small number of trials have been performed, most of them in patients with end stage renal failure and in conjunction with a kidney transplant. So far, islet transplantation, although able to correct hyperglycemia in experimental models of diabetes in animals, has consistently failed in man, certainly due to the fact that there is no method for isolating an adequate number of intact islets from a single human donor pancreas. A recent paper demonstrated a need to transplant 5000 islets/kg body weight to correct hyperglycemia in diabetic dogs (1). This would yield 300,000 islets for a human adult, which would represent about one third of the islets of a human pancreas.

Purification of human myeloperoxidase by Concanavalin A-Sepharose affinity chromatography.

Myeloperoxidase (E.C. 1.11.1.7. Donor: H2O2 oxidoreductase), an enzyme of the azurophil granule of polymorphonuclear leukocytes, has been purified from the blood of a single human donor. Leukocytes were harvested by dextran sedimentation and contaminating erythrocytes were removed by hypotonic lysis. Myeloperoxidase was extracted from the leukocytes by dissolution in cetyltrimethyl-ammonium bromide (CETAB). The extract was adsorbed to a Con A-Sepharose gel, eluted with alpha-methyl-D-mannoside and rechromatographed on Sephadex G-200. Approximately 100% of the initial peroxidase activity was recovered in the final preparation, which was homogeneous by polyacrylamide disc gel electrophoresis at pH 4.5 and displayed the typical oxidized and reduced absorption spectra of purified myeloperoxidase. The oxidized enzyme showed a Soret maximum at 430 nm which shifted to 470 nm upon reduction. The molecular weight was estimated by G-200 Sephadex gel filtration chromatography to be 146,000 daltons.

Enhanced representation of HL-A antigens on human lymphocytes after mitogenesis induced by phytohemagglutinin or Epstein-Barr virus.

The amount of HL-A antigens present on the peripheral blood lymphocytes of a single human donor was increased about 11-fold after stimulation with phytohemagglutinin and 36-fold after transformation with Epstein-Barr virus. This increase applied to all four HL-A specificities of these cells. The response to phytohemagglutinin was dependent on dose and was first observed at 12 hr of incubation. Measurements of the amount of surface membranes by geometry, by radioiodinatable surface proteins, and by 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) assay all indicated that the enhanced representation of HL-A antigens after stimulation by phytohemagglutinin or transformation by Epstein-Barr virus must be due to an increase in the density of these antigens on the cell surface.