Cardiopulmonary bypass surgery-cold alert!

The term "cold agglutinin (CA)" refers to a group of disorders caused by anti-erythrocyte autoantibodies that preferentially bind RBCs at cold temperatures (4°C-18°C). CAs contribute to 10 to 15% of autoimmune hemolytic anemia. We report a case of CAs diagnosed intraoperatively during emergency mitral valve replacement.

Vaccination in a humanized mouse model elicits highly protective PfCSP-targeting anti-malarial antibodies.

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

Polysaccharide enabled biogenic fabrication of pH sensing fluorescent gold nanoclusters as a biocompatible tumor imaging probe.

A biocompatible natural polysaccharide (PSP001) isolated from the fruit rind of Punica granatum was conjugated with L-cysteine (Y) to be used as a skeleton for the fabrication of fluorescent gold nanoclusters (AuNCs) represented as PSP-Y-AuNCs. With an average size of ~ 6 nm, PSP-Y-AuNCs demonstrated high quantum yield (31%), with a pH-sensitive fluorescence emission behavior. An emission maximum of 520 nm was obtained at acidic pH, which was blue shifted with increasing pH. This feature provides the possibilities for accurate ratiometric pH imaging. The PSP-Y-AuNCs not only demonstrated excellent biocompatibility with cancer cells and isolated peripheral lymphocytes and red blood cells but also demonstrated to be an active molecular imaging probe with appealing cellular uptake efficiency. The investigations with BALB/c mice further confirmed the non-toxic nature and in vivo imaging potential of the AuNCs. Estimation of the bio-distribution on solid tumor bearing syngeneic murine models revealed a tumor-targeted enhanced fluorescence emission pattern which is attributed to the pH responsive fluorescence behavior and the acidic microenvironment of the tumor. These findings were further confirmed with an impressive tumor accumulation pattern displayed in a xenograft of human cancer bearing nude mice. On account of their impressive biocompatibility and photophysical features, PSP-Y-AuNCs can exploited for the real-time fluorescence imaging of cancer tissues. Graphical abstract Fluorescent gold nanoclusters (PSP-Y-AuNCs) fabricated using a non-toxic natural polysaccharide (PSP001) demonstrated pH sensitive fluorescence emission pattern. The increased fluorescence readouts at acidic conditions and excellent biocompatibility made the PSP-Y-AuNCs an appealing candidate for in vivo tumor imaging applications.

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.

The attenuation of cockroach allergy by DNA vaccine encoding cockroach allergen Bla g 2.

Bla g 2 is one of the most potent cockroach allergens. No effective treatment or vaccination strategies are yet available. We evaluated the prophylactic efficacy of Bla g 2 DNA vaccination in a mouse model of allergic airway inflammation. C57/BL6 mice were given Bla g 2 DNA vaccine prior to sensitization with recombinant Bla g 2 (rBla g 2) antigens, followed by nebulized rBla g 2 challenge. Bla g 2 vaccine could express at both transcriptional and translational levels in mammalian cells. Moreover, Bla g 2 vaccine significantly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid, and markedly decreased allergen-induced inflammatory infiltrates in the lungs and Bla g 2-specific IgE in serum upon challenge with rBla g 2. Importantly, Bla g 2 vaccine could induce the production of antigen-specific IFN-γ and downregulated Th2 pro-inflammatory cytokines IL-4, IL-5, and IL-13. Thus, DNA vaccination showed protective efficacy against a clinically relevant allergen, Bla g 2.

Structural determination of the phosphorylation domain of the ryanodine receptor.

The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum membrane protein that is essential for Ca(2+) cycling in both skeletal and cardiac muscle. Genetic mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central core disease. One phosphorylation site (Ser 2843) has been identified in a segment of RyR1 flanked by two RyR motifs, which are found exclusively in all RyR isoforms as closely associated tandem (or paired) motifs, and are named after the protein itself. These motifs also contain six known MH mutations. In this study, we designed, expressed and purified the tandem RyR motifs, and show that this domain contains a putative binding site for the Ca(2+)/calmodulin-dependent protein kinase ß isoform. We present a 2.2 Å resolution crystal structure of the RyR domain revealing a two-fold, symmetric, extended four-helix bundle stabilized by a ß sheet. Using mathematical modelling, we fit our crystal structure within a tetrameric electron microscopy (EM) structure of native RyR1, and propose that this domain is localized in the RyR clamp region, which is absent in its cousin protein inositol 1,4,5-trisphosphate receptor.

Two ZnF-UBP domains in isopeptidase T (USP5).

Human ubiquitin-specific cysteine protease 5 (USP5, also known as ISOT and isopeptidase T), an 835-residue multidomain enzyme, recycles ubiquitin by hydrolyzing isopeptide bonds in a variety of unanchored polyubiquitin substrates. Activation of the enzyme's hydrolytic activity toward ubiquitin-AMC (7-amino-4-methylcoumarin), a fluorogenic substrate, by the addition of free, unanchored monoubiquitin suggested an allosteric mechanism of activation by the ZnF-UBP domain (residues 163-291), which binds the substrate's unanchored diglycine carboxyl tail. By determining the structure of full-length USP5, we discovered the existence of a cryptic ZnF-UBP domain (residues 1-156), which was tightly bound to the catalytic core and was indispensable for catalytic activity. In contrast, the previously characterized ZnF-UBP domain did not contribute directly to the active site; a paucity of interactions suggested flexibility between these two domains consistent with an ability by the enzyme to hydrolyze a variety of different polyubiquitin chain linkages. Deletion of the known ZnF-UBP domain did not significantly affect rate of hydrolysis of ubiquitin-AMC and suggested that it is likely associated mainly with substrate targeting and specificity. Together, our findings show that USP5 uses multiple ZnF-UBP domains for substrate targeting and core catalytic function.

Administering human adipose-derived mesenchymal stem cells to prevent and treat experimental arthritis.

Rheumatoid arthritis is a chronic autoimmune disease and affecting approximately 1% of the population. Human adipose-derived mesenchymal stem cells (hASCs) were recently found to suppress effector T cell and inflammatory responses and, thus, to have beneficial effects in various autoimmune diseases. In this study, we examined whether hASCs could play a protective and/or therapeutic role in collagen-induced arthritis (CIA). We showed that hASCs both prevented and treated CIA by significantly reducing the incidence and severity of experimental arthritis. We further demonstrated that treatment with hASCs inhibited the production of various inflammatory mediators, decreased antigen-specific Th1/Th17 cell expansion, and induced the production of anti-inflammatory cytokine interleukin-10. Moreover, hASCs could induce the generation of antigen-specific Treg cells with the capacity to suppress collagen-specific T cell responses.

Roles of the lipid-binding motifs of Atg18 and Atg21 in the cytoplasm to vacuole targeting pathway and autophagy.

Atg18 and Atg21 are homologous WD-40 repeat proteins that bind phosphoinositides via a novel conserved Phe-Arg-Arg-Gly motif and function in autophagy-related pathways. Atg18 is required for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas Atg21 is only required for the Cvt pathway. Currently, the functions of both proteins are poorly understood. Here, we examined the relationship between the phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding abilities of Atg18 and Atg21 and autophagy by expressing variants of these proteins that have mutations in their phosphoinositide-binding motifs. Cells expressing PtdIns(3)P-binding mutants of both these proteins showed highly reduced autophagy. Furthermore, the localization of components of two related ubiquitin-like protein conjugation systems, Atg8 and Atg16, to the phagophore assembly site is affected. Consistent with the aberrant localization of the above Atg proteins, precursor Ape1, a cargo of the Cvt pathway and autophagy, is partially protease-sensitive in starvation conditions. This finding suggests a requirement for the PtdIns(3)P binding capability of Atg18 and Atg21 in efficient completion of the sequestering autophagic vesicles. Finally, using a multiple knock-out strain, we found that Atg18 and Atg21 facilitate the recruitment of Atg8-PE to the site of autophagosome formation and protect it from premature cleavage by Atg4, which represents a key aspect of post-translational autophagy regulation. Taken together, our results suggest that PtdIns(3)P binding by at least Atg18 or Atg21 is required for robust autophagic activity and that the PtdIns(3)P-binding motifs of Atg18 and Atg21 can compensate for one another in the recruitment of Atg components that are dependent on PtdIns(3)P for their phagophore assembly site association.

Crystal structures of monomeric actin bound to cytochalasin D.

The fungal toxin cytochalasin D (CD) interferes with the normal dynamics of the actin cytoskeleton by binding to the barbed end of actin filaments. Despite its widespread use as a tool for studying actin-mediated processes, the exact location and nature of its binding to actin have not been previously determined. Here we describe two crystal structures of an expressed monomeric actin in complex with CD: one obtained by soaking preformed actin crystals with CD, and the other obtained by cocrystallization. The binding site for CD, in the hydrophobic cleft between actin subdomains 1 and 3, is the same in the two structures. Polar and hydrophobic contacts play equally important roles in CD binding, and six hydrogen bonds stabilize the actin-CD complex. Many unrelated actin-binding proteins and marine toxins target this cleft and the hydrophobic pocket at the front end of the cleft (viewing actin with subdomain 2 in the upper right corner). CD differs in that it binds to the back half of the cleft. The ability of CD to induce actin dimer formation and actin-catalyzed ATP hydrolysis may be related to its unique binding site and the necessity to fit its bulky macrocycle into this cleft. Contacts with residues lining this cleft appear to be crucial to capping and/or severing. The cocrystallized actin-CD structure also revealed changes in actin conformation. An approximately 6 degrees rotation of the smaller actin domain (subdomains 1 and 2) with respect to the larger domain (subdomains 3 and 4) results in small changes in crystal packing that allow the D-loop to adopt an extended loop structure instead of being disordered, as it is in most crystal structures of actin. We speculate that these changes represent a potential conformation that the actin monomer can adopt on the pathway to polymerization or in the filament.

Characterisation of the immune response to type I collagen in scleroderma.

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSElow). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4+, activated (CD25+), memory (CD45RO+) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-gamma but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-gamma, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-alpha, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls.

Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag.

The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(-) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(-) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.

Atg9 cycles between mitochondria and the pre-autophagosomal structure in yeasts.

Autophagy is a degradative process conserved among eukaryotic cells. It allows the elimination of cytoplasm including aberrant protein aggregates and damaged organelles. Accordingly, it is implicated in normal developmental processes and also serves a protective role in tumor suppression and elimination of invading pathogens, whereas defects in autophagy are associated with various human diseases including cancer and neurodegeneration. Atg proteins mediate the sequestration event that occurs at the preautophagosomal structure (PAS) by catalyzing the formation of double-membrane vesicles, termed autophagosomes. In Saccharomyces cerevisiae, the integral membrane protein Atg9 that is required for autophagy cycles through the PAS. Here, we demonstrate that Atg9 shuttles between this location and mitochondria. These data support a new model where mitochondria may provide at least part of the autophagosomal lipids and suggest a novel cellular function for this well-studied organelle.

Intracellular staining for HIV-specific IFN-gamma production: statistical analyses establish reproducibility and criteria for distinguishing positive responses.

The cellular immune response plays a pivotal role in controlling the spread of HIV-1 infection by lysing virally infected cells and producing potent antiviral cytokines, such as interferon-gamma (IFN-gamma). Flow cytometric methods have been established to evaluate the contribution of both CD4 and CD8 subsets of T lymphocytes to the immune response to HIV by measuring their production of intracellular IFN-gamma following brief antigenic stimulation. We present a statistical treatment of intracellular cytokine staining (ICS) data that is aimed at establishing the reproducibility and robustness of this assay for use in HIV clinical trials. Comparisons of responses from HIV-seronegative and seropositive individuals were used to establish a 2-fold criterion for distinguishing positive responses with a low probability of false positives (<1%). Additional comparisons established that the reproducibility of the assay is between 1.4 and 2.0-fold depending on the magnitude of the response. Little variability was demonstrated between multiple operators for both the execution and analysis components of these experiments (<10% difference with 95% confidence). We conclude that the statistical criteria established by these analyses allow for the accurate detection and comparison of positive responses. Using these statistical criteria, the ICS assay is sufficiently robust for use in HIV-specific vaccine trials.