Fractionation of Tetanus Toxoid by Sephadex Gel Filtration and Immunochemical Characterization of the Fractions

Three different ty;pes of tetanus toxoid were fractionated on Sephadex G - 200 gel. The distribution of the fractions was determined spectrophotometrically at 280 mfL . Specific activity of the fractions in vitro was determined by flocculation with standard tetanus antitoxin and by means of immunodiffusion and immunoelectrophoresis. In all three types of the toxoid the active component appeared in the first three fractions ; about 750/o was found in the third fraction. The purity of this fraction, in terms of Lf/mg. of protein nitrogen (PN), was increased about twic e in comparison with the starting material. The highest purity, 2400 Lf/ mg. PN, was achieved in the third fraction of the toxoid type B-1, which w as p repurified, first by ultrafiltration of the toxin, then, after detoxication, by fractional precipitation with ammonium sulfate. This fraction was homogeneous according to immunodiffusion and immunoelectrophoresis te sts. It has been concluded that fractional purification of t etanus toxoid can be effectively achieved by gel filtration on Sephadex G-200, but the effect of purification highly depends on the purity of the starting material

A Gel-Chromatographic and Light Scattering Study of the Salmonella typhi Endotoxin

The endotoxin of Salmonella typhi, strain 0-901, was isolated by extraction with hypertonic (1 M) sodium chloride solutions and studied by gel-chromatography and light scattering methods. The gel-chromatographic separation was performed on Sepharose 2B, Sepharose 4B and Sephadex G-200 gels, and the fractionated material was monitored by ultraviolet and phenol-sulfuric acid colorimetry as well as by a photometric latex agglutination test. The extracted material consisted of two components: one was the high molecular weight endotoxin and the other a protein-polysaccharide complex of a molecular weight lower than 66,000 dalton. The light scattering experiments of endotoxin extracts showed the average molecular weights from 1.9 to 4.9 million dalton. The separation of the low molecular weight proteinic component was attempted by thermal denaturation, but this had to be abandoned owing to the denaturation and degradation of endotoxin. A high molecular weight endotoxin component was isolated by elution on a Sephadex G-200 column and had the molecular weight of 5.6 million dalton, which was in good accord with the value previously determined for a Boivin extraction sample. The high molecular weight endotoxin sample proved to be a highly polydispersed material. From the estimates of various averages of gyration radii it has been concluded that the particles of this sample have a more compact structurE than those of the Boivin extraction sample, possibly due to the tertiary structuring of polypeptide chains in the protein-lipopolysaccharide complex of the endotoxin particle

Centriole polarisation to the immunological synapse directs secretion from cytolytic cells of both the innate and adaptive immune systems.

BACKGROUND: Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release. RESULTS: NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse. CONCLUSIONS: These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological synapse. Morphologically, the overall structure of the immunological synapses formed by NK and iNKT cells are very similar to those formed by CTLs, with both exocytic and endocytic organelles polarised towards the centrosome at the plasma membrane, which forms a focal point for exocytosis and endocytosis within the immunological synapse. We conclude that centrosomal polarisation provides a rapid, responsive and precise mechanism for secretory lysosome delivery to the immunological synapse in CTLs, NK cells and iNKT cells.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Killer Ig-like receptors (kirs). their role in nk cell modulation and developments leading to their clinical exploitation

Natural killer (NK) cells contribute to the first line of defense against viruses and to the control of tumor growth and metastasis spread. The discovery of HLA class I specific inhibitory receptors, primarily of killer Ig-like receptors (KIRs), and of activating receptors has been fundamental to unravel NK cell function and the molecular mechanisms of tumor cell killing. Stemmed from the seminal discoveries in early ‘90s, in which Alessandro Moretta was the major actor, an extraordinary amount of research on KIR specificity, genetics, polymorphism, and repertoire has followed. These basic notions on NK cells and their receptors have been successfully translated to clinical applications, primarily to the haploidentical hematopoietic stem cell transplantation to cure otherwise fatal leukemia in patients with no HLA compatible donors. The finding that NK cells may express the PD-1 inhibitory checkpoint, particularly in cancer patients, may allow understanding how anti-PD-1 therapy could function also in case of HLA class Ineg tumors, usually susceptible to NK-mediated killing. This, together with the synergy of therapeutic anti-checkpoint monoclonal antibodies, including those directed against NKG2A or KIRs, emerging in recent or ongoing studies, opened new solid perspectives in cancer therapy

Cell-laden hydrogel as a clinical-relevant 3D model for analyzing neuroblastoma growth, immunophenotype, and susceptibility to therapies

High risk Neuroblastoma (NB) includes aggressive, metastatic solid tumors of childhood. The survival rate improved only modestly, despite the use of combination therapies including novel immunotherapies based on the antibody mediated targeting of tumor-associated surface ligands. Treatment failures may be due to the lack of adequate in vitro models for studying, in a given patient, the efficacy of potential therapeutics, including those aimed to enhance anti-tumor immune responses. We here propose a 3D alginate-based hydrogel as extracellular microenvironment to evaluate the effects of the three-dimensionality on biological and immunological properties of NB cells. NB cell lines grown within the 3D alginate spheres presented spheroid morphology, optimal survival, and proliferation capabilities, and a reduced sensitivity to the cytotoxic effect of imatinib mesylate. 3D cultured NB cells were also evaluated for the constitutive and IFN-y-induced expression of surface molecules capable of tuning the anti-tumor activity of NK cells including immune checkpoint ligands. In particular, IFN-y induced de novo expression of high amounts of HLA-I molecules, which protected NB cells from the attack mediated by KIR/KIR-L matched NK cells. Moreover, in the 3D alginate spheres, the cytokine increased the expression of the immune checkpoint ligands PD-Ls and B7-H3 while virtually abrogating that of PVR, a ligand of DNAM-1 activating receptor, whose expression correlates with high susceptibility to NK-mediated killing. Our 3D model highlighted molecular features that more closely resemble the immunophenotypic variants occurring in vivo and not fully appreciated in classical 2D culture conditions.Thus, based on our results, 3D alginate-based hydrogels might represent a clinical-relevant cell culture platform where to test the efficacy of personalized therapeutic approaches aimed to optimize the current and innovative immune based therapies in a very systematic and reliable way

Inhibitory 2B4 contributes to NK cell education and immunological derangements in XLP1 patients

X-linked lymphoproliferative disease 1 (XLP1) is an inherited immunodeficiency, caused by mutations in SH2D1A encoding Signaling Lymphocyte Activation Molecule (SLAM)-associated protein (SAP). In XLP1, 2B4, upon engagement with CD48, has inhibitory instead of activating function. This causes a selective inability of cytotoxic effectors to kill EBV-infected cells, with dramatic clinical sequelae. Here, we investigated the NK cell education in XLP1, upon characterization of killer Ig-like receptor (KIR)/KIR-L genotype and phenotypic repertoire of self-HLA class I specific inhibitory NK receptors (self-iNKRs). We also analyzed NK-cell cytotoxicity against CD48+ or CD48− KIR-ligand matched or autologous hematopoietic cells in XLP1 patients and healthy controls. XLP1 NK cells may show a defective phenotypic repertoire with substantial proportion of cells lacking self-iNKR. These NK cells are cytotoxic and the inhibitory 2B4/CD48 pathway plays a major role to prevent killing of CD48+ EBV-transformed B cells and M1 macrophages. Importantly, self-iNKR defective NK cells kill CD48− targets, such as mature DCs. Self-iNKR− NK cells in XLP1 patients are functional even in resting conditions, suggesting a role of the inhibitory 2B4/CD48 pathway in the education process during NK-cell maturation. Killing of autologous mature DC by self-iNKR defective XLP1 NK cells may impair adaptive responses, further exacerbating the patients’ immune defect

Genetic predisposition to hemophagocytic lymphohistiocytosis: report on 500 patients from the Italian registry

Background Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease affecting mostly children but also adults and characterized by hyperinflammatory features. A subset of patients, referred to as having familial hemophagocytic lymphohistiocytosis (FHL), have various underlying genetic abnormalities, the frequencies of which have not been systematically determined previously. Objective This work aims to further our understanding of the pathogenic bases of this rare condition based on an analysis of our 25 years of experience. Methods From our registry, we have analyzed a total of 500 unselected patients with HLH. Results Biallelic pathogenic mutations defining FHL were found in 171 (34%) patients; the proportion of FHL was much higher (64%) in patients given a diagnosis during the first year of life. Taken together, mutations of the genes PRF1 (FHL2) and UNC13D (FHL3) accounted for 70% of cases of FHL. Overall, a genetic diagnosis was possible in more than 90% of our patients with FHL. Perforin expression and the extent of degranulation have been more useful for diagnosing FHL than hemophagocytosis and the cytotoxicity assay. Of 281 (56%) patients classified as having "sporadic" HLH, 43 had monoallelic mutations in one of the FHL-defining genes. Given this gene dosage effect, FHL is not strictly recessive. Conclusion We suggest that the clinical syndrome HLH generally results from the combined effects of an exogenous trigger and genetic predisposition. Within this combination, different weights of exogenous and genetic factors account for the wide disease spectrum that ranges from HLH secondary to severe infection to FHL

A Gel-Chromatographic and Light Scattering Study of the Salmonella typhi Endotoxin

The endotoxin of Salmonella typhi, strain 0-901, was isolated by extraction with hypertonic (1 M) sodium chloride solutions and studied by gel-chromatography and light scattering methods. The gel-chromatographic separation was performed on Sepharose 2B, Sepharose 4B and Sephadex G-200 gels, and the fractionated material was monitored by ultraviolet and phenol-sulfuric acid colorimetry as well as by a photometric latex agglutination test. The extracted material consisted of two components: one was the high molecular weight endotoxin and the other a protein-polysaccharide complex of a molecular weight lower than 66,000 dalton. The light scattering experiments of endotoxin extracts showed the average molecular weights from 1.9 to 4.9 million dalton. The separation of the low molecular weight proteinic component was attempted by thermal denaturation, but this had to be abandoned owing to the denaturation and degradation of endotoxin. A high molecular weight endotoxin component was isolated by elution on a Sephadex G-200 column and had the molecular weight of 5.6 million dalton, which was in good accord with the value previously determined for a Boivin extraction sample. The high molecular weight endotoxin sample proved to be a highly polydispersed material. From the estimates of various averages of gyration radii it has been concluded that the particles of this sample have a more compact structurE than those of the Boivin extraction sample, possibly due to the tertiary structuring of polypeptide chains in the protein-lipopolysaccharide complex of the endotoxin particle

Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity.

none15We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)-like receptors (KIR) ligand-mismatched donors. We showed that, in most transplantation patients, variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype, as well as through phenotypic and functional analyses of NK cells, both at the polyclonal and clonal level. Donor-derived KIR2DL1(+) NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3(+) NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3(+) (or by NKG2A(+)) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether, these results may have important clinical implications for the selection of optimal donors for haplo-HSCT.openPENDE D; MARCENARO S; FALCO M; MARTINI S; BERNARDO ME; MONTAGNA D; ROMEO E; COGNET C; MARTINETTI M; MACCARIO R; MINGARI MC; VIVIER E; MORETTA L; LOCATELLI F; MORETTA A.Pende, D; Marcenaro, S; Falco, M; Martini, S; Bernardo, Me; Montagna, Daniela; Romeo, E; Cognet, C; Martinetti, M; Maccario, R; Mingari, Mc; Vivier, E; Moretta, L; Locatelli, Franco; Moretta, A

Genetic predisposition to hemophagocytic lymphohistiocytosis: Report on 500 patients from the Italian registry.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease affecting mostly children but also adults and characterized by hyperinflammatory features. A subset of patients, referred to as having familial hemophagocytic lymphohistiocytosis (FHL), have various underlying genetic abnormalities, the frequencies of which have not been systematically determined previously. OBJECTIVE: This work aims to further our understanding of the pathogenic bases of this rare condition based on an analysis of our 25 years of experience. METHODS: From our registry, we have analyzed a total of 500 unselected patients with HLH. RESULTS: Biallelic pathogenic mutations defining FHL were found in 171 (34%) patients; the proportion of FHL was much higher (64%) in patients given a diagnosis during the first year of life. Taken together, mutations of the genes PRF1 (FHL2) and UNC13D (FHL3) accounted for 70% of cases of FHL. Overall, a genetic diagnosis was possible in more than 90% of our patients with FHL. Perforin expression and the extent of degranulation have been more useful for diagnosing FHL than hemophagocytosis and the cytotoxicity assay. Of 281 (56%) patients classified as having "sporadic" HLH, 43 had monoallelic mutations in one of the FHL-defining genes. Given this gene dosage effect, FHL is not strictly recessive. CONCLUSION: We suggest that the clinical syndrome HLH generally results from the combined effects of an exogenous trigger and genetic predisposition. Within this combination, different weights of exogenous and genetic factors account for the wide disease spectrum that ranges from HLH secondary to severe infection to FHL.Supported by grants from Associazione Italiana Ricerca Istiocitosi (AIRI), Associazione Ciemmeesse-Girotondo per il Meyer, Ministero della Salute (Bando Malattie Rare RF-TOS-2008-1219488), and the Seventh Framework Programme (FP7) of the European Commission (“FIGHT-HLH” Project no. 306124 to M.A.). Disclosure of potential conflict of interest: D. Pende receives royalties paid to her institution. G. M. Griffiths has received research support from the Wellcome Trust. L. Luzzatto is on the Alexion Pharmaceuticals SAB and has received consultancy fees from GlaxoSmithKline.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.jaci.2015.06.04