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

Prinos poznavanju bakarnih katalizatora za hidriranje

Inaktiviranje t. zv. bakarnih kromita kod katalitickih hidriranja pripisuje se obično redukciji CuO u Cu2O, odnosno mozda i u metal Cu. Hidriranjem estera uljne kiseline ovdje je dokazano, da kako metalicki bakar (Raney-Cu), tako i specijalno pripravljeni Cu2O mogu dobro sluziti za proizvodnju oktadekanola. Za ovu reakciju se dosad smatralo, da je bakarni kromit jedini prikladni oblik bakarnih katalizatora. Nasi nalazi govore u prilog pretpostavci R. Schencka, da je kataliticki aktivna tvar bakarnog kromita metalni bakar, koji nastaje in situ kod samog hidriranja

Prinos poznavanju bakarnih katalizatora za hidriranje

Inaktiviranje t. zv. bakarnih kromita kod katalitickih hidriranja pripisuje se obično redukciji CuO u Cu2O, odnosno mozda i u metal Cu. Hidriranjem estera uljne kiseline ovdje je dokazano, da kako metalicki bakar (Raney-Cu), tako i specijalno pripravljeni Cu2O mogu dobro sluziti za proizvodnju oktadekanola. Za ovu reakciju se dosad smatralo, da je bakarni kromit jedini prikladni oblik bakarnih katalizatora. Nasi nalazi govore u prilog pretpostavci R. Schencka, da je kataliticki aktivna tvar bakarnog kromita metalni bakar, koji nastaje in situ kod samog hidriranja

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

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

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

The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans

Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity

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