Papers by Stephan Ladisch
The Journal of pediatrics, Jan 11, 2017
To develop and validate a diagnostic score that assists in discriminating primary hemophagocytic ... more To develop and validate a diagnostic score that assists in discriminating primary hemophagocytic lymphohistiocytosis (pHLH) from macrophage activation syndrome (MAS) related to systemic juvenile idiopathic arthritis. The clinical, laboratory, and histopathologic features of 362 patients with MAS and 258 patients with pHLH were collected in a multinational collaborative study. Eighty percent of the population was assessed to develop the score and the remaining 20% constituted the validation sample. Variables that entered the best fitted model of logistic regression were assigned a score, based on their statistical weight. The MAS/HLH (MH) score was made up with the individual scores of selected variables. The cutoff in the MH score that discriminated pHLH from MAS best was calculated by means of receiver operating characteristic curve analysis. Score performance was examined in both developmental and validation samples. Six variables composed the MH score: age at onset, neutrophil co...
Biochimica et biophysica acta, Apr 1, 1991
Gangliosides shed by tumor cells are implicated in modulating tumor formation. For example, rapid... more Gangliosides shed by tumor cells are implicated in modulating tumor formation. For example, rapid progression of human neuroblastoma tumors is associated with high circulating levels of shed GD2 ganglioside. To elucidate the kinetic and qualitative characteristics of tumor cell ganglioside shedding, which is difficult to accomplish in vivo, we examined this process in LAN-5 human neuroblastoma cells in vitro. Three major gangliosides, GD2, GM2 and GT1b, comprise 82% of the mean total of 38 nmol LBSA/10(8) cells. These molecules are shed very rapidly (1-3 pmol/10(6) cells per h, or approx. 0.5% of the total cell gangliosides per h). Quantitative and qualitative characteristics of ganglioside shedding are remarkably constant over a 40-fold range of cell density. Not only GD2, but every major carbohydrate species is shed, in proportion to its concentration in the cell, with slightly greater shedding of ceramide subspecies containing shorter chain fatty acids. These findings were confirmed in three other neuroblastoma cell lines, LAN-1, IMR and KCNR. We suggest that the high expression and rapid generalized shedding of human neuroblastoma tumor gangliosides results in significant in vivo accumulation of these biologically active molecules during tumor growth.
Journal of the National Cancer Institute, Jun 7, 2000
Background: Gangliosides are immunosuppressive cell surface molecules that are often present in h... more Background: Gangliosides are immunosuppressive cell surface molecules that are often present in high concentrations in and shed actively by tumor cells. These molecules inhibit the antitumor immune response that is implicated in tumor rejection. We therefore determined the ability of tumor cells pharmacologically depleted of gangliosides to form tumors in mice. Methods: We tested a ganglioside-rich subline of B16 murine melanoma, MEB4, and MEB4 cells that had been depleted of endogenous gangliosides by incubation with 0.5 M 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1propanol, a specific inhibitor of the enzyme glucosylceramide synthase. Tumor formation was assessed twice a week for 10 weeks after the intradermal injection of tumor cells, and metastatic potential was assessed 4 weeks after tail vein injection of tumor cells. All P values are from two-sided tests. Results: Reduction of the ganglioside content of MEB4 cells, which was not cytotoxic to cells and did not inhibit cell proliferation in vitro, markedly reduced their ability to form tumors. Only 40% of the mice given an intradermal injection of 10 5 ganglioside-depleted MEB4 cells developed tumors compared with 100% of the mice given an injection of 10 5 control MEB4 cells (P<.001). Ganglioside depletion also reduced metastasis: A mean of five pulmonary metastases was detected per mouse given an injection of 2 × 10 5 gangliosidedepleted MEB4 cells compared with a mean of 25 per mouse given an injection of 2 × 10 5 control MEB4 cells. Conclusion: Tumor cells with a pharmacologically decreased concentration of gangliosides produce fewer tumors in mice than do untreated cells, suggesting that pharmacologic depletion of gangliosides should be explored further as a therapeutic approach to cancer. [J Natl Cancer Inst 2000;92:912-7] MATERIALS AND METHODS Cell Culture MEB4 and GM95 cells (20), two sublines of the B16 murine melanoma, were obtained from the RIKEN Cell Bank, Saitama, Japan. The cells were maintained in Dulbecco's modified Eagle medium (product 12430-054; Life Technologies, Inc. [GIBCO], Gaithersburg, MD) supplemented with 10% fetal bovine serum. Cultures were maintained in an atmosphere of 5% CO 2 /95% air in a humidified incubator at 37°C.
Biochemical and Biophysical Research Communications, Sep 1, 1994
Structural variations among gangliosides significantly influence their immunosuppressive activity... more Structural variations among gangliosides significantly influence their immunosuppressive activity. By total chemical synthesis, a sialic acid-containing glycoconjugate, 2-(tetradecylhexadecyl)-O-(5-acetamido-3,5-dideoxy-D-glycero-alpha -D-galacto-2-nonulopyranosylonic acid)-(2--&gt;3)-O-beta-D-galactopyrannosyl-(1--&gt;4)-beta-D- glucopyrannoside was synthesized. This glycoconjugate has the same carbohydrate structure as does GM3 ganglioside and a branched alkane in place of ceramide. It markedly inhibits the tetanus toxoid-induced human lymphoproliferative response in vitro (ID90 &lt; 7 microM) and is five-fold more active than d18:1-C18:0 GM3 ganglioside, to which it is structurally related. This glycoconjugate is also a potent inhibitor of the murine alloimmune response in vivo: 10 nmol of the molecule injected subcutaneously together with an allogeneic cell challenge markedly inhibits the cellular immune response in the draining popliteal lymph node. In fact, the effect is quantitatively similar to that of systemically administered cyclosporin A, a well-studied immunosuppressive agent.
Biochemistry, 1995
New chemical synthetic methods have permitted the synthesis of a spectrum of glycosphingolipid mo... more New chemical synthetic methods have permitted the synthesis of a spectrum of glycosphingolipid molecular species, some of which are not naturally occurring. Here we have studied a number of chemically synthesized gangliosides for immunosuppressive activity, using a human in vitro specific antigen (tetanus toxoid)-induced assay of the cellular immune response. Chemically synthesized G M~ and G M~ had the same high degree of immunosuppressive activity as did natural G M~ and G M~ gangliosides, verifying that inhibition is intrinsic to the ganglioside molecules and not caused by other molecules sometimes found in natural preparations (e.g., proteins). Studies of modified molecular species of G M~ and &4, also prepared by chemical synthesis, have shown the influence of certain structural details upon the immunosuppressive activity of gangliosides: (i) the inverse relationship between fatty acyl chain length and immunosuppressive activity is extended to even shorter chain lengths, with the synthetic gangliosides d18: l-C2:0-G~3 and d18: 1-C14:0-G~3 being more immunosuppressive than d18: 1-C18:0-G~3 and d18: 1-C24:0-G~3; (ii) hydroxylation of the fatty acyl group decreases immunosuppressive activity; (iii) substitution of an S-glycosidic bond for an 0-glycosidic bond in the sialic acid ketosidic linkage in G M~ does not alter its activity; and (iv) modifications of the sialic acid group variably influence immunosuppressive activity, since KDN-GM~ and-G M~ ganglioside analogues, which contain a 3-deoxy-D-glycero-D-galacto-2nonulopyranosonic acid in place of N-acetylneuraminic acid, retain activity, while other modifications such as 8-epi-G~3, and to a lesser extent 9-deoxy-G~3, reduce immunosuppressive activity. In conclusion, gangliosides produced by total chemical synthesis have the immunosuppressive properties of the natural molecules and are useful in probing which elements of ganglioside structure are critical to their biological activity.
Angiogenesis, Oct 29, 2013
Tumor cells shed gangliosides and populate their microenvironment with these biologically active ... more Tumor cells shed gangliosides and populate their microenvironment with these biologically active membrane glycosphingolipids. In vitro, ganglioside enrichment amplifies receptor tyrosine kinase signaling and activation of vascular endothelial cells. However, a long-standing question is whether in the actual microenvironment of a neoplasm, in vivo, tumor cell ganglioside shedding stimulates angiogenesis. Here we tested the hypothesis that tumor gangliosides have a critical proangiogenic role in vivo using novel murine tumor cells (DKO) genetically completely incapable of ganglioside synthesis and impaired in tumor growth vs. wild-type (WT) gangliosiderich cells. We studied angiogenesis during tumor formation by these ganglioside-depleted cells, quantifying vessel formation, angiogenic factor production/release, and consequences of reconstitution with purified WT gangliosides. DKO cells formed virtually avascular tumors, much smaller than ganglioside-rich WT tumors and displaying a striking paucity of blood vessels, despite levels of VEGF and other angiogenic factors that were similar to those of WT cells. Transient enrichment of the ganglioside milieu of the DKO cell inoculum by adding purified WT gangliosides partially restored angiogenesis and tumor growth. We conclude that tumor gangliosides trigger robust angiogenesis important for tumor growth. Our findings suggest strategies to eliminate their synthesis and shedding by tumor cells should be pursued.
Blood, Jun 7, 2002
for the Histiocyte Society Hemophagocytic lymphohistiocytosis (HLH) comprises familial (primary) ... more for the Histiocyte Society Hemophagocytic lymphohistiocytosis (HLH) comprises familial (primary) hemophagocytic lymphohistiocytosis (FHL) and secondary HLH (SHLH), both clinically characterized by fever, hepatosplenomegaly, and cytopenia. FHL, an autosomal recessive disease invariably fatal when untreated, is associated with defective triggering of apoptosis and reduced cytotoxic activity, resulting in a widespread accumulation of T lymphocytes and activated macrophages. In 1994 the Histiocyte Society initiated a prospective international collaborative therapeutic study (HLH-94), aiming at improved survival. It combined chemotherapy and immunotherapy (etoposide, corticosteroids, cyclosporin A, and, in selected patients, intrathecal methotrexate), followed by bone marrow transplantation (BMT) in persistent, recurring, and/or familial disease. Between July 1, 1994, and June 30, 1998, 113 eligible patients aged no more than 15 years from 21 countries started HLH-94. All had either an affected sibling (n ؍ 25) and/or fulfilled the Histiocyte Society diagnostic criteria. At a median follow-up of 3.1 years, the estimated 3-year probability of survival overall was 55% (95% confidence interval ؎ 9%), and in the familial cases, 51% (؎ 20%). Twenty enrolled children were alive and off therapy for more than 12 months without BMT. For patients who received transplants (n ؍ 65), died prior to BMT (n ؍ 25), or were still on therapy (n ؍ 3), the 3-year survival was 45% (؎ 10%). The 3-year probability of survival after BMT was 62% (؎ 12%). HLH-94 is very effective, allowing BMT in most patients. Survival of children with HLH has been greatly improved.
ABSTRACT Background: Hemophagocytic lymphohistiocytosis (HLH) comprises familial (primary) hemoph... more ABSTRACT Background: Hemophagocytic lymphohistiocytosis (HLH) comprises familial (primary) hemophagocytic lymphohistiocytosis (FHL) and secondary HLH (SHLH), both clinically characterized by fever, hepatosplenomegaly, and cytopenia. FHL, an autosomal recessive disease invariably fatal when untreated, is associated with defective triggering of apoptosis and reduced cytotoxic activity, resulting in a widespread accumulation of T-lymphocytes and activated macrophages. Methods: In 1994 the Histiocyte Society initiated a prospective international collaborative therapeutic study (HLH-94), aiming at improved survival. It combined chemotherapy and immunotherapy (etoposide, corticosteroids, cyclosporin A, and, in selected patients, intrathecal methotrexate), followed by bone marrow transplantation (BMT) in persistent, recurring and/or familial disease. Results: 113 eligible patients aged 15 years from 21 countries started HLH-94 between July 1, 1994 and June 30, 1998. They all either had an affected sibling (n=25) and/or fulfilled the Histiocyte Society diagnostic criteria. At a median follow-up of 3.1 years, the estimated 3-year probability of survival overall was 55% (95% confidence interval +- 9%) and in the familial cases 51% (+/-20%). Twenty enrolled children were alive and off-therapy for >12 months without BMT. For patients who were transplanted (n=65), died prior to BMT (n=25) or were still on therapy (n=3), the 3-yearsurvival was 45% (+/-10%). The 3-year probability of survival after BMT was 62% (+/-12%). Conclusions: HLH-94 is very effective, allowing BMT in most patients. Survival of children with HLH has been greatly improved. E-mail to the corresponding author: [email protected] words: Familial hemophagocytic lymphohistiocytosis, treatment, etoposide, cyclosporin A, dexamethasone, methotrexate, bone marrow transplantation, survival From bloodjournal.hematologylibrary.org by guest on June 7, 2013. For personal use only.
Cancer Letters, Nov 1, 1997
Shedding of gangliosides by tumor cells may enhance tumor development. We recently showed that ce... more Shedding of gangliosides by tumor cells may enhance tumor development. We recently showed that cells of the human brain tumor, medulloblastoma, shed gangliosides in vitro and have therefore examined ganglioside shedding by pediatric brain tumors into the cerebrospinal fluid (CSF). GD3, a major ganglioside in medulloblastoma and astrocytoma, was the target for detection in the CSF by immunostaining using the monoclonal antibody R24 and enhanced chemiluminescence detection. Mean CSF GD3 levels in patients with medulloblastomas (n = 9) and astrocytomas (n = 10) were significantly higher than those of controls (mean +/- SD 44.7 +/- 8.4 versus 18.2 +/- 1.9 pmol/ml, n = 20, P &lt; 0.0002). Mass spectrometric analysis showed that tumor-derived ganglioside GD3 contained heterogeneous ceramide structures and, interestingly, the ceramide subspecies with shorter fatty acyl chains were selectively shed. The elevated CSF GD3 concentrations in patients with medulloblastoma and astrocytoma support the concept that ganglioside shedding, which may have significant biological consequences, is characteristic of human brain tumors.
Nature, Aug 1, 1967
ABSTRACT
Biochimica et biophysica acta, Sep 1, 1996
Gangliosides, immunosuppressive molecules shed by tumor cells, are potent inhibitors of monocyte ... more Gangliosides, immunosuppressive molecules shed by tumor cells, are potent inhibitors of monocyte accessory cell function. However, the specific monocyte cellular defect caused by gangliosides is unknown. Here we sought to delineate whether this abnormality is in the induction of suppressor cells, in intracellular antigen processing, or in intercellular antigen presentation. Three sets of studies of the tetanus toxoid (TT)-induced lymphoproliferative response, which is dependent upon monocyte accessory function, address this issue: (1) Antigen (TT)-primed human monocytes incubated with 50-100 ~zM human brain gangliosides for 24-48 h, washed, and then combined with T-cells, were inhibited in triggering T-cell proliferation, showing that the effect was occurring after antigen processing was complete. (2) T-cell responses to immobilized anti-CD3 or to antigen-primed control monocytes in the presence of ganglioside-exposed monocytes were unaffected, showing that ganglioside-exposed monocytes did not act as suppressor cells. (3) Stimulation by TT peptide fragment 830-843, which does not require processing, was completely inhibited by exposure of monocytes to gangliosides. These findings identify ganglioside interference with monocyte accessory cell function at the level of antigen presentation. We conclude that tumor gangliosides may inhibit host anti-tumor cellular immune responses by preventing the effective cellular interactions of the antigen-primed monocyte with the responding T-lymphocyte.
Journal of Clinical Investigation, Jun 1, 1987
The role of tumor cell membrane gangliosides in tumor formation was probed using a series of clon... more The role of tumor cell membrane gangliosides in tumor formation was probed using a series of cloned murine AKR lymphoma cell lines. Tumor formation was directly related to high expression and shedding of membrane gangliosides. In vivo, as little as I pmol of purified total gangliosides of highly tumorigenic cells, injected intradermally with poorly tumorigenic cells (which lacked and did not shed gangliosides), markedly increased the tumorigenicity of these cells in syngeneic normal mice. Thus, gangliosides shed by tumor cells are a previously unrecognized, extremely potent enhancer of tumor formation in vivo.
ABSTRACT Hemophagocytic lymphohistiocytosis (HLH) used to have a dismal prognosis. We report the ... more ABSTRACT Hemophagocytic lymphohistiocytosis (HLH) used to have a dismal prognosis. We report the final results of HLH-94, the largest prospective diagnostic/therapeutic HLH study so far. The treatment includes immunosuppressive and cytotoxic therapy aiming at clinical remission, followed by HSCT in patients with familial, persistent, or recurrent disease. Altogether 249 patients fulfilled inclusion criteria and started HLH-94 therapy July 1994-December 2003; 227 (91%) with follow-up ≥5-years. At a median follow-up of 6.2 years, estimated 5-year probability-of-survival was 54±6%. Seventy-two patients (29%) died before HSCT; sixty-four within the first year, 97% of which had active disease. In 124 patients that underwent HSCT, 5-year-survival was 66±9% with tendency to increased survival (p=0.064) in patients with non-active disease at HSCT. Patients with familial disease had a 5-year-survival of 50%±13%; none survived without HSCT. Patients deceased during the first two months more often had jaundice, edema and elevated creatinine. Forty-nine non-transplanted patients (20%) were alive without signs of HLH activity and off-therapy >1-year; they presented at older age (p<0.001), were more often female (p=0.011) and less often had CNS-disease (p<0.001) or hepatomegaly (p=0.007). To conclude, HLH-94 chemo-immunotherapy has considerably improved outcome in HLH. Collaborative efforts are needed to further reduce early mortality, HSCT-related mortality, and neurological late-effects. From bloodjournal.hematologylibrary.org by guest on September 27, 2012. For personal use only.
British Journal of Cancer, Jun 8, 2004
Recent findings link increased expression of the structurally complex 'b' pathway gangliosides GD... more Recent findings link increased expression of the structurally complex 'b' pathway gangliosides GD1b, GT1b, GQ1b (CbG) to a favourable clinical and biological behaviour in human neuroblastoma (NB). Seeking a model to probe these observations, we evaluated four human NB cell lines. Very low CbG content (4-10%) in three of the four cell lines (LAN-5, LAN-1, SMS-KCNR) reflected the ganglioside pattern observed in the most aggressive NB tumours. Pharmacological alterations of complex ganglioside synthesis in vitro by a 5-7 day exposure to 5-10 mM retinoic acid, which is employed in maintenance therapy of disseminated NB, included markedly increased (i) relative expression of CbG (6.672.0-fold increase, P ¼ 0.037), (ii) relative expression of the analogous 'a' pathway gangliosides, termed CaG (6.471.4-fold increase in GM1a and GD1a; P ¼ 0.010), and (iii) total cellular ganglioside content (2.0-6.3-fold), which in turn amplified the accumulation of structurally complex gangliosides. Substantial increases (2.7-2.9-fold) in the activity of GD1b/GM1a synthase (b-1,3-galactosyltransferase), which initiates the synthesis of CbG and CaG, accompanied the all-trans retinoic acid (ATRA)-induced ganglioside changes. Thus, increased CbG synthesis in NB cell lines is attributable to a specific effect of ATRA, namely induction of GD1b/GM1a synthase activity. Since the shift towards higher expression of CbG and CaG during retinoic acid-induced cellular differentiation reflects a ganglioside pattern found in clinically less-aggressive tumours, our studies suggest that complex gangliosides may play a role in the biological and clinical behaviour of NB.
Glycoconjugate Journal, Jun 1, 1996
Shedding of neuroblastoma gangliosides is positively correlated with tumour progression in patien... more Shedding of neuroblastoma gangliosides is positively correlated with tumour progression in patients with neuroblastoma. In assessing the biological activity of these ganglioside molecules, we recently found that total human neuroblastoma gangliosides inhibit cellular immune responses. Here, we have studied the major neuroblastoma gangli.oside, Gin. GD2 was purified by high performance liquid chromatography and structurally characterized by mass spectrometry, hmnunoregulatory effects of GD2 in vivo were then determined in an established murine model. GD2 significantly downregulated the local cellular immune response to an allogeneic cell challenge; the usual increase in mass of the lymph node draining the injection site was reduced by 88%, from 1.52 to 0.19 mg (control versus GD2-treated mice; p < 0.01). In parallel, lymphocyte recovery from each node was also reduced from 2.4 to 1.2 × 106 cells, and lymphocyte DNA synthesis was reduced to half of the control level. These results show that certain shed tumour gangliosides, such as GD2, function as intercellular signalling molecules, downregulate the cellular immune response, and may thereby enhance tumour formation and progression.
Cancer Letters, Jul 1, 2005
Low (≤35%) or absent expression of the complex 'b' pathway gangliosides GD1b, GT1b and GQ1b (CbG)... more Low (≤35%) or absent expression of the complex 'b' pathway gangliosides GD1b, GT1b and GQ1b (CbG) correlates with an aggressive biological phenotype in human neuroblastoma tumors. To develop an in vitro model to probe mechanisms by which CbG may contribute to neuroblastoma behavior, we have comprehensively evaluated ganglioside expression in nine well-established human neuroblastoma cell lines, all derived from poor prognosis tumors. Total cellular ganglioside content ranged from 8 to 69 nmol/10 8 cells. High performance thin layer chromatography revealed that the simple disialoganglioside GD2 was prominent in eight of the cell lines (up to 60% of total gangliosides), whereas CbG were low (1-21%) in all nine cell lines. The structurally most complex 'b' pathway species, GQ1b, was not detected in any of the cell lines. The prominence of GD2 in neuroblastoma cell lines mirrors the high expression of GD2 that characterizes human neuroblastoma tumors, and the low CbG expression in the cell lines is analogous to that found in clinically and biologically unfavorable neuroblastoma tumors, thus establishing these neuroblastoma cell lines as valuable model systems for study of the role of CbG in the pathobiology of human neuroblastoma. Keywords Ganglioside; Neuroblastoma; Glycosphingolipid Gangliosides, membrane-bound glycosphingolipid molecules that are frequently aberrantly expressed in tumors, have been implicated in the biology of various cellular processes, and linked to the behavior of many types of tumors [1], Extensive studies by ourselves and others have established the diagnostic and prognostic potential of specific ganglioside changes in neuroblastoma, a pediatric malignancy of neural crest origin [2-6]. Ganglioside molecules consist of a sialic acid-containing carbohydrate portion and a hydrophobic lipid backbone (ceramide), embedded in the outer leaflet of the cell membrane [7]. Ganglioside biosynthesis occurs as a set of stepwise glycosylations via two main pathways designated 'a' (GM2, GM1a, GD1a) and 'b' (GD3, GD2, GD1b, GT1b, GQ1b), from a common precursor (GM3) derived from lactosylceramide (Fig. 1) [8]. Human neuroblastoma consists of distinct disease categories, with unfavorable disease marked by early dissemination, older age at diagnosis and biological anomalies such as MYCN ☆ Supported by National Institutes of Health Grants CA-90362 (K.K.
Cancer Letters, Nov 1, 2003
Aberrant ganglioside metabolism is linked to tumor progression. Since ganglioside depletion reduc... more Aberrant ganglioside metabolism is linked to tumor progression. Since ganglioside depletion reduced tumorigenicity of MEB4 murine melanoma cells, we studied N-butyldeoxynojirimycin (N B-DNJ), an imino sugar administered orally to inhibit glucosylceramide (GlcCer) synthase in patients with glycosphingolipid storage diseases, for effects on MEB4 melanoma tumor cell ganglioside metabolism, cell biology, and tumorigenesis. Here we show that 50 mM N B-DNJ reduced MEB4 cell GlcCer synthase activity (by 70%), ganglioside synthesis (by 61%), and shedding (by 37%) while ceramide concentrations and cell viability were unaffected. Partial ganglioside depletion caused a delay in tumor onset but not in tumor incidence, possibly because of rapid (48 h) ganglioside recovery. The delay in tumor development by N B-DNJ treatment of MEB4 cells provides further support for the concept of tumor cell ganglioside metabolism as a therapeutic target in cancer.
Current Opinion in Pediatrics, Feb 1, 1990
Primary neuroblastomas obtained before therapy from 36 patients were studied to determine the fre... more Primary neuroblastomas obtained before therapy from 36 patients were studied to determine the frequency of tumors expressing a specific glycosphingolipid, Go: ganglioside. Total tissue gangliosides were purified by a new partition method, quantitated, and analyzed by high-performance thin-layer chro
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Papers by Stephan Ladisch