Age-dependent reference intervals in children for the disialoganglioside, GD2, a circulating tumor biomarker for the childhood cancer, neuroblastoma.

BACKGROUND: The disialoganglioside GD2 is a circulating tumor biomarker for the childhood cancer, neuroblastoma. This study establishes reference intervals for GD2 concentration in children within the age range where neuroblastoma commonly occurs. METHODS: Leftover plasma samples taken for routine clinical laboratory tests from children without cancer were collected and assayed for the 18-carbon fatty acid chain length lipoform of GD2 using a validated high-pressure liquid chromatography tandem mass spectrometry method with a lower limit of quantification of 3 nM. Samples were stratified into 5 age cohorts (0-6 months, 6-12 months, 12-36 months, 3-10 years and > 10 years). Non-parametric statistical methods were used to define the upper bound of the reference interval for each age cohort. RESULTS: GD2 was measurable in 90% of samples from children < 10 years of age and GD2 concentration was age-dependent, peaking at 9 months followed by a gradual decline. GD2 was below the lower limit of quantification in 55% of samples in the > 10 years cohort. Upper bounds of reference intervals were 15.5 nM in 0-6 month cohort, 35.1 nM in 6-12 month cohort, 24.9 nM in 12-36 month cohort, 18.4 in 3-10 year cohort and 10.4 nM in > 10 year cohort. CONCLUSIONS: Age-dependent reference intervals were defined for circulating GD2 in children. GD2 concentration was highest in the 6-12 month age cohort, which is below the age of most children with high-risk neuroblastoma. The peak GD2 concentration at 9 months may reflect neurodevelopmental events in the brain.

An integrated Qual/Quan strategy for ganglioside lipidomics using high-resolution mass spectrometry and Skyline software.

RATIONALE: Gangliosides (GS) are attractive targets in biomarker discovery because of their physiological significance in numerous human diseases including certain cancers and developmental and metabolic disorders. The robust strategy described here enables the profiling of numerous GS while obtaining quantitative data of exploratory biomarkers present in human plasma and whole blood. METHOD: The GS from human blood, human plasma, and several cell lines were extracted using a mixture of methanol and isopropanol/0.1% formic acid followed by direct analysis of the supernatant. The simultaneous Qualitative and Quantitative (Qual/Quan) approach involves micro flow (20 µL/min) high pressure liquid chromatography (HPLC)/high-resolution mass spectrometry (HRMS) and post-acquisition data processing with Skyline software for profiling numerous GS in biological matrices. The quantitative assay involves reverse-phase liquid chromatography/HRMS and calibration curves using commercially available GS. RESULTS: Protein precipitation resulted in ~60%-80% GS recovery from biological matrices. Direct injection of the extract allowed for quantification of targeted GS in human blood, plasma, and cancer cell lines. The lower limit of detection for the target analytes, GM1, GT1, GD1, spiked into 1% BSA/PBS, ranged from 1 to 10 ng/mL. Human lung cancer cell lines contained variable amounts (1-130 ng/mL) of soluble Fuc-GM1 analogs, potential biomarkers of lung cancer. CONCLUSIONS: A combination of simple extraction and micro-HPLC/HRMS allowed for quantification of GS in human serum and whole blood. Integration of HRMS with Skyline allowed for GS profiling in the same samples using post-acquisition HRMS data without the need for reanalysis. The strategy presented here is expected to play an important role in profiling exploratory GS biomarkers in discovery bioanalytical research.

Combinatory Data-Independent Acquisition and Parallel Reaction Monitoring Method for Deep Profiling of Gangliosides.

Ganglioside is an important class of lipid species involved in intercellular signaling and various diseases, especially for neurodegenerative diseases. Systematic ganglioside profiling is challenging because of their naturally low abundance and highly diverse species. Herein, a new data-independent acquisition and parallel reaction monitoring (DIA/PRM) method with superior sensitivity was developed. The untargeted DIA acquisition consecutively records all the precursor ion and fragment ions at the same time, while the targeted PRM analysis with versatile higher collisional dissociation generates full MS/MS spectra for structure elucidation and verification. As compared with traditional data-dependent acquisition (DDA), the DIA/PRM method unbiasedly detected the majority of abundant ganglioside species and as low as 50 pg of ganglioside in an untargeted manner. Gangliosides in four kinds of biological samples including the mouse brain, mouse plasma, HeLa cell, and human colon cancer tissue were systematically identified, and low-abundance ganglioside species were further extended on the basis of linear chromatography retention rules of the most frequently detected ganglioside species. A total of 383 ganglioside features were defined with 329 of them derived from 32 ganglioside species. Taking advantage of the high-resolution MS analysis, rare ganglioside species were further elucidated according to their characteristic fragment ions and neutral losses. In total, 18 gangliosides with a ceramide carbon number from 20 to 25 and modified gangliosides, including 18 acetylated, 8 diacetylated, 1 phosphorylated, 36 N-glycolyneuraminic acid (NeuGc)-containing, and 7 di-NeuGc-containing gangliosides, were newly identified. The developed DIA/PRM method therefore generated a rich ganglioside resource for further functional exploration and is a unique alternative for DDA analysis for global ganglioside profiling in various biological systems.

The ganglioside GD2 as a circulating tumor biomarker for neuroblastoma.

BACKGROUND: GD2 is a ganglioside that is ubiquitously expressed in the plasma membrane of neuroblastoma and is shed into the circulation. PROCEDURE: GD2 was measured with a high-pressure liquid chromatography/tandem mass spectrometry assay in serum or plasma from 40 children without cancer (controls) and in biobanked samples from 128 (73 high-risk) children with neuroblastic tumors at diagnosis, 56 children with relapsed neuroblastoma, 14 children with high-risk neuroblastoma after treatment, and 8 to 12 children each with 10 other common childhood cancers at diagnosis. RESULTS: The C18 (18 carbon fatty acid) lipoform was the predominant circulating form of GD2 in controls and in patients with neuroblastoma. The median concentration of GD2 in children with high-risk neuroblastoma at diagnosis was 167 nM (range, 16.1-1060 nM), which was 30-fold higher than the median concentration (5.6 nM) in controls. GD2 was not elevated in serum from children with the differentiated neuroblastic tumors, ganglioneuroma (n = 10) and ganglioneuroblastoma-intermixed subtype (n = 12), and in children with 10 other childhood cancers. GD2 concentrations were significantly higher in serum from children with MYCN-amplified tumors (P = 0.0088), high-risk tumors (P < 0.00001), International Neuroblastoma Staging System (INSS) stage 4 tumors (P < 0.00001), and in children who died (P = 0.034). CONCLUSIONS: Circulating GD2 appears to be a specific and sensitive tumor biomarker for high-risk/high-stage neuroblastoma and may prove to be clinically useful as a diagnostic or prognostic circulating tumor biomarker. GD2 will be measured prospectively and longitudinally in children enrolled on a high-risk neuroblastoma treatment trial to assess its ability to measure response to treatment and predict survival.

Antiganglioside antibodies in neurological diseases.

Gangliosides are sialylated glycosphingolipids, highly abundant in our nervous system. Antibodies targeting gangliosides are usually developed as a consequence of molecular mimicry following infections. Antiganglioside antibodies are implicated in many neurological disorders such as acute and chronic polyradiculoneuropathies which includes different variants of Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuropathy and multifocal motor neuropathy. Presence of such antibodies in paraneoplastic peripheral neuropathy, neurodegenerative disorders, multiple sclerosis, myasthenia gravis and amyotrophic lateral sclerosis have also been reported. Recent evidence supports a role of antiganglioside antibodies in the pathogenesis of acute vestibular syndrome. Binding of antibodies to gangliosides on axonal membranes, nodes of Ranvier, myelin sheath components, Schwann cells, neuromuscular junctions or other neural cell surfaces may elicit inflammatory damage through complement-dependent and independent mechanisms, resulting in nerve conduction blocks and subsequent axonal degeneration. Gangliosides are essential for proper cell signaling, transduction and influences neuroplasticity, all of which are affected by autoimmune mediated damage. Better insight into the pathophysiological role of antiganglioside antibodies in different neurological diseases may improve their utility as diagnostic and prognostic biomarkers.

Gangliosides profiling in serum of breast cancer patient: GM3 as a potential diagnostic biomarker.

Gangliosides altered during the pathological conditions and particularly in cancers. Here, we aimed to profile the gangliosides in breast cancer serum and propose potential biomarkers. LC-FTMS method was first used to identify all the ganglioside species in serum, then LC-MS/MS-MRM method was employed to quantitate the levels of gangliosides in serum from healthy volunteers and patients with benign breast tumor or breast cancer. 49 ganglioside species were determined, including GM1, GM2, GM3, GD1, GD3 and GT1 species. Compared to healthy volunteers, the levels of GM1, GM2, GM3, GD1 and GD3 displayed a rising trend in breast cancer patients. In particular, as the major glycosphingolipid component, GM3 showed excellent diagnostic accuracy in cancer serum (AUC > 0.9). PCA profile of the GM3 species showed clear distinction between normal and cancer serum. What's more, ROC curve proved great diagnostic accuracy of GM3 between cancer and benign serum. In addition, GM3 was discovered as a diagnostic marker to differentiate luminal B subtype from other subtypes. Furthermore, a positive correlation between GM3 and Ki-67 status of patients was identified. In conclusion, our results introduced the alteration patterns of serum gangliosides in breast cancer and suggested serum GM3 as a potential diagnostic biomarker in breast cancer diagnosis and luminal B subtype distinction.

Post-infectious sensory neuropathy with anti-GT1a and GQ1b antibodies associated with cold urticaria.

A 64 years-old woman presented subacute onset distal paraesthesia concurrently with cold-induced urticaria, a rare form of physical urticaria. Both the disturbances developed a fortnight after an upper respiratory tract infection. EMG confirmed an exclusively sensory polyneuropathy, with prolongation of distal latencies and reduction of amplitudes. Anti-GQ1b and anti-GT1a antigangliosides antibodies were found in serum. The clinical workout included CSF analysis, cryoglobulin and paraprotein search, neurotropic infective agents, neoplastic markers and extensive autoimmune disease antibodies analysis, all of which resulted negative. Intravenous immunoglobulins were administered, leading to progressive resolution of the sensory disturbance, while a combination of steroid and anti-histaminics treatment was used for the urticaria. The positivity for anti-ganglioside search with an EMG pattern characterized by a mixture of demyelinating and axonal features may suggest a nodo-paranodopathy at early stages. This is the first case of an association between an acute sensory neuropathy and cold urticaria, two immune mediated conditions apparently due to very different hypersensitivity pathways. A proposed mechanism for the co-occurence of these two conditions is presented, whereas this case expands the clinical spectrum of autoimmune diseases associated with anti-GQ1b and anti-GT1a antibodies.

A validated HPLC-MS/MS method for estimating the concentration of the ganglioside, GD2, in human plasma or serum.

GD2 is a ganglioside found in the plasma membrane of the neural crest-derived cancer, neuroblastoma. GD2 is shed into the circulation of patients with neuroblastoma and could serve as a tumor biomarker to monitor tumor burden or response to treatment. We developed and validated a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method to quantify the D18:1-18:0 (C18) and the D18:1-20:0 (C20) lipoforms of GD2 in human plasma and serum. Human brain derived GD2 containing a mixture of C18 and C20 was used as the analytical standard. Samples were extracted with methanol containing dueterated-GM1 (internal standard), and analytes were separated on a Phenomenex Kinetex C18 column eluted with a gradient mobile phase composed of ammonium acetate buffer, methanol and isopropanol. An AB Sciex 4500 QTRAP mass spectrometer in negative ion mode was used to quantify the doubly charged GD2 C18 and C20 lipoform precursor ions (m/z 836.8 and m/z 850.8) that both yield a product ion of m/z 290.0. The calibration curves were linear from 4-1000 ng/mL and 6-1500 ng/mL for GD2 C18 and C20 lipoforms respectively. Inter-day and intra-day accuracy were within the acceptable validation range in plasma and serum.

The association of the lipidomic profile with features of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) affects up to 18% of reproductive-aged women with reproductive and metabolic complications. While lipidomics can identify associations between lipid species and metabolic diseases, no research has examined the association of lipid species with the pathophysiological features of PCOS. The aim of this study was to examine the lipidomic profile in women with and without PCOS. This study was a cross-sectional study in 156 age-matched pre-menopausal women (18-45 years, BMI >20 kg/m2; n = 92 with PCOS, n = 64 without PCOS). Outcomes included the association between the plasma lipidomic profile (325 lipid species (24 classes) using liquid chromatography mass spectrometry) and PCOS, adiposity, homeostasis assessment of insulin resistance (HOMA), sex hormone-binding globulin (SHBG) and free androgen index (FAI). There were no associations of the lipidomic profile with PCOS or testosterone. HOMA was positively associated with 2 classes (dihydroceramide and triacylglycerol), SHBG was inversely associated with 2 classes (diacylglycerol and triacylglycerol), FAI was positively associated with 8 classes (ceramide, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, diacylglycerol and triacylglycerol) and waist circumference was associated with 8 classes (4 positively (dihydroceramide, phosphatidylglycerol, diacylglycerol and triacylglycerol) and 4 inversely (trihexosylceramide, GM3 ganglioside, alkenylphosphatidylcholine and alkylphosphatidylethanolamine)). The lipidomic profile was primarily related to central adiposity and FAI in women with or without PCOS. This supports prior findings that adiposity is a key driver of dyslipidaemia in PCOS and highlights the need for weight management through lifestyle interventions.

Plasma Content Variation and Correlation of Plasmalogen and GIS, TC, and TPL in Gastric Carcinoma Patients: A Comparative Study.

BACKGROUND We studied the variation in plasma content of plasmalogen and ganglioside, total cholesterol (TC), and total phospholipid (TPL) in gastric carcinoma patients. The plasma plasmalogen levels were determined according to the vinyl ester bond method. MATERIAL AND METHODS Plasma ganglioside level was determined according to the method of Sevennerholm. The total cholesterols and total phospholipid were determined by routine methods. RESULTS The plasma plasmalogen level of gastric carcinoma patients was significantly higher than in the control (normal) group, and the difference was markedly significant (p<0.01). The plasma total sialic acid (TSA) and lipid-bound sialic acid (LSA) of gastric carcinoma patients were higher than those of the normal control group (p<0.05). The total cholesterol content was higher than those in the normal control group (p<0.02), but the total phospholipid content was lower than in the normal control group and the difference was markedly significant (p<0.05). In the gastric carcinoma patients group, the plasmalogen and ganglioside-TSA levels were positively correlated (r=0.01, P<0.01). Plasmalogen and total cholesterols were also positively correlated (r=0.82, P<0.01), and plasmalogen and total phospholipid were negatively correlated (r=-0.82, p<0.01). CONCLUSIONS In gastric carcinoma patients, the plasma plasmalogen content was significantly elevated and was positively correlated with elevated level of gangliosides and total cholesterols, but it was negatively correlated with level of total phospholipids.

Diagnosis value of membrane glycolipids biochemistry index in intracranial and gastrointestinal tumors.

The diagnostic value of membrane glycolipid biochemistry index, the lipid-bound sialic acid (LSA) and total sialic acid (TSA) in cerebrospinal fluid (CSF) was evaluated in 30 intracranial and 65 gastrointestinal tumors. The plasma LSA, TSA and red cell membrane sialic acid (R-SA) in were determined according to the method of Sevenmerhulm. Our results showed that the levels of LSA and TSA in CSF of intracranial tumor patients was higher than that of normal group(p<0.01). The concentration of TSA and LSA in patients with malignant glioma was higher than that of benign meningioma patients(P<0.01). No significance was found between intracranial halmatoma patients and normal control group for levels of membrane glycolipids (p>0.05). Results also found that the plasma LSA, TSA and R-SA of gastric carcinoma were significantly higher than those of control group (p<0.05); while no significant difference was found in the plasma LSA, TSA and R-SA levels between chronic gastritis, gastrohelcoma and normal control group (p>0.05). Plasma LSA, TSA and R-SA levels of gastric carcinoma patient were significantly higher than those of chronic gastritis patients and gastrohelcoma patients(p<0.05). It was also found that plasma LSA, TSA and R-SA contents were significantly higher in large intestine carcinoma patients than in benign in stestine tumor patients (p<0.05) while no significant difference was found between intestine benign tumor and normal control group (p>0.05). The levels of LSA, TSA and R-SA were obviously higher in the patients with metastasis than in the ones without (p<0.05.) The membrane glycolipid biochemistry index LSA and TSA in CSF are sensive markers for diagnosing intracranial tumors. For gastrointestinal malignant tumors the plasma LSA TSA and red blood cell membrane SA may be considered as auxiliary indicators for diagnosis. They can be used for distinguishing benign from malignant tumors.

Rapid and sensitive LC-ESI-MS of gangliosides.

Gangliosides are a class of sphingolipids characterized by a ceramide lipid chain attached to an anionic oligosaccharide moiety that varies in complexity based on the level of sialylation. Heterogeneity in the oligosaccharide chain of gangliosides is a direct result of the monosaccharide structure, content, sequence, and connections. Gangliosides are highly concentrated in the central nervous system, and are cell type-specific as well as development-dependent and their quantities and species can undergo drastic changes during cell differentiation. Specific localization of gangliosides also allows for interaction with a variety of bioeffectors, including glycoproteins, antibodies, peptide hormones, and growth factors. There are currently no rapid analytical assays capable of identifying and quantifying gangliosides. The aim of this study is to establish a reliable chromatographic mass spectrometry based assay capable of profiling ganglioside levels in complex biological samples at high sensitivity. We describe here a chromatographic method using an amino column on which the separation is based on hydrophilic interaction with the sugar moiety of gangliosides. Several gangliosides, including GM1-3, GD1a,b, GD2-3, and GT1a,b, were efficiently separated in less than 10 min at a limit of detection ranging between 10-50 pg on column with a concentration dynamic range extending over 4 orders of magnitude. The developed method allowed the sensitive quantitation of gangliosides derived from the blood serum of patients with different esophagus diseases, including, adenocarcinoma, high-grade dysplasia, and Barrett's.

Síndrome anti-GQ1b: Descripción de cuatro pacientes y revisión de la literatura / Anti-GQ1b syndrome: Report of four cases

Anti-GQ1b syndrome includes Miller Fisher Syndrome (MFS), Guillain Barré Syndrome (GBS), Bickerstaff`s brain stem encephalitis (BBE) and Acute Ophtamoplegia (AO). We report four patients aged 16 to 76 years, with anti-GQ1b syndrome. All presented with MFS, one of them evolved to GBS pharyngeal-cervical-brachial variant and other to GBS with BBE. All had a previous history of diarrhea or upper respiratory tract infection. All had positive anti-GQ1b serum antibodies. Both brain magnetic resonance imaging and cerebrospinal fluid analysis were normal. Electrophysiology studies were compatible with a demyelinating disease. Two patients needed airway protection with an orotracheal tube and developed dysautonomia. All four patients were treated with immunomodulation. On the sixth month follow-up, patients had only minimal alterations in the neurological examination.

Peripheral blood mononuclear cell gene expression in chronic obstructive pulmonary disease.

Although most cases of chronic obstructive pulmonary disease (COPD) occur in smokers, only a fraction of smokers develop the disease. We hypothesized distinct molecular signatures for COPD and emphysema in the peripheral blood mononuclear cells (PBMCs) of current and former smokers. To test this hypothesis, we identified and validated PBMC gene expression profiles in smokers with and without COPD. We generated expression data on 136 subjects from the COPDGene study, using Affymetrix U133 2.0 microarrays (Affymetrix, Santa Clara, CA). Multiple linear regression with adjustment for covariates (gender, age, body mass index, family history, smoking status, and pack-years) was used to identify candidate genes, and ingenuity pathway analysis was used to identify candidate pathways. Candidate genes were validated in 149 subjects according to multiplex quantitative real-time polymerase chain reaction, which included 75 subjects not previously profiled. Pathways that were differentially expressed in subjects with COPD and emphysema included those that play a role in the immune system, inflammatory responses, and sphingolipid (ceramide) metabolism. Twenty-six of the 46 candidate genes (e.g., FOXP1, TCF7, and ASAH1) were validated in the independent cohort. Plasma metabolomics was used to identify a novel glycoceramide (galabiosylceramide) as a biomarker of emphysema, supporting the genomic association between acid ceramidase (ASAH1) and emphysema. COPD is a systemic disease whose gene expression signatures in PBMCs could serve as novel diagnostic or therapeutic targets.

Cancer-associated carbohydrate antigens as potential biomarkers for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Therefore, developing the early, high-sensitivity diagnostic biomarkers to prevent HCC is urgently needed. Serum a-fetoprotein (AFP), the clinical biomarker in current use, is elevated in only ~60% of patients with HCC; therefore, identification of additional biomarkers is expected to have a significant impact on public health. In this study, we used glycan microarray analysis to explore the potential diagnostic value of several cancer-associated carbohydrate antigens (CACAs) as biomarkers for HCC. We used glycan microarray analysis with 58 different glycan analogs for quantitative comparison of 593 human serum samples (293 HCC samples; 133 chronic hepatitis B virus (HBV) infection samples, 134 chronic hepatitis C virus (HCV) infection samples, and 33 healthy donor samples) to explore the diagnostic possibility of serum antibody changes as biomarkers for HCC. Serum concentrations of anti-disialosyl galactosyl globoside (DSGG), anti-fucosyl GM1 and anti-Gb2 were significantly higher in patients with HCC than in chronic HBV infection individuals not in chronic HCV infection patients. Overall, in our study population, the biomarker candidates DSGG, fucosyl GM1 and Gb2 of CACAs achieved better predictive sensitivity than AFP. We identified potential biomarkers suitable for early detection of HCC. Glycan microarray analysis provides a powerful tool for high-sensitivity and high-throughput detection of serum antibodies against CACAs, which may be valuable serum biomarkers for the early detection of persons at high risk for HCC.

Serum total gangliosides level: clinical prognostic implication.

PURPOSE: The gangliosides overexpression contributes to the development of skin melanoma. The purpose of this study was to determine if the total gangliosides serum levels might predict the tumor growth in patients with melanoma or if the transfer of shed cell gangliosides reflects the implication in the clinical prognostic of these patients. PATIENTS AND METHODS: Total gangliosides serum levels were measured in the cryopreserved serum by estimating lipid-associated sialic acid in 761 patients before surgical resection of melanoma, in 406 patients with precancerous pigmentary lesions, and in 410 healthy individuals. This study was performed at the Dermatovenereological Research Center, Bucharest, Romania, during 1991-2010. All sera obtained after surgical resection of melanocytic tumors were analyzed to see if adjuvant therapy (chemo-, immuno-, immunochemo-therapy) induced gangliosides changes in melanoma patients and if the responses were correlated with survival. RESULTS: Total gangliosides serum levels were higher in melanoma patients than in precancerous melanocytic lesions patients or in healthy individuals. Larger tumors in Breslow index and more advanced stage of disease were correlated with higher total gangliosides serum values. Augmented total gangliosides serum levels after melanoma adjuvant treatment were predictive for decreased overall survival, whereas decreased total gangliosides serum levels were predictable for improved overall survival. CONCLUSIONS: A marker for early melanoma complications and survival may be the total gangliosides serum level.

[Identification and characterization of a monoclonal IgM reacting with disialylated gangliosides recognizing the CANOMAD syndrome]. / Identification et caractérisation d'une IgM monoclonale sérique à activité anti-gangliosides disialylés évoquant un Canomad syndrome.

We reported the laboratory phenotype of a monoclonal IgM-lambda against disialylated gangliosides, in a 81-year-old man admitted to a neurological department because of the progressive development of distal paresthesias, gait unsteadiness, difficulty to walk and having falls. Serological studies revealed an IgM monoclonal protein with lambda light chain component of MGUS type. IgM level was 4 g/L. The positive laboratory studies showed high titers of IgM antibodies in excess of 1/10(5) against specific disialylated gangliosides including GD1b, GD3, GT1b and GQ1b. There was no serum IgM binding to MAG and SGPG/SGLPG. Clonality by in-house immunodot of ganglioside antibodies was demonstrated using kappa and lambda light chain specific antibodies. Light chain subtype of the anti-ganglioside antibody activity and monoclonal IgM was lambda subtype. The reactivity at high titers was against gangliosides containing the disialosyl epitope. The clinical and laboratory features have been described under the acronym CANOMAD: Chronic Ataxic Neuropathy with Ophthalmoplegia, M proteins, cold Agglutinins and Disialosyl antibodies. Administration of IVIg produced a significant neurological improvement during six years. Then the neuropathy became refractory in the IVIg and worsened in severity, a cure by Rituximab® was established. The patient died from a pneumopathy only two months later. Monoclonal IgM binding to disialylated gangliosides have high level of specificity for diagnosis of the CANOMAD syndrome.

The anti-tumor effect of resveratrol alone or in combination with immunotherapy in a neuroblastoma model.

We investigated the anti-tumor effect of peritumoral resveratrol in combination with immunotherapy in vivo in neuroblastoma-bearing mice. Subcutaneous NXS2 tumors were induced in A/J mice. On day 10, some mice received 15 mcg of intravenous immunocytokine for 5 days, mice received 20 mg of peritumoral resveratrol twice a week (starting on day 12) for a total of 5 injections, and a separate group received a combination of both regimens. Tumor progression and survival were assessed every 3-4 days. Blood and primary tumor tissue samples were collected on day 20 for Complete Blood Count and CD45 immunohistochemistry and histology, respectively. The primary tumor regressed in all mice receiving peritumoral resveratrol. Most of these mice receiving peritumoral resveratrol alone developed metastatic tumors and recurrence of the primary tumor after cessation of therapy. When resveratrol and immunocytokine regimens were combined, 61% of the mice receiving this combination therapy resolved their primary tumors and survived without developing metastatic tumors, compared to 15 and 13% receiving resveratrol or immunocytokine alone, respectively. None of the therapeutic regimes prevented lymphocyte infiltration or affected the complete blood count. Greater necrosis was observed microscopically in tumors from mice receiving the combination therapy. These results demonstrate that the combination therapy of peritumoral resveratrol plus intravenous immunocytokine provides better anti-tumor effects in this model than either therapy alone.

The role of 9-O-acetylated ganglioside D3 (CD60) and {alpha}4{beta}1 (CD49d) expression in predicting the survival of patients with Sezary syndrome.

BACKGROUND: Sézary syndrome is a rare and very aggressive leukemic variant of cutaneous T-cell lymphoma characterized by extensive skin involvement and a malignant circulating CD4(+) T-cell clone which homes to the skin, over-expresses CD60, and lacks CD7, CD26 and CD49d. So far prognostic markers in this disease are limited to treatment with systemic steroids, age, serum lactate dehydrogenase, and a white blood cell count of 20×10(9)/L or higher: no other biological marker with prognostic value, especially related to malignant cells, has been described. DESIGN AND METHODS: We used flow activated cell sorting analysis to compare the distribution of the T-cell receptor-Vß repertoire and several surface molecules (CD7, CD26, CD49d and CD60) within the circulating CD4(+) T-cell population in 62 patients with Sézary syndrome, 180 with mycosis fungoides, 6 with B-cell lymphomas, and 19 with chronic eczema. We calculated the 5-year overall survival of patients with Sézary syndrome after first hospital admission using Kaplan-Meier product-limit estimates and hazard ratios from the Cox proportional hazards model. RESULTS: We found that both higher number of CD60(+) and lower number of CD49d(+) cells within circulating CD4(+) T cells at disease presentation were significantly associated with a lower probability of survival. An exceedingly high risk of death was observed for patients with a combination of a high proportion of CD4(+)CD60(+) cells (≥ 0.5×10(9)/L) and low proportion of CD4(+)CD49d(+) cells (<0.5×10(9)/L) (hazard ratio = 12.303, 95% confidence interval 1.5-95.9; P<0.02). In addition, a skewed usage of T-cell receptor-Vß subfamilies was observed in the circulating T-cell clone for 61.9% of all patients with Sézary syndrome, T-cell receptor-Vß 2 and 5.1 subfamilies being the most frequently represented (42.8%), followed by T-cell receptor-Vß 12 and 13.1. CONCLUSIONS: In this study we showed that up-regulation of CD60 and down-regulation of CD49d on circulating CD4(+) T cells are two useful markers for predicting a very poor outcome in patients with Sézary syndrome.