Effect of simulated gastrointestinal digestion on sialic acid and gangliosides present in human milk and infant formulas.

The effects of simulated gastrointestinal digestion upon sialic acid and gangliosides in infant and follow-on formulas and human milk, as well as their bioaccessibility, have been evaluated. The gastric stage is the step that causes a greater decrease in sialic acid and ganglioside contents. The intestinal stage only decreases the total and individual contents of gangliosides. After gastrointestinal digestion, neither sialic acid nor gangliosides were found in the nonbioaccessible fraction. The highest bioaccessibility (100 × content in soluble fraction after gastrointestinal digestion/total content) of sialic acid is found in human milk (87%), followed by infant formula (77%) and follow-on formula (16%). In the case of gangliosides, the highest bioaccessibility is present in the follow-on formula (51%), followed by human milk (29%) and infant formula (5%).

GD3, an overexpressed tumor-derived ganglioside, mediates the apoptosis of activated but not resting T cells.

We previously elucidated an important role for gangliosides in renal cell carcinoma-mediated T lymphocyte apoptosis, although the mechanism by which they mediated lymphocyte death remained unclear. Here, we show that when added in purified form, GD3 is internalized by activated T cells, initiating a series of proapoptotic events, including the induction of reactive oxygen species (ROS), an enhancement of p53 and Bax accumulation, an increase in mitochondrial permeability, cytochrome c release, and the activation of caspase-9. GD3-induced apoptosis of activated T cells was dose dependent and inhibitable by pretreating the lymphocytes with N-acetylcysteine, cyclosporin A, or bongkrekic acid, emphasizing the essential role of ROS and mitochondrial permeability to the process. Ganglioside-induced T-cell killing was associated with the caspase-dependent degradation of nuclear factor-kappaB-inducible, antiapoptotic proteins, including RelA; this suggests that their loss is initiated only after the cascade is activated and that their disappearance amplifies but not triggers GD3 susceptibility. Resting T cells did not internalize appreciable levels of GD3 and did not undergo any of the proapoptotic changes that characterize activated T lymphocytes exposed to the ganglioside. RelA overexpression endows Jurkat cells with resistance to GD3-mediated apoptosis, verifying the role of the intact transcription factor in mediating protection from the ganglioside.

Preferential binding of the anticancer drug rViscumin (recombinant mistletoe lectin) to terminally alpha2-6-sialylated neolacto-series gangliosides.

Production of biochemically defined recombinant mistletoe lectin was achieved by cloning and separate expression of the single catalytically active A-chain and the B-chain with carbohydrate binding properties in Escherichia coli, yielding an active heterodimeric protein named rViscumin (Eck et al. [1999] Eur. J. Biochem., 265, 788-797). Employing solid phase binding assays, rViscumin was shown to preferentially bind to terminally alpha2-6-sialylated neolacto-series gangliosides IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer isolated from human granulocytes. Only marginal binding of rViscumin to galactose-terminated neutral GSLs was determined, whereas reinvestigation of ricin specificity demonstrated this lectin as a galactose-binding protein. Human promyelotic HL-60 cells exhibited an IC(50) value (half maximum cytotoxicity) of 1.16 pM and human bladder carcinoma 5637 cells of 12.1 pM rViscumin; CHO-K1 cells were resistant to rViscumin treatment up to a concentration of 5.26 nM tested. Quantification of the predominant receptor ganglioside IV(6)Neu5Ac-nLc4Cer by means of a specific anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibody revealed 3.68 x 10(6) and 1.54 x 10(6) receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking alpha2-6-sialylated gangliosides. The data imply a direct correlation of rViscumin cytotoxicity and the expression of receptor ganglioside. Moreover, CHO-K1 cells were rendered susceptible toward rViscumin cytotoxicity after exogenous application of human granulocyte gangliosides. Thus, (1) rViscumin has to be considered as a sialic acid-specific rather than a galactose-specific type II ribosome-inactivating protein, and (2) neolacto-series gangliosides with Neu5Acalpha2-6Galbeta1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.

Studies on a soft glycopyrrolate analog, SG-1.

A short-acting soft drug analog (SG-1) of glycopyrrolate (G) was developed by retrometabolic design in order to minimize systemic side effects and optimize the therapeutic index. SG-1,3-(1'-hydroxycyclopentyl)phenylacetoxy-1-methyl-1- methoxycarbonylpyrrolidinium bromide, was synthesized by: (a) esterification of phenylacetic acid with N-methyl-3-pyrrolidinol by DCC to obtain N-methyl-3-pyrrolidinyl phenylacetate; (b) reaction of lithium salt of above phenylacetates with cyclopentanone to obtain N-methyl-3-pyrrolidinyl 3-(1'-hydroxycyclopentyl)phenylacetate; and (c) quarternization with methyl bromoacetate in acetonitrile to give the designed product. To evaluate the pharmacological effect of SG-1, its mydriatic activity in rabbit eyes was compared to that of glycopyrrolate. At the pharmacodynamically equivalent doses (the lowest dose that induces the maximum response) of SG-1 (1%) and glycopyrrolate (0.1%), the mydriatic activities lasted for 5 and 100 h, respectively. Compared to glycopyrrolate, the intrinsic pupil dilation potency of SG-1 was lower (approximately 1/10th) but the duration of action was much shorter (< 1/20th) as SG-1 is susceptible to facile enzymatic hydrolysis/deactivation in the rabbit eyes. In vitro metabolism and stability investigations further supported this finding. In vitro half lives of SG-1 in rat plasma, blood, and 20% liver and lung tissue homogenates were 15.62, 53.86, 263.43, and 318.35 min, respectively. In human plasma and blood, half-lives were 19.93 and 88.32 min, respectively. SG-1 was relatively stable under acidic conditions (pH 5 and lower). SG-1 is a promising, clinically useful short acting anticholinergic.

Ganglioside GD1b supresses immunoglobulin production by human peripheral blood mononuclear cells.

Gangliosides are sialic acid-containing glycolipids, that have various immunomodulatory effects. We previously reported that various gangliosides in vitro either inhibited or enhanced spontaneous immunoglobulin (Ig) production by human peripheral blood mononuclear cells (PBMC). GD1b was one of the inhibitory gangliosides. In this study, we further examined the mechanism for the inhibitory effect of GD1b. The inhibitory effect of GD1b was revealed at 0.1 microM, increased dose dependently, and was maximized at 10 microM, which reduced spontaneous IgG, IgM, and IgA production of human PBMC by 50.5%, 52.0%, and 48.3% compared with controls, respectively. GD1b did not affect the proliferation and viability of PBMC. GD1b did not alter Ig production of B cells alone. Interleukin 6 (IL-6) and IL-10 each partially reversed the GD1b-induced inhibition of Ig production by PBMC, and the addition of both cytokines completely reversed the inhibition. When endogenous IL-6 and IL-10 were neutralized by specific antibodies, GD1b did not reveal inhibitory effects on the Ig production. GD1b inhibited IL-6 and IL-10 production of CD4+ T cells, without affecting those of CD8+ T cells, monocytes, or B cells. When CD4+ T cells were preincubated with GD1b and washed and cultured with B cells and monocytes, Ig production was also suppressed. These results suggest that GD1b may indirectly suppress Ig production of B cells in whole PBMC by reducing IL-6 and IL-10 production of CD4+ T cells. GD1b may act as an important inhibitor of human humoral immune responses.

Exogenous gangliosides. How do they cross the blood-brain barrier and how do they inhibit cell proliferation.

Gangliosides have been used to treat specific central nervous system lesions and to inhibit proliferation of neuroblastoma cells in vitro. However, the mechanisms by which they (1) cross the blood-brain barrier and (2) inhibit cell proliferation have not been clearly defined. Evidence is presented in support of the hypotheses that (1) serum albumin functions in the transport of gangliosides across the blood-brain barrier, and (2) when gangliosides inhibit cell proliferation, they do so by inhibiting the activity of DNA polymerases alpha and beta.

Pharmacokinetics and acute toxicology of intraventricular 131 I-monoclonal antibody targeting disialoganglioside in non-human primates.

Tumors metastatic to the leptomeninges are often incurable despite current aggressive treatment modalities. Regional therapy by intrathecal administration of monoclonal antibodies (MoAbs) can maximize their concentration to tumor sites while reducing systemic toxicities. Anti-GD2 antibody 3F8 has successfully targeted human neuroectoderm derived tumors. Disialoganglioside GD2 expression in the central nervous system is identical between humans and cynomolgus monkeys. We studied the pharmacokinetics and the acute and subacute toxicities of intraventricular 131I-3F8 in 8 cynomolgus monkeys. Four animals were purposely immunized with intravenous 3F8 administered 2-4 weeks prior to injections. All animals remained clinically stable. Toxicities included weight loss, fever and CSF leukocytosis. One animal developed a left-sided hemiparesis following his seventh injection, presumably due to a local drug accumulation in the setting of an intermittently patent catheter. The estimated radiation dose to the CSF was 19-48 Gy in the immunized monkeys and 19-82 Gy in the nonimmunized monkeys, and to blood was 0.11-0.98 Gy and 0.29-2.03 Gy, respectively. Histopathology revealed chronic reactive changes adjacent to the region of catheter placement and a focal vasculitis in 2 animals. Peripheral blood counts and bone marrow examinations remained normal. Because of the blood-brain barrier, CSF monkey-anti-mouse antibody titers were less than 10 per cent of those in the serum. In contrast to the CSF radioactivity clearance which was similar in all animals, blood clearance was substantially accelerated in 3F8-immunized animals versus controls. Correspondingly, the CSF to blood dose ratio was improved 1.3 to 6.6 fold (mean 3.5). We conclude that intraventricular administration of 131I-3F8 in primates is tolerable. It can deliver very high doses of radiation to the CSF space with minimal toxicity to blood and bone marrow. Serum anti-mouse antibody accelerates the clearance of 131I-3F8 in blood and may improve the therapeutic index.

Co-existence of serum-dependent and serum-independent mechanisms for liposome clearance and involvement of non-Kupffer cells in liposome uptake by mouse liver.

The effect of serum on liver uptake of liposomes with different compositions was investigated using a single-pass liver perfusion technique. Among the liposomes tested are those containing CL, PA, DPGS, PE or glycolipids such as PI, GD, GT1b and aGM1. Liposomes containing PA, CL and DPGS showed high level of liver uptake in the absence of serum. Presence of serum decreased the total liver uptake for liposomes containing CL and PA by 50% and did not affect the level of liver uptake for DPGS-containing liposomes. The presence of serum, however, significantly increased the liposome uptake by the perfused liver for PG, PE and aGM1 liposomes. Liposomes containing PI showed a minimal liver uptake regardless of serum presence. Fluorescence microscopic studies using a dual fluorescence label system in combination with Kupffer cell elimination technique showed that, in addition to the dominant role of Kupffer cells in taking up liposomes, non-Kupffer cells may also be involved in taking up CL and DCP-containing liposomes. Competition experiments using various liposome compositions indicated that liposome uptake by the liver cells may involve different receptors. Serum activity in enhancing the liver uptake for PE- and aGM1-containing liposomes can be blocked by treatment of serum with EDTA, EGTA/Mg2+ and high temperature (56 degrees C), suggesting the involvement of complement system. Results from this study support the conclusion that blood clearance of liposomes by the liver involves two independent mechanisms, one requires serum opsonins and the other does not.

Entry of newly synthesized gangliosides into myelin.

Ganglioside synthesis and transport to myelin was studied in brainstem slices prepared from 19-21-day-old rats. The slices were incubated for up to 2 h in the presence of [3H]glucosamine to label primarily the hexosamine portion of complex gangliosides. The amount of radioactivity incorporated into gangliosides during slice incubations was only 10-15% of the amount of the label incorporated during in vivo labeling of brainstem gangliosides using equivalent amounts of [3H]glucosamine. Among individual gangliosides this inhibition was greater for the more complex gangliosides. When labeled gangliosides were isolated from homogenate and myelin fractions prepared from brain slices, the complex total gangliosides of both fractions showed a lag in labeling kinetics but with a lower specific radioactivity for the myelin fraction, reflecting the larger pool size and slower turnover rate exhibited by myelin components. Chase experiments showed that more complex gangliosides in homogenate exhibited almost no effect of chase after 30 min. Addition of the Golgi-disrupting agent monensin to slice incubations inhibited the labeling of all gangliosides except GM3, GM2, and GD3, and transport to myelin of all complex gangliosides except GM2. These results show that a monensin-sensitive mode of transport is responsible for the translocation of most newly synthesized gangliosides into myelin.

Axonal transport of intraocularly injected [3H-Sph]-GD1a in the chicken optic system and the fate of the exogenous ganglioside distributed by blood.

Twelve-day-old chicks (White Leghorn) received an injection of 481 kBq (8.1 nmol) of [3H-Sph]-GD1a, which was labeled in its sphingoid, into the right eye. Structures of the injected and the non-injected (control) optic system (retinae, optic nerves, chiasm, optic lobes), the cerebrum, blood liver, kidney, and fly-muscle were analyzed 1, 4, 8 and 14 days later, with respect to total non-volatile radioactivity and to that bound to lower-phase lipids and gangliosides. It was demonstrated that exogenous [3H-Sph]-GD1a was taken up by the retina and mainly catabolized. 3H-label, reincorporated into the lower-phase lipids and gangliosides as well as authentic exogenous [3H-Sph]-GD1a were transported rapidly anterogradely in the entire optic system. [3H-Sph]-GD1a, distributed via the blood stream, was taken up by liver, kidney and muscle and was metabolized faster in these organs than in the retina. The cerebrum and the brain structures of the control optic system incorporated 3H-radioactivity to a much lower extent than the non-neural organs.