Suramin exerts in vivo cytokine modulatory properties on splenocytes from experimental allergic encephalomyelitis-induced SJL mice: implications for autoimmune disease therapy.

The present study sought to examine the immunopharmacologic effects of suramin on splenocytes from experimental allergic encephalomyelitis (EAE)-induced mice, taken in line with a previous observed action of suramin in ameliorating EAE. Suramin, a polysulfonated napthylurea, decreased the proliferation of T cells, in the presence of various stimuli, such as guinea pig myelin basic protein (MBP), mouse spinal cord homogenate (MSCH), bovine proteolipid protein (PLP), P1 (synthetic peptide 139-151 of PLP), and Mycobacterium tuberculosis (MTB). Suramin inhibited T cell proliferation in a dose-dependent manner. However, cytokine assays revealed that suramin increased antigen-induced levels of IL-4, whilst IFN-gamma levels were decreased. Using various doses of suramin (25, 15, and 5 micrograms/g), its cytokine modulatory effect displayed a consistent dose-dependent activity in vivo. This cytokine modulation commenced on week 2 after immunization and persisted all throughout the drug administration period, up to the 4th week. These results indicate that the prospects of using suramin in the treatment of multiple sclerosis may be feasible.

In vitro immunopharmacologic effect of suramin on modifying Th-subset cytokine levels in splenocytes and T-cell clones: a therapeutic application for autoimmune disease.

The in vitro immunopharmacologic effect of suramin, a polysulfonated napthylurea, was examined on splenocytes and T helper (Th) 1 and 2 clones. Cytokine capture assays revealed that suramin inhibited the production of IFN-gamma, whilst IL-4 is increased in splenocytes. To examine whether suramin preferentially acts on Th cells, clones SP39A1 and SP41D5 were used. The former is a Th1 clone, whereas the latter is Th2. Suramin also inhibited IFN-gamma production by SP39A1, whilst IL-4 production by SP41D5 was enhanced. The action of suramin was of a dose-dependent nature. With regards to T cell proliferation, suramin inhibited the proliferation of both SP39A1 and SP41D5. The ability of suramin to manipulate Th subset cytokine balance can render it a feasible therapeutic agent in autoimmune disorders such as multiple sclerosis.

Chelerythrine inhibits the secretory response of human blood platelets without specifically inhibiting protein kinase C.

Chelerythrine (chloride) has previously been documented to be a potent and selective inhibitor of the serine/threonine-specific protein kinase C (PKC). In this study, it was shown that 10 microM chelerythrine completely inhibited serotonin secretion and partially inhibited phosphatidic acid formation in human blood platelets activated by thrombin (1U/ml). However, there was no effect on PKC activity as assessed by the level of phosphorylation of the 47K protein. Therefore, chelerythrine has been shown not to be a specific inhibitor of PKC. Without specifically affecting PKC activity, it is nevertheless capable of completely inhibiting platelet secretion, indicating that it may affect the signal transduction pathway responsible for platelet secretion at a point downstream or independent of PKC.

Production and characterization of separate monoclonal antibodies to human brain and erythrocyte acetylcholinesterases.

Four murine monoclonal antibodies (MAbs) of the IgM class were raised against human acetylcholinesterase (AChE; Ec 3.1.1.7). The MAbs BMS-3E4, BMS-7G10, and BMS-9F4 all recognized human erythrocyte AChE, while BMS-6D6 bound specifically to human soluble brain AChE, on the basis of immunobinding assays. Dose-response studies gave an ELISA ED50 titer of 4.5 x 10(-4) M for BMS-6D6, while BMS-3E4 gave the best titer at 8.8 x 10(-4) M. Sucrose density gradients demonstrated sedimentation of antigen-antibody complexes, consistent with earlier findings (i.e., BMS-6D6 bound with brain AChE while BMS-3E4 preferred erythrocyte (AChE). No cross-reactivity between the two MAbs against the two antigens was noted.

Purification of murine immunoglobulin M from spent tissue culture supernatant by one-step gel filtration chromatography procedure.

Optimal purification of immunoglobulin M (IgM) grown as spent tissue culture supernatant can be achieved by a straightforward size-exclusion chromatography procedure. Preparative isolation of IgM was achieved by precipitation with saturated ammonium sulphate to achieve a 45% solution. IgM was then purified by gel filtration chromatography with a solution of 100 mM Tris + 150 mM NaCl at pH 8. Denaturing electrophoresis and Western immunoblotting revealed two prominent bands at 80 and 25 kDa, indicative of the heavy and light chains of IgM respectively. The specific immunoreactivity of this IgM was assessed by a modified enzyme antigen capture assay. In contrast to affinity and ion-exchange chromatographies, gel filtration allows retention of IgM immunoreactivity.

Comparison of standard chromatographic procedures for the optimal purification of soluble human brain acetylcholinesterase.

With the view of purifying soluble human brain acetylcholinesterase (AChE) into its separate isoforms, various preparative chromatographic procedures were compared. Chromatofocusing of cerebrospinal fluid (CSF) AChE revealed two major activity peaks, whilst that of caudate nucleus AChE showed one major peak. Both CSF and caudate nucleus AChE eluted at isoelectric points (pI) of between 5.5 and 5.2. Chromatofocusing failed to separate AChE into its individual isoforms, based on qualitative isoelectric focusing. Preparative purification by affinity chromatography showed a better AChE yield with the use of procainamide as a ligand, vis-à-vis acridinium. Maximum recovery for CSF and caudate nucleus AChE was 10 and 43% using acridinium and procainamide, respectively. Qualitative analysis by SDS-PAGE of affinity-purified AChE revealed four major bands between 50 and 62 kDa, corresponding to the catalytic subunits of AChE as verified by an anti-AChE polyclonal antibody. A size-exclusion column also allowed brain AChE purification, with the latter eluting at a putative molecular mass of 310 kDa. Unfortunately, cation-exchange using the state-of-the-art SMART system failed to separate AChE into its isoforms. AChE aggregation is given as one major obstacle precluding good resolution of isoforms.

Characterization of immunoglobulin M antibodies raised by in vitro immunization specific to soluble human caudate nucleus and cerebrospinal fluid acetylcholinesterase.

Using as antigen a 16 amino acid peptide corresponding to the C-terminus of soluble human brain acetylcholinesterase (AChE), four monoclonal antibodies were raised by the novel procedure of in vitro immunization. MAbs BMS-5, -6, -7, and -8 all demonstrated positive binding to the isoforms of cerebrospinal fluid (CSF) and foetal bovine serum (FBS) AChE. In contrast, no binding was seen with "membrane-bound" caudate nucleus and erythrocyte AChEs. With CSF samples from Alzheimer's patients, immunoblotting of isoelectric focussing (IEF) gels with the four mAbs failed to demonstrate any positive binding to the anomalous AChE band. Immunoblotting of SDS-reduced AChE showed binding of the four mAbs with "soluble" caudate nucleus and FBS AChE, but not with erythrocyte AChE. Hence, mAbs BMS-5, BMS-6, BMS-7 and BMS-8 all bind specifically to tetrameric AChE under non-denaturing and denaturing conditions.

Comparative study of d-diltiazem and its analogues as calcium antagonists in snail neurones.

The blocking effects of d-diltiazem, its metabolites, deacetyl-d-diltiazem (d-M1), deacetyl-N-demethyl-d-diltiazem (d-M2), deacetyl-O-demethyl-d-diltiazem (d-M4) and deacetyl-N, O-demethyl-d-diltiazem (d-M6) and 1-diltiazem were investigated on the voltage-gated calcium current (ICa) of an Achatina neurone. Based on the IC50 values, the order of potency was: d-diltiazem (0.426 mM), d-M2 (0.456), d-M1 (0.491), 1-diltiazem (0.759) greater than d-M4 (1.212) greater than d-M6 (greater than 2.000). Assuming that the IC50 reflects the KD for resting Ca2+ channels (Kr), steady state activation studies gave KD values for the inactivated channels (Ki) and Kr/Ki ratios of 0.122 mM (Ki) and 3.52 (Kr/Ki) (d-diltiazem), 0.112 and 6.98 (1-diltiazem), 0.083 and 6.07 (d-M1) and 0.156 and 2.97 (d-M2). All drugs tested showed a certain degree of voltage-dependence. The further percentage reduction in the normalized ICa after high frequency stimulation demonstrated the use-dependence of: 1-diltiazem (27.5%), d-diltiazem (26.3) greater than d-M2 (19.2) greater than d-M1 (16.7) greater than d-M6 (9.8). The voltage- and use-dependence of these drugs are consistent with their Ca2+ antagonistic properties.

Suppression of calcium current in a snail neurone by eperisone and its analogues.

The effects of eperisone, tolperisone and isoperisone on the calcium current (ICa) were studied in an identified neurone of Achatina fulica under voltage clamping. At a holding voltage of -50 mV, these drugs inhibited ICa dose dependently without affecting activation time. Eperisone (IC50 = 0.348 mM) and isoperisone (0.226 mM) were significantly more effective than tolperisone (1.089 mM). Eperisone binds competitively with Ca2+ to the Ca2+ channels, based on its effects seen with various extracellular Ca2+ concentrations. The three drugs shifted the steady state inactivation curves in the hyperpolarizing direction. The mean dissociation constants for inactivated Ca2+ channels were calculated to be 0.070 mM (eperisone), 0.162 mM (tolperisone) and 0.014 mM (isoperisone). These values were much lower than their IC50 at Vh of -50 mV, which are reflected as the dissociation constants for resting Ca2+ channels. High frequency stimulation did not potentiate ICa suppression, suggesting that the drugs hardly bind to activated Ca2+ channels. These findings indicate that the selectivities of the Ca2+ channels to the drugs are in the order of their inactivated, resting and open states.

Achatin-I, an endogenous neuroexcitatory tetrapeptide from Achatina fulica Férussac containing a D-amino acid residue.

A tetrapeptide named achatin-I was purified from the suboesophageal and cerebral ganglia of the African giant snail Achatina fulica Férussac, and evoked a potent neuroexcitatory effect. The amino acid sequence of achatin-I is Gly-D-Phe-Ala-Asp. Achatin-I induced a voltage-dependent inward current, due to Na+, on the identifiable giant neuron, periodically oscillating neuron (PON), of the same snail. All possible isomers of achatin-I were synthesized using the solid-phase method. The sensitivity of the neuron to achatin-I and its isomers was strictly stereospecific; among the various isomers, only achatin-I showed marked effects (ED50 = 2.29 x 10(-6)M), while Gly-D-Phe-D-Ala-Asp, the synthetic D-Ala-isomer, was less than 10(-3) active.

Effects of compounds related to beta-hydroxyglutamic acid (BHGA) on identifiable giant neurones of an African giant snail (Achatina fulica Férussac).

The present study aimed to further elucidate the pharmacological features, with respect to sensitivity to L-BHGA agonists, of the receptors sensitive to beta-hydroxy-L-glutamic acid (L-BHGA) in five Achatina giant neurones: PON (periodically oscillating neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone), RAPN (right anterior pallial neurone) and v-RCDN (ventral-right cerebral distinct neurone). Of these neurones, d-RPLN and RAPN were depolarized by L-BHGA, while PON, VIN and v-RCDN were inhibited. Threo-beta-hydroxy-DL-aspartic acid markedly depolarized d-RPLN and RAPN (effective potency quotient (EPQ) in relation to the more effective L-BHGA isomer: 1 for d-RPLN and 0.3 for RAPN). This compound produced only slight inhibitory effects on PON, VIN and v-RCDN with EPQs calculated to be less than 0.03, less than 0.03 and 0.03, respectively. On the other hand, erythro-beta-hydroxy-DL-aspartic acid at 10(-3) M was almost ineffective, except on v-RCDN where it elicited some slight inhibitory effects (EPQ: 0.01). L-Aspartic and D-aspartic acid at 10(-3) M, also had almost no effect except for slight effects of D-aspartic acid on d-RPLN (EPQ: 0.1). N-Methyl-L- and N-methyl-D-aspartic acid were slightly effective only on v-RCDN (EPQ: less than 0.01 and 0.01, respectively). The other compounds, including beta-hydroxypyrroglutamic acid (cyclic BHGA) and proline derivatives, were almost ineffective at 10(-3) M; very weak effects were occasionally observed on some neurones.(ABSTRACT TRUNCATED AT 250 WORDS)