Transplantation of purified islet cells in diabetic rats. III. Immunosuppressive effect of cyclosporin.

This study examined the effect of cyclosporin on the survival of islet beta-cell allografts in streptozocin (STZ)-induced diabetic rats. At a daily oral dose of 5 mg/kg, the agent prevented the rejection of isolated islets, provided they were little contaminated by other pancreatic tissue. The immunosuppressive effect rapidly disappeared after discontinuation of the drug, except when the donor tissue had been pretreated to reduce its nonendocrine content. All recipients of cultured and selected islets maintained a normalized state for greater than 15 wk beyond the 5-wk drug course; this was not the case for shorter periods of treatment. A long-term beneficial effect was also observed in all recipients of purified islet beta-cell grafts, which reversed without treatment in half of the cases. Cyclosporin markedly reduced the mononuclear cell infiltration in each type of islet beta-cell allograft; aggregates of mixed endocrine islet cells were kept virtually infiltration free. Conditions with minimal initial infiltration were associated with long-term graft survival without the need for continuous pharmacological immunosuppression. We conclude that a short-term cyclosporin treatment can induce long-term survival of allografted islet beta-cells, provided the grafts are only slightly contaminated by nonendocrine elements. In rodents, sufficient immunosuppression was achieved by circulating cyclosporin levels of 100-400 ng/ml. Higher concentrations were cytotoxic for cultured islet beta-cells and islet non-beta-cells. A 5-wk treatment with the immunomodulator ciamexone also resulted in long-term survival of purified beta-cell allografts but not of cultured islets.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure/function analyses of IL-2 binding proteins on human B cell precursor acute lymphoblastic leukemias.

We have studied the expression and function of interleukin-2 (IL-2) receptors on B cell precursor acute lymphoblastic leukemias (ALL). After incubation of B cell precursor ALL in vitro for 24 hr, 11 out of 17 leukemic bone marrow aspirates expressed the Tac/CD25 protein (greater than 10% positive blasts). Expression of Tac/CD25 on the leukemic cells was confirmed by two color flow cytometric analysis using anti-Tac/CD25 and anti-CALLA/CD10 monoclonal antibodies. The molecular mass of the B cell precursor ALL Tac/CD25 protein was 55 kilodaltons (kD), identical to that on activated T cells. Binding of radiolabeled IL-2 in two leukemic bone marrow aspirates demonstrated the presence of high affinity IL-2 receptors. Cross-linking of 125I-labeled IL-2 to TPA activated B cell precursor ALL revealed the 55 kD Tac/CD25 protein and an additional protein of 75 kD. Recombinant IL-2 in concentrations of 10-1,000 U/ml had essentially no proliferative effect in 10 patients tested, whereas low molecular weight B cell growth factor (L-BCGF) induced proliferation in 8 of 10 patients. L-BCGF also induced expression of CD20 in 3 of 7 CD20 negative B cell precursor ALL. IL-2 did not induce CD20, but enhanced its expression in the 3 patients who responded to L-BCGF. We conclude that IL-2 has essentially no proliferative effect on B cell precursor ALL, despite the presence of high affinity IL-2 receptors and the presence of the IL-2 binding cell surface molecules similar to those on activated T cells. IL-2 may, however, induce a phenotypic change (CD20 acquisition) consonant with differentiation in synergy with L-BCGF.

Liquid levodopa/carbidopa produces significant improvement in motor function without dyskinesia exacerbation.

We performed the first double-blind, crossover comparison between levodopa/carbidopa (LD/CD) in optimized liquid versus tablet doses to measure plasma LD levels and relative effects on disabilities (motor function, fluctuations, and dyskinesias) in patients with Parkinson's disease. Twenty-three subjects with motor fluctuations were optimized with open-label LD/CD tablets and liquid. In a double-dummy design, patients randomly received 2 weeks of liquid and 2 weeks of tablet LD/CD. Twice during each arm, we evaluated patients hourly 9 AM to 4 PM with the use of plasma LD levels, the Unified Parkinson's Disease Rating Scale, a dyskinesia rating scale, and "on-off" ratings. Patients receiving liquid LD/CD ingested significantly higher doses and had significantly improved motor function and total "on" time, without an increase in dyskinesia severity. The number of motor fluctuations in the two phases was not significantly different. LD levels and variability were also equivalent with the two formulations. At optimized dosing, liquid LD/CD offers a means to significantly improve motor disability in patients with Parkinson's disease without exacerbating dyskinesia.

Alpha-tocopherol in the ventricular cerebrospinal fluid of Parkinson's disease patients: dose-response study and correlations with plasma levels.

OBJECTIVE: To determine if ventricular cerebrospinal fluid (vCSF) alpha-tocopherol levels in Parkinson's disease (PD) patients can be increased by oral alpha-tocopherol supplementation and whether vCSF levels are linearly related to plasma alpha-tocopherol levels. BACKGROUND: In spite of its putative neuroprotective properties, alpha-tocopherol has failed to alter PD clinical progression. However, the ability of supplemental alpha-tocopherol to affect brain or vCSF levels has never been assessed in humans nor has a dose response curve for alpha-tocopherol in vCSF been established. METHODS: Five PD patients with Ommaya catheters received oral dl-alpha-tocopherol over 5 months. Each patient ingested alpha-tocopherol daily with monthly dosage increases (400, 800, 1,600, 3,200, 4,000 IU/day). Plasma and vCSF samples were obtained at baseline and at the end of each month. Alpha-tocopherol levels were determined in triplicate by high-pressure liquid chromatography with fluorometric and electrochemical detection. RESULTS: At baseline, endogenous alpha-tocopherol was detected in plasma and vCSF, with a greater than one-hundred-fold difference between the fluid compartments (mean plasma level 18.76 microM/l (SD +/- 4.69) versus mean CSF level 0.114 microM/l (SD +/- 0.084). A clear dose-response curve occurred in plasma, with statistically significant increases over baseline developing even with 400 IU/d. With higher doses, a significant increase continued without evidence of saturation. However, there was no significant increase in vCSF alpha-tocopherol levels at any dose, including the supraclinical (4,000 IU/d). There was no correlation between plasma and vCSF alpha-tocopherol levels. CONCLUSION: Oral alpha-tocopherol supplementation, even at supraclinical doses, fails to increase vCSF alpha-tocopherol levels. This lack of change may be due to limited passage across the blood-brain barrier or very rapid alpha-tocopherol metabolism. All prior negative studies on efficacy of alpha-tocopherol in PD may need reevaluation in light of these pharmacologic data.

Intravenous levodopa in hallucinating Parkinson's disease patients: high-dose challenge does not precipitate hallucinations.

In five nondemented Parkinson's disease patients with daily visual hallucinations, we tested whether high-dose IV levodopa (LD) infusions precipitated hallucinations. Two infusion paradigms were studied, each with 1.5-mg/kg hourly dose for 4 hours--steady infusion and pulse infusion of the full hour dose over 5 minutes each hour. In both protocols, plasma LD levels changed significantly during the infusion protocol. The cumulative area under the curve was equivalent for the two infusions. All patients remained alert, and none developed visual hallucinations. The two patients with peak-dose dyskinesias on oral LD developed prominent dyskinesias during the infusion. Visual hallucinations do not relate simply to high levels of LD or to sudden changes in plasma levels.

Animal model of posthypoxic myoclonus: effects of serotonergic antagonists.

OBJECTIVE: To study specific serotonin (5-hydroxytryptamine [5-HT]) receptor subtype antagonists in an animal model of posthypoxic myoclonus. BACKGROUND: Although serotonergic system dysfunction is implicated in posthypoxic myoclonus, anatomic specificity and linkage to receptor subtypes are not delineated. METHODS: The authors performed a pharmacologic study to identify specific serotonin receptor subtype antagonists effective in inhibiting myoclonus in posthypoxic rats. Sprague-Dawley rats underwent cardiac arrest for 8 minutes and were resuscitated. On the day of pharmacologic testing, animals were rated every 10 minutes at -30 minutes to time 0 (drug injection) and from +60 to +150 minutes. Using a blinded methodology, animals were injected with normal saline, vehicle, or one of seven serotonin antagonists given at a dose that maintains serotonin receptor subtype specificity: WAY100135 (5-HT1A), methiothepin mesylate (5-HT1B/1D/2), mesulergine hydrochloride (5-HT2A/2B), GR 127935 (5-HT1D), SR 46349 (5-HT2), ondansetron (5-HT3), or GR 125487 (5-HT4). Drugs that produced a significant decrease in myoclonus compared with the control were studied in a dose-response study with six doses across a range from the original dose studied to 10% of that dose. RESULTS: Two drugs were significantly different from placebo: methiothepin mesylate and mesulergine hydrochloride. GR 127935 showed a trend toward reducing myoclonus. Dose-response studies showed that all doses of methiothepin mesylate and the three highest doses of mesulergine hydrochloride inhibited myoclonus effectively. CONCLUSIONS: 5-HT1B, 5-HT2A/2B, and possibly 5-HT1D receptor subtypes likely play a role in posthypoxic myoclonus. More specific 5-HT antagonists that affect these receptor subtypes are candidates for future testing in this model and in Lance-Adams syndrome.

Peptide inhibitors of caspase-3-like proteases attenuate 1-methyl-4-phenylpyridinum-induced toxicity of cultured fetal rat mesencephalic dopamine neurons.

Multiple aspartate-specific cysteine proteases have been identified and specific members of this family have been implicated in the apoptotic death of many mammalian cell types. Caspase-3-like proteases seem to play a pivotal role in neuronal apoptosis since mice with germline inactivation of the caspase-3 gene manifest profound alterations in neurogenesis. Moreover, inhibitors of caspase-3-related proteases have been shown to inhibit neuronal apoptosis. Here we extend recent work from our laboratory on the mechanisms mediating the neurotoxic actions of 1-methyl-4-phenylpyridinium using ventral mesencephalon cultures containing dopamine neurons. We demonstrate that low concentrations of 1-methyl-4-phenylpyridinium induce apoptosis in dopamine neurons by morphological and biochemical criteria. Moreover, pretreatment of ventral mesencephalon cultures with the tetrapeptide inhibitors of the caspase-3-like proteases zVAD-FMK or Ac-DEVD-CHO specifically inhibit death of dopamine neurons induced by low concentrations of 1-methyl-4-phenylpyridinium, whereas the caspase-1-like inhibitor Ac-YVAD-CHO was without effect. Our data indicate that exposure of cultured ventral mesencephalon dopamine neurons to low concentrations of 1-methyl-4-phenylpyridinium results in apoptotic death and that caspase-3-like proteases may mediate the neurotoxic apoptotic actions of 1-methyl-4-phenylpyridinium.

Combined toxicity of prenatal bacterial endotoxin exposure and postnatal 6-hydroxydopamine in the adult rat midbrain.

We previously reported that injection of the Gram (-) bacteriotoxin, lipopolysaccharide (LPS), into gravid females at embryonic day 10.5 led to the birth of animals with fewer than normal dopamine (DA) neurons when assessed at postnatal days (P) 10 and 21. To determine if these changes continued into adulthood, we have now assessed animals at P120. As part of the previous studies, we also observed that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) was elevated in the striatum, suggesting that these animals would be more susceptible to subsequent DA neurotoxin exposure. In order to test this hypothesis, we injected (at P99) 6-hydroxydopamine (6OHDA) or saline into animals exposed to LPS or saline prenatally. The results showed that animals exposed to prenatal LPS or postnatal 6OHDA alone had 33% and 46%, respectively, fewer DA neurons than controls, while the two toxins combined produced a less than additive 62% loss. Alterations in striatal DA were similar to, and significantly correlated with (r(2)=0.833) the DA cell losses. Prenatal LPS produced a 31% increase in striatal TNFalpha, and combined exposure with 6OHDA led to an 82% increase. We conclude that prenatal exposure to LPS produces a long-lived THir cell loss that is accompanied by an inflammatory state that leads to further DA neuron loss following subsequent neurotoxin exposure. The results suggest that individuals exposed to LPS prenatally, as might occur had their mother had bacterial vaginosis, would be at increased risk for Parkinson's disease.

The effects of storage conditions and trophic supplementation on the survival of fetal mesencephalic cells.

It is estimated that only 5-10% of dopamine (DA) neurons implanted into the striatum of patients undergoing fetal-nigral transplantation as a treatment for Parkinson's Disease survive. Because it is often necessary to store fetal tissue prior to transplantation, we evaluated various storage parameters that could influence DA neuron viability in rostral mesencephalic tegmentum (RMT) cultures using tyrosine hydroxylase immunoreactive (THir) cell counts as an index of DA neuron survival. A high K+ hibernation media (HM) was used in all studies. We found that RMT cell viability and THir cell counts decreased as storage duration increased (up to 120 h). Storage at 37 degrees C in HM killed all cells, while storage at 10 degrees C yielded higher survival rates than 4 degrees C. In comparison to trypsinization, mechanical dissociation of tissue increased cell viability. Neutral pH and a storage density of at least 1 x 10(6) cells/mL were found to be optimal, while striatal coculture of RMT cells with striatal feeder layers increased THir viability up to 16-fold in comparison to monocultures. The nurturing effect of striatal coculture may be explained by the release of autotrophic factors, and we tested this hypothesis by supplementing the HM with human placental cord serum (HPCS, 8%), glial-derived neurotrophic factor (GDNF; 10 microg/mL), and brain-derived neurotrophic factor (BDNF; 10 microg/mL). GDNF and HPCS supplements increased RMT cell viability by 10-15%, while GDNF, BDNF, and HPCS increased viability of THir cells by approximately 40% at all time points studied. As Klenow enzyme labeling technique indicated that 33% of stored RMT cells were undergoing apoptosis, we found that GDNF, BDNF, and HPCS reduced apoptosis by 50%. DNA laddering and DAPI nuclear stain confirmed the presence of apoptosis in hibernated RMT cells, leading us to postulate that the high viability counts seen with trypan blue exclusion are misleading.

Partial purification of a pramipexole-induced trophic activity directed at dopamine neurons in ventral mesencephalic cultures.

We previously demonstrated that media conditioned by exposure to ventral mesencephalic (VM) cultures in the presence of pramipexole (PPX) and other drugs with dopamine (DA) D3 properties, increased the growth and survival of DA neurons in recipient VM cultures. This trophic activity was heat-labile and not present in parietal cortex cultures or cultures pretreated with the DA neuron toxin MPP+. In an effort to begin to identify the protein(s) responsible for this trophic effect, we compared the conditioned media from normal VM cultures, VM cultures incubated with PPX, and VM cultures pretreated with MPP+ and treated with PPX. Neutralization studies using anti-GDNF and anti-BDNF failed to reduce the conditioned media transfer effect, and Millipore Ultrafree centrifugation studies placed the mol.wt. of the activity around 30 kDa. SDS separation revealed three potential bands of interest. A 35-kDa band was present in normal cultures, increased in PPX-incubated cultures, and absent in MPP+-pretreated/PPX-incubated cultures. This conforms to the effect the protein concentrates used to produce these gels had on the growth of DA neurons in VM cultures. Since VM cultures grown in neural basal media, which inhibits the growth of glia, still responded to PPX in a dose-dependent fashion, the trophic activity may be a DA autotrophic factor. However, the gels also revealed two bands at approximately 31 and 55 kDa that were reduced by exposure to PPX and present in MPP+-pretreated cultures. The possibility that these are neuroinhibitory factors that are also regulated by PPX therefore cannot be ruled out.

Striatal trophic activity is reduced in the aged rat brain.

Our previous studies demonstrated that the survival of a mesencephalic graft was reduced in aged animals suggesting an age-related decline in target-derived neurotrophic activity. We tested this hypothesis by examining dopamine (DA) and trophic activities from the striatum of intact or unilateral 6-hydroxydopamine (6-OHDA) lesioned rats of increasing age. Fisher 344 rats were 4, 12, 18, and 23 months old (m.o.) at sacrifice. Half the animals had received unilateral 6-OHDA lesions of the mesostriatal DA pathway 8 weeks earlier. Striatal tissue punches were analyzed for DA, homovanillic acid (HVA), and DA activity (HVA/DA) using HPLC. The remainder of the striatal tissue was homogenized to generate tissue extracts which were added to E14.5 ventral mesencephalic cultures to test trophic activity. In the non-lesioned animals, striatal DA was reduced and striatal DA activity was increased in the 18 and 23 m.o. animals relative to the 4 and 12 m.o. animals. Striatal trophic activity was inversely related to age. In the lesioned animals, striatal DA ipsilateral to 6-OHDA infusion was below detection limits while the contralateral striatum exhibited age-related changes in DA similar to those seen in the non-lesioned animals. In 4 m.o. lesioned rats, striatal trophic activity ipsilateral to 6-OHDA infusion was elevated by 26% relative to the contralateral side. The ipsi/contra-lateral differences in striatal trophic activity were reduced in 12 m.o. animals and absent in the 18 and 23 m.o. groups. These data suggest that advancing age is associated with a reduction in striatal DA as well as trophic activity. Moreover, the aged striatum loses its ability to biochemically and trophically compensate for DA reduction and therefore may represent a more challenging environment for the survival, growth, and function of a fetal graft.

The influence of recombinant mediators on in vitro IgG subclass secretion by peripheral blood mononuclear cells from patients with hypogammaglobulinemia and from normal donors.

Patients with hypogammaglobulinemia have recurrent infections and fail to produce protective antibodies. In order for B cells to mature into antibody producing cells, several other cell types such as macrophages and helper T lymphocytes must be involved. They secrete several mediators such as interleukin-1 (IL-1), IL-4, IL-5, IL-6 and interferon-gamma (IFN-gamma). These factors, as recombinant mediators, were tested to assess their ability to correct the immunoglobulin production defect in vitro from pokeweed mitogen (PWM) stimulated cultures of peripheral blood mononuclear cells (PBMC) from 11 patients with hypogammaglobulinemia, and from 10 normals as controls. In general, PBMC from hypogammaglobulinemic patients secreted very little Ig in cultures, and no mediator induced a statistically significant increase in the secretion of any IgG subclass. When assessed on an individual basis, one patient demonstrated a variable pattern of increase in total IgG, IgG1, and IgG3, secretion induced by various mediators, to within one standard deviation of the average secretion of normal PBMC cultured in PWM. In the case of the normal cells, IL-4, IL-6 and IL-1 plus IL-4 were able to increase IgG2 secretion in culture with PWM. No increase in secretion of IgG1, IgG3 and IgG4 or total IgG was demonstrable however. Hence, although there is variability in responsiveness amongst the patients, there does not appear to be any one of these recombinant mediators which will correct the defect.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the neurotoxicity of dihydroxyphenylalanine stereoisomers in cultured dopamine neurons.

Oxidant stress resulting from excess dopamine (DA) may contribute to the development and progression of Parkinson's disease (PD). Free radicals resulting from the enzymatic metabolism of DA are most often discussed in this regard. However, levodopa (L-DOPA) and DA can also undergo autooxidation, producing free radicals as well as cytotoxic metabolites. We evaluated the neurotoxic effects of the two stereoisomers of L-DOPA to differentiate between enzyme-mediated and autooxidation mechanisms. Various concentrations of D- or L-DOPA (1 mM through 10 nM) were added to freshly harvested rostral mesencephalic tegmentum cultures. After 72 h, the cultures were fixed and stained for tyrosine hydroxylase (TH). The number of TH-immunoreactive (THir) neurons was then assessed and used as an index of DA neuron survival. Both D- and L-DOPA induced a dose-dependent loss of THir neurons (F10,21 = 135.75, p < 0.0001 and F10,21 = 142.53, p < 0.0001, respectively) with ED50 values of 10(-5.3) and 10(-5.2) M, respectively. The dose-response curves for each drug were not significantly different from one another (F1,43 = 0.09, p > 0.05). Moreover, both drugs killed THir as well as non-THir cells at high concentrations, suggesting a nonspecific toxic effect. These data are most consistent with an enzyme-independent, autooxidation-mediated mechanism for DA neuron loss.

Prenatal cocaine exposure and postnatal hypoxia independently decrease carotid body dopamine in neonatal rats.

The effects of prenatal cocaine exposure on the levels of carotid body dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were investigated in 5-day-old rat pups exposed to normoxic and hypoxic conditions. Timed-pregnant Sprague-Dawley rats were injected b.i.d. with either cocaine HCl (30 mg/kg) or isotonic saline (1 ml/kg) from gestational days 7-21. On the fifth postnatal day, pups were subjected to either 20 min of 0.21 or 0.08 fractional inspired oxygen (FlO2). Under a strictly timed protocol, both carotid bodies were removed from each pup, placed in an antioxidant solution to prevent DA breakdown, and subsequently analyzed via HPLC with electrochemical detection to determine carotid body DA and DOPAC content. Two-way ANOVA revealed decreases in DA in cocaine-exposed pups. No HVA was detectable in any of the samples. The 0.08 FlO2 condition decreased DA compared to 0.21 FlO2. The additive consequences of DA depletion resulting from the combination of prenatal cocaine and postnatal hypoxia decreased carotid body DA to 14% of control levels, with several animals exhibiting DA content below detection limits. Considering the role of the carotid body in the ventilatory response to hypoxia, these data suggest that prenatal cocaine exposure may adversely affect the normal chemoreceptive function of the carotid body.

Intra-parenchymal injection of tumor necrosis factor-alpha and interleukin 1-beta produces dopamine neuron loss in the rat.

Inflammatory processes are thought to underlie the dopamine (DA) neuron loss seen in Parkinson's disease (PD). However, it is not known if the inflammation precedes that loss, or is a consequence of it. We injected tumor necrosis factor alpha (TNFalpha) and interleukin 1 beta (IL-1beta) into the median forebrain bundle to determine if these pro-inflammatory cytokines could induce DA neuron loss in the substantia nigra (SN) by themselves. The magnitude of the DA cell loss as well as the decreases in striatal DA, were both dose and time to sacrifice dependent. Injecting both cytokines together produced greater cell losses and DA reductions than that seen when the cytokines were injected alone. The DA neuron loss seen was more pronounced in the lateral nigra and its ventral tier and similar to that seen when other toxins are injected. These data suggest that TNFalpha and IL-1beta can induce DA neuron loss by themselves and could produce DA neuron loss independent of other inflammatory events.

6-Hydroxydopamine-induced alterations in blood-brain barrier permeability.

Vascular inflammation is well known for its ability to compromise the function of the blood--brain barrier (BBB). Whether inflammation on the parenchymal side of the barrier, such as that associated with Parkinson's-like dopamine (DA) neuron lesions, similarly disrupts BBB function, is unknown. We assessed BBB integrity by examining the leakage of FITC-labeled albumin or horseradish peroxidase from the vasculature into parenchyma in animals exposed to the DA neurotoxin 6-hydroxydopamine (6OHDA). Unilateral injections of 6OHDA into the striatum or the medial forebrain bundle produced increased leakage in the ipsilateral substantia nigra and striatum 10 and 34 days following 6OHDA. Microglia were markedly activated and DA neurons were reduced by the lesions. The areas of BBB leakage were associated with increased expression of P-glycoprotein and beta 3-integrin expression suggesting, respectively, a compensatory response to inflammation and possible angiogenesis. Behavioural studies revealed that domperidone, a DA antagonist that normally does not cross the BBB, attenuated apomorphine-induced stereotypic behaviour in animals with 6OHDA lesions. This suggests that drugs which normally have no effect in brain can enter following Parkinson-like lesions. These data suggest that the events associated with DA neuron loss compromise BBB function.

Attenuation of levodopa-induced toxicity in mesencephalic cultures by pramipexole.

The direct-acting dopamine (DA) agonist pramipexole (2 amino-4,5,6,7-tetrahydro-6-propyl-amino-benzthiazole-dihydrochlori de) was evaluated for its ability to attenuate levodopa-induced loss of tyrosine hydroxylase immunoreactive (THir, a marker for dopamine neurons) cells in mesencephalic cultures. Pramipexole reduced levodopa-induced THir cell loss in a dose-dependent and saturable fashion (ED50 = 500 pM), its inactive stereoisomer was significantly less potent in this regard and pergolide and bromocriptine had negligible cytoprotective effects. Culture media from mesencephalic cultures incubated with pramipexole for 6 days increased THir cell counts in freshly harvested recipient cultures. The magnitude of this effect was directly proportional to the amount of pramipexole in the donor cultures and heat-inactivation of the media abolished the growth promoting effect. The results from this exploratory set of experiments suggest that pramipexole may be cytoprotective to dopamine neurons in tissue culture. Pramipexole's affinity for DA receptors, its antioxidant action or its ability to enhance mesencephalic trophic activity could be responsible for this effect.

Neuroprotective effects of D3 dopamine receptor agonists.

The effects of D(3) receptor activation are unresolved at this time, but may have practical implications in the treatment of Parkinson's disease (PD). As a result of assessing the neuroprotective effects of the direct-acting D(3) preferring dopamine (DA) agonist pramipexole (PPX), we have observed that drugs which psossess D(3) affinity increase the production of a DA neurotrophic factor in tissue culture. This molecule is increased by treatment with PPX, is constitutively produced by DA neurons in culture, and possesses a molecular weight of approximately 35kDa. It is hypothesized that this molecule may be the so-called DA autotrophic factor referred to by many authors over the past two decades. Interestingly, the protein is oxidant-labile and, therefore, D(3) agonists which increase its production and also possess antioxidant capacity would provide unique neuroprotective benefits to patients with PD. However, many questions remain. Although the data supporting this notion are strong, it is clear that other unknown characteristics of DA agonists, including increased production of anti-apoptotic proteins, are also involved. This manuscript will review this concept in the context of tissue culture strategies of neuroprotection. Although no conclusion can be made at this time, it is clear that direct comparisons of the neuroprotective effects of direct-acting DA agonists in mesencephalic culture can provide considerable insight into the mechanistic actions of anti-dopaminergic drugs.

Cytokine-induced conversion of mesencephalic-derived progenitor cells into dopamine neurons.

We have previously shown that a combination of the cytokines interleukin (IL)-1, IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF) can convert rat fetal (E14.5) mesencephalic progenitor cells into tyrosine hydroxylase (TH)-immunoreactive (ir) neurons in vitro. The experiments described here characterize the mesencephalic progenitor cells and their cytokine-induced conversion into dopamine (DA) neurons. For all experiments, we used bromodeoxyuridine (BrdU)-ir cultures of (E14.5) mesencephalic progenitor cells that had been expanded at least 21 days. We first demonstrated that IL-1 induced DA neuron conversion in mesencephalic progenitors, but not in striatal progenitors (P < 0.001). Thus, these cells should be classified as lineage-restricted progenitors, and not omnipotent stem cells. To further characterize cell populations in these cultures, we used monoclonal antibodies against Hu (an early marker for neurons), growth-associated protein (GAP)-43 (a marker for neuronal process extension), TH (a marker for DA neurons), and glial fibrillary acidic protein (GFAP, a marker for astrocytes). We assessed (E14.5) mesencephalic progenitor cell cultures (plated at 125,000 cells/cm2) incubated in the cytokine mixture (described above) or in complete media (CM, negative control). Following 7 days incubation, GFAP-positive cells formed a nearly confluent carpet in both types of cultures. However, numbers of Hu-ir and GAP-43-ir cells in the cytokine-incubated cultures far exceeded those in CM-incubated controls (P = 0.0003, P = 0.0001, respectively), while numbers of TH-ir cells were 58-fold greater in the cytokine-incubated cultures versus CM-incubated controls. The TH phenotype persisted for 7 days following withdrawal of the differentiation media. Numerous double-labeled cells that were BrdU-ir and also TH-ir, or Hu-ir and also TH-ir, were observed in the cytokine-incubated cultures. These data suggest that cytokines "drive" the conversion of progenitor cells into DA neurons.

Aggregated immunoglobulin and Fc fragment of IgG induce IL-6 release from human monocytes.

The Fc fragment of immunoglobulin (Ig) has been shown to play an important role in the regulation of humoral immunity, cellular immunity, lymphocyte and monocyte activation, and immune mediator secretion. We wished to determine if Ig or Fc fragments would induce IL-6 production from monocytes. Incubation of monocytes purified from human peripheral blood mononuclear cells with aggregated Ig or Fc fragments of Ig induced interleukin-6 (IL-6) activity in the supernatants. Monomeric Ig taken from an intravenous preparation of Ig, from which all aggregated Ig are removed, would not induce IL-6 production from monocytes whereas as a heat-treated aliquot, presumably containing aggregates, did induce IL-6. The supernatants were assayed according to their ability to induce growth in a murine hybridoma cell line B9, or enhance Ig secretion of B cells stimulated with Staphylococcus aureus Cowan 1 (SAC). The IL-6 activity in the supernatants could be neutralized by a polyclonal rabbit anti-human IL-6 antiserum in both assays of IL-6 activity. Exposure of T-enriched or B-enriched lymphocyte subpopulations to Fc fragments did not induce the release of any IL-6 after 12 hr of incubation, but small amounts of IL-6 were produced by B-enriched cells after 60 hr of exposure to Fc fragments. Hence Fc fragments and aggregated Ig induce peripheral blood monocytes to rapidly secrete large quantities of interleukin-6.