Microglia and astrocyte activation in the frontal cortex of rats with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for the human disease multiple sclerosis (MS), a demyelinating and neurodegenerative pathology of the central nervous system. Both diseases share physiopathological and clinical characteristics, mainly associated with a neuroinflammatory process that leads to a set of motor, sensory, and cognitive symptoms. In MS, gray matter atrophy is related to the emergence of cognitive deficits and contributes to clinical progression. In particular, injury and dysfunction in certain areas of the frontal cortex (FrCx) have been related to the development of cognitive impairments with high incidence, like central fatigue and executive dysfunction. In the present work we show the presence of region-specific microglia and astrocyte activation in the FrCx, during the first hours of acute EAE onset. It is accompanied by the production of the pro-inflammatory cytokines IL-6 and TNF-α, in the absence of detectable leukocyte infiltration. These findings expand previous studies showing presynaptic neural dysfunction occurring at the FrCx and might contribute to the understanding of the mechanisms involved in the genesis and prevalence of common MS symptoms.

Treatment with a hybrid between the synapsin ABC domains and the B subunit of E. coli heat-labile toxin reduces frequency of proinflammatory cells and cytokines in the central nervous system of rats with EAE.

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are crucially dependent on the invasion of activated autoreactive lymphocytes and blood macrophages into the central nervous system (CNS). Proinflammatory mononuclear cells and activated local microglia mediate inflammation, demyelination and axonal damage at the target organ. Previously, we observed that the administration of a hybrid between the synapsin ABC domains and the B subunit of Escherichia coli heat labile-enterotoxin (LTBABC) to rats with EAE ameliorated disease by modulating the peripheral Th1 response to myelin basic protein (MBP). In the present study, we investigated the effect of LTBABC administration on proinflammatory cell frequency in the CNS of rats with EAE. Treatment with the hybrid in the inductive phase of EAE attenuated disease severity and diminished histological inflammatory infiltrates and demyelination in the spinal cord of rats with acute EAE. Lower frequencies of infiltrating and local macrophages as well as CD4+ T cells that produce the proinflammatory cytokines interferon-gamma (IFN-γ) and interleukin (IL)-17 were found at the target organ. Concomitantly, low levels of INF-γ and IL-17 and increased levels of IL-10 were measured in cultures of CNS infiltrating cells and spinal cord tissue. An increased frequency of CD4+CD25+Foxp3 cells was observed at the disease peak and at the beginning of the recovery stage. These results provide further evidence for the immunomodulatory properties of the fusion protein LTBABC in autoimmune demyelinating disease affecting the central nervous system.

Ventilation during cardiopulmonary bypass: impact on heat shock protein release.

AIM: Cardiopulmonary bypass (CPB), utilized in on-pump coronary artery bypass graft procedures (CABG) induces generalized immune suppression, release of heat shock proteins (HSP), inflammatory markers and apoptosis-specific proteins. We hypothesized that continued mechanical ventilation during cardiopulmonary bypass attenuates immune response and HSP liberation. METHODS: Thirty patients undergoing conventional coronary artery bypass graft (CABG) operation were randomized into a ventilated on CPB (VG; N.=15) and a non-ventilated CPB group (NVG; N.=15). Blood samples were drawn at the beginning and end of surgery, as well as on the five consecutive postoperative days (POD). Molecular markers were measured by ELISA. Data are given as mean ± (SD). Mann-Whitney-U-test was used for statistical analysis. RESULTS: Serum concentrations of HSP70 were significantly lower in VG compared to NVG on POD-1 (VG: 1629±608 vs. NVG: 5203±2128.6 pg/mL, P<0.001). HSP27 and HSP60 depicted a minor increase in both study groups at the end of surgery without any intergroup differences (HSP27: VG 6207.9±1252.5 vs. NVG 7424.1±2632.5; HSP60: VG 1046.2±478.8 vs. NVG 1223.5±510.1). IL-8 and CK-18 M30 evidenced the highest serum concentrations at the end of surgery (IL-8: VG 119.5±77.9 vs. NVG 148.0±184.55; CK-18 M30: VG 62.1±39.2 vs. NVG 67.5±33.9) with no differences between groups. Decreased ICAM-1 serum concentrations were detected postoperatively, however ICAM-1 concentrations on POD-1 to POD-5 showed slightly elevated concentrations in both study groups with no intergroup differences. CONCLUSION: Significantly less HSP70 was detectable in patients receiving uninterrupted mechanical lung ventilation on CPB, indicating either different inflammatory response, cellular stress or cell damage between the ventilated and non-ventilated group. These data suggest that continued mechanical ventilation has a modulatory effect on the immune response in patients after CABG surgery.

Molecular mechanisms involved in interleukin 1-beta (IL-1ß)-induced memory impairment. Modulation by alpha-melanocyte-stimulating hormone (α-MSH).

Pro-inflammatory cytokines can affect cognitive processes such as learning and memory. Particularly, interleukin-1ß (IL-1ß) influences the consolidation of hippocampus-dependent memories. We previously reported that administration of IL-1ß in dorsal hippocampus impaired contextual fear memory consolidation. Different mechanisms have been implicated in the action of IL-1ß on long-term potentiation (LTP), but the processes by which this inhibition occurs in vivo remain to be elucidated. We herein report that intrahippocampal injection of IL-1ß induced a significant increase in p38 phosphorylation after contextual fear conditioning. Also, treatment with SB203580, an inhibitor of p38, reversed impairment induced by IL-1ß on conditioned fear behavior, indicating that this MAPK would be involved in the effect of the cytokine. We also showed that IL-1ß administration produced a decrease in glutamate release from dorsal hippocampus synaptosomes and that treatment with SB203580 partially reversed this effect. Our results indicated that IL-1ß-induced impairment in memory consolidation could be mediated by a decrease in glutamate release. This hypothesis is sustained by the fact that treatment with d-cycloserine (DCS), a partial agonist of the NMDA receptor, reversed the effect of IL-1ß on contextual fear memory. Furthermore, we demonstrated that IL-1ß produced a temporal delay in ERK phosphorylation and that DCS administration reversed this effect. We also observed that intrahippocampal injection of IL-1ß decreased BDNF expression after contextual fear conditioning. We previously demonstrated that α-MSH reversed the detrimental effect of IL-1ß on memory consolidation. The present results demonstrate that α-MSH administration did not modify the decrease in glutamate release induced by IL-1ß. However, intrahippocampal injection of α-MSH prevented the effect on ERK phosphorylation and BDNF expression induced by IL-1ß after contextual fear conditioning. Therefore, in the present study we determine possible molecular mechanisms involved in the impairment induced by IL-1ß on fear memory consolidation. We also established how this effect could be modulated by α-MSH.

Lipocalin-2 serum levels are increased in acute hepatic failure.

Lipocalin-2 (LCN-2), which is expressed in immunocytes as well as hepatocytes, is upregulated in cells under stress from infection or inflammation with increase in serum levels. We sought to investigate the relevance of LCN-2 in the setting of acute hepatic failure, particularly when addressed with the molecular adsorbent recirculating system (MARS). We measured serum LCN-2 concentrations with enzyme-linked immunosorbent assay (ELISA) in 8 patients with acute-on-chronic-liver failure (ACLF) and acute liver failure (ALF) who were treated with MARS. The controls were 14 patients with stable chronic hepatic failure (CHF). LCN-2 was determined immediately before and after the first MARS session. Baseline LCN-2 serum concentrations were significantly increased among ACLF and ALF patients as compared with CHF (P = .004 and P = .0086, respectively). There was no significant difference between the ALF and ACLF group. Moreover, serum LCN-2 levels did not change significantly during the MARS treatment. Serum LCN-2 levels, therefore, may be useful to discern acute from chronic hepatic failure and to monitor the course as well as the severity of the disease.

The effect of the molecular adsorbent recirculating system on moxifloxacin and meropenem plasma levels.

BACKGROUND: Adequate plasma antibiotic concentrations are necessary for effective elimination of invading microorganism; however, extracorporeal organ support systems are well known to alter plasma concentrations of antibiotics, requiring dose adjustments to achieve effective minimal inhibitory concentrations in the patient's blood. METHODS: A mock molecular adsorbent recirculating system (MARS) circuit was set using 5000 ml of bovine heparinized whole blood to simulate an 8-h MARS treatment session. After the loading dose of 400 mg of moxifloxacin or 2 g of meropenem had been added, blood was drawn from the different parts of the MARS circuit at various time points and analyzed by high-performance liquid chromatography. The experiments were performed in triplicate. Additionally, meropenem concentrations were determined in the plasma of one patient treated with MARS suffering from acute liver failure due to an idiosyncratic reaction to immunosuppressive medication. RESULTS: In our single-compartment model, a significant decrease in the quasi-systemic concentration of moxifloxacin and meropenem could be detected as early as 15 min after the commencing of the MARS circuit. Moreover, within 60 min the moxifloxacin and meropenem concentrations were less than 50% of the initial value. The activated charcoal removed the majority of moxifloxacin and meropenem in the albumin circuit. In our patient, the meropenem concentrations in the return line after MARS were constantly lower than in the access line, indicating a likely removal of meropenem through MARS. CONCLUSION: Our data provide evidence that moxifloxacin and meropenem are effectively removed from the patient's blood by MARS, leading to low plasma levels. Dose adjustments of both antibiotic compounds may be required.

Serum 20S proteasome is elevated in patients with renal cell carcinoma and associated with poor prognosis.

BACKGROUND: To date, no reliable serum marker for clear cell renal cell carcinoma (CCRCC) is available. The aim of this study was to evaluate the putative significance of circulating 20S proteasome levels. METHODS: Preoperative 20S proteasome serum levels were determined in 113 CCRCC patients and 15 healthy controls by a sandwich enzyme-linked immunosorbent assay. Associations with CCRCC, pathological variables, disease-specific survival (DSS), and response to sunitinib were evaluated. RESULTS: Median 20S proteasome levels were higher in CCRCC patients than in healthy controls (4.66 vs 1.52 µg ml(-1), P<0.0001). The area under the receiver operating characteristics curve curve was 87.1%. The 20S proteasome levels were associated with symptoms (P=0.0008), distant metastases (P=0.0011), grade (P=0.0247), and necrosis (P=0.0462). The 20S proteasome levels were identified as a prognostic factor for DSS in both univariable (hazards ratio 1.21, P<0.001) and multivariable (hazards ratio 1.17, P=0.0015) survival analysis. In patients responding to sunitinib, 20S proteasome levels were lower than in patients with stable disease and progressive disease. CONCLUSION: This study demonstrates for the first time that increased 20S proteasome levels are associated with CCRCC, advanced disease, and poor prognosis. Routine use of this marker may allow better diagnosis, risk stratification, risk-adjusted follow-up, and identification of patients with a greater likelihood of response to targeted therapy.

Inhibitory role of diazepam on autoimmune inflammation in rats with experimental autoimmune encephalomyelitis.

Glutamate and GABA are the main excitatory and inhibitory neurotransmitters in the CNS, and both may be involved in the neuronal dysfunction in neurodegenerative conditions. We have recently found that glutamate release was decreased in isolated synaptosomes from the rat cerebral cortex during the development of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. In contrast to control animals where GABA induced a decrease in the evoked glutamate release, which was abolished by picrotoxin (a GABA(A) antagonist), synaptosomes from EAE rats showed a loss in the inhibition of the glutamate release mediated by GABA with a concomitant diminution of the flunitrazepam-sensitive GABA(A) receptor density. We have presently further evaluated the relevance of the GABAergic system in EAE by treating rats challenged for the disease with the GABA agonist diazepam. Administration of diazepam during 6 days starting at day 6 or 11 after EAE active induction led to a marked decrease of the disease incidence and histological signs associated with the disease. Cellular reactivity and antibody responses against the encephalitogenic myelin basic protein were also diminished. Beyond the effects of diazepam on the autoimmune, inflammatory response, we report also a positive effect on neurotransmission. Treatment with diazepam inhibited the previously described reduction in glutamate release in the frontal cortex synaptosomes from EAE animals. These data suggest that an endogenous inhibitory GABAergic system within the immune system is involved in the diazepam effect on EAE and indicate that increasing GABAergic activity potently ameliorates EAE.

Participation of the GABAergic system on the glutamate release of frontal cortex synaptosomes from Wistar rats with experimental autoimmune encephalomyelitis.

We previously found that the glutamate release was decreased in synaptosomes from rat cerebral cortex during the development of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Various other reports have shown a deficit in the expression of proteins associated with GABAergic neurotransmission in the neocortex of patients with multiple sclerosis and it was also demonstrated that the activation of GABAA receptors leads to an inhibition of glutamate release. Now, in order to evaluate the events that may affect the neuronal function in EAE synaptosomes, we analyzed the participation of the GABAergic system in glutamate release and in the flunitrazepam-sensitive GABAA receptor density. This revealed alterations in the GABAergic system of the frontal cortex synaptosomes from EAE animals. GABA induced a decrease in the 4-aminopyridine-evoked glutamate release in control synaptosomes which was abolished by picrotoxin, a GABAA receptor antagonist. In contrast, synaptosomes from EAE rats showed a loss in the inhibition of glutamate release mediated by GABA. Furthermore, the flunitrazepam-sensitive GABAA receptor density was decreased during the acute stage of the disease in synaptosomes from EAE rats. We also observed a loss of inhibition in the Ca2+-dependent phosphorylation of synapsin I mediated by GABA in nerve terminals from EAE animals, which could explain the loss of GABAergic regulation on evoked glutamate release. The changes observed in the GABAA receptor density as well as the loss of GABAergic inhibition of glutamate release were partially reverted in cortical synaptosomes from recovered EAE animals. These results suggest that the decrease in the flunitrazepam-sensitive GABAA receptor density may explain the observed failure of GABAergic regulation in the glutamate release of synaptosomes from EAE rats, which might contribute to the appearance of clinical symptoms and disease progression.

Enhanced monocyte migration and pro-inflammatory cytokine production by Porphyromonas gingivalis infection.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis, a major periodontal pathogen, has been reported to be involved in atherogenesis. In order to further understand this pathogen's link with systemic inflammation and vascular disease, we investigated its influence on murine monocytes and macrophages from three different sources. MATERIAL AND METHODS: Concanavalin A-elicited peritoneal macrophages, peripheral blood monocyte-derived macrophages and WEHI 274.1 monocytes were infected with either P. gingivalis 381 or its non-invasive fimbriae-deficient mutant, DPG3. RESULTS: Infection with P. gingivalis 381 markedly induced monocyte migration and significantly enhanced production of the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-6. Consistent with a role for this pathogen's major fimbriae and/or its invasive capacity, infection with DPG3 had a minimal effect on both monocyte attraction and pro-inflammatory cytokine production. CONCLUSION: Since monocyte recruitment and activation are important steps in the development of vascular inflammation and atherosclerosis, these results suggest that P. gingivalis infection may be involved in these processes.

Nucleosome serum levels in acute hepatic failure and MARS treatment.

Serum nucleosomes have been suggested to be markers for cell death and apoptosis. Increased hepatocyte apoptosis can be demonstrated in acute liver failure (ALF) as well as acute-on-chronic liver failure (ACLF). We investigated the relevance of nucleosomes in the setting of acute hepatic failure. Further, we studied the effects of the molecular adsorbent recirculating system (MARS) on this marker of cell death. We measured serum nucleosome concentrations with ELISA in 12 patients with ACLF and 7 patients suffering from ALF, with 14 patients experiencing stable chronic hepatic failure (CHF) as controls. In a subset of 8 ACLF and ALF patients treated with MARS, nucleosomes were determined immediately before and after the first MARS session. Baseline nucleosome serum concentrations were significantly increased in ACLF and ALF patients as compared with CHF patients (P = .0161 and P = .0037, respectively). There was no significant difference between the ALF and ACLF groups. Moreover, serum nucleosome levels did not change significantly during MARS treatment in ALF and ACLF patients. Serum nucleosome levels therefore may be useful to discern acute from chronic hepatic failure or to monitor the course and the severity of the disease. Our results, however, warrant further larger clinical studies regarding the clearance of nucleosome in artificial liver-assist devices and to assess their role in acute hepatic failure.

Pro-inflammatory interleukin-18 and Caspase-1 serum levels in liver failure are unaffected by MARS treatment.

BACKGROUND: The pro-inflammatory cytokine IL-18 and its activator Caspase-1 are involved in acute liver failure and acute-on-chronic-liver-failure. In acute liver failure and acute-on-chronic-liver-failure, the MARS system has been used to support liver function. Enhancement of IL-18, as seen in other extracorporeal-support systems like hemodialysis might thus have mitigated beneficial effects of the MARS system in acute hepatic failure. PATIENTS AND METHODS: We measured serum concentrations of IL-18 and Caspase-1 in 10 patients with acute liver failure and 10 patients suffering from acute-on-chronic-liver-failure, who were all treated with MARS. Thirteen patients suffering from chronic hepatic failure and 15 healthy individuals served as controls. Data are given as mean with 95% CI. RESULTS: Baseline IL-18 serum concentrations were significantly increased in acute liver failure and acute-on-chronic-liver-failure patients as compared to chronic hepatic failure (P=0.0039 and P=0.0011, respectively) and controls (P=0.0028 and P=0.0014, respectively). Caspase-1 serum concentrations were as well significantly elevated in the acute liver failure and acute-on-chronic-liver-failure groups as compared to chronic hepatic failure patients (P=0.0039 and P=0.0232, respectively) and controls P<0.0001 and P<0.0007, respectively). IL-18 and Caspase-1 did not change significantly during MARS treatment in acute liver failure and acute-on-chronic-liver-failure patients. CONCLUSIONS: MARS had no effect on IL-18 and Caspase-1 serum concentrations in acute liver failure and acute-on-chronic-liver-failure, providing no evidence of harmful effects by the increase of these potentially hepatocidal cytokines.

Effects of periodontal therapy on serum C-reactive protein, sE-selectin, and tumor necrosis factor-alpha secretion by peripheral blood-derived macrophages in diabetes. A pilot study.

BACKGROUND AND OBJECTIVE: Diabetes is associated with an increased risk for vascular disease and periodontitis. The aim of this study was to assess the effects of periodontal treatment in diabetes with respect to alterations in the pro-inflammatory potential of peripheral blood mononuclear cells. MATERIAL AND METHODS: Ten patients with diabetes and moderate to severe periodontitis received full-mouth subgingival debridement. Blood samples for serum/plasma and mononuclear cell isolation were collected prior to and 4 wk after therapy. Mononuclear cells were analyzed by flow cytometry and stimulated with lipopolysaccharide or ionomycin/phorbol ester to determine the pro-inflammatory capacity of macrophages and lymphocytes, respectively. RESULTS: Following periodontal treatment, all patients demonstrated a significant improvement in clinical periodontal status (p < 0.05), despite only modest reduction in subgingival bacterial load or homologous serum immunoglobulin G titers. CD14(+) blood monocytes decreased by 47% (p < 0.05), and the percentage of macrophages spontaneously releasing tumor necrosis factor-alpha decreased by 78% (p < 0.05). There were no significant changes in the capacity of lymphocytes to secrete interferon-gamma. Among a number of serum inflammatory markers tested, high-sensitivity-C-reactive protein significantly decreased by 37% (p < 0.01) and soluble E-selectin decreased by 16.6% (p < 0.05). CONCLUSION: These data suggest a reduced tendency for monocyte/macrophage-driven inflammation with periodontal therapy and a potential impact on atherosclerosis-related complications in diabetic individuals.

Infection with a periodontal pathogen induces procoagulant effects in human aortic endothelial cells.

BACKGROUND: Multiple studies have demonstrated a link between periodontal infections and vascular disease. Porphyromonas gingivalis, a major periodontal pathogen, has been shown to adhere to and invade endothelial cells. OBJECTIVE: In order to dissect mechanisms underlying these observations, we assessed the role of P. gingivalis infection in modulating properties of endothelial cells linked to atherothrombosis. METHODS: Primary human aortic endothelial cells (HAEC) were infected with either P. gingivalis 381 or its non-invasive fimbriae-deficient mutant, DPG3. Markers of coagulation and thrombosis were assessed 8 h and 18 h postinfection in cell lysates and supernatants. RESULTS: Infection with P. gingivalis 381 significantly enhanced tissue factor expression and activity, and suppressed levels of tissue factor pathway inhibitor. Furthermore, P. gingivalis infection decreased levels and activity of tissue plasminogen activator, and enhanced plasminogen activator inhibitor-1 antigen and activity. Consistent with an important role for bacterial adhesion/invasion in this setting, infection with DPG3 failed to induce procoagulant properties in HAEC. Most of the above effects of P. gingivalis 381 were more apparent at the later time point (18 h postinfection). This suggests that P. gingivalis infection, rather than having an immediate and direct effect, might activate pathways that, in turn, trigger endothelial procoagulant mechanisms. CONCLUSIONS: Taken together these data demonstrate for the first time that infection with a periodontal pathogen induces procoagulant responses in HAEC.

Heightened levels of circulating 20S proteasome in critically ill patients.

BACKGROUND: Recently, circulating proteasome core particles (20S proteasome) have been suggested as a marker of cell damage and immunological activity in autoimmune diseases. Aberrant leucocyte activation and increased lymphocyte apoptosis with consecutive T-cell unresponsiveness is deemed to play a pivotal role in the sepsis syndrome. Moreover sepsis-induced muscle proteolysis mainly reflects ubiqutin proteasome-dependent protein degradation. We therefore sought to investigate serum levels of 20S proteasome in critical ill patients. MATERIAL AND METHODS: Case-control-study at a university hospital intensive care unit; 15 patients recruited within 24-48 h of diagnosis of sepsis, 13 trauma patients recruited within 24 h of admission to the ICU, a control group of 15 patients who underwent abdominal surgery, and 15 healthy volunteers. ELISA was used to measure the concentration of 20S proteasome in the sera of the patients and controls. Data are given as mean +/- SEM. Mann-Whitney U-test was used to calculate significance and a P-value of 0.05 was considered to be statistically significant. RESULTS: Marked increase of 20S proteasome was detected in the sera of septic patients (33 551 +/- 10 034 ng mL-1) as well as in trauma patients (29 669 +/- 5750 ng mL-1). In contrast, significantly lower concentrations were found in the abdominal surgery group (4661 +/- 1767 ng mL-1) and in the healthy control population (2157 +/- 273 ng mL-1). CONCLUSION: Detection of 20S proteasome may represent a novel marker of immunological activity and muscle degradation in sepsis and trauma patients, and may be useful in monitoring the clinical effect of proteasome-inhibitors.

Time course of IgM antibodies which block anti-myelin basic protein IgG antibodies associated with development of experimental autoimmune encephalomyelitis in rabbits.

Several authors have demonstrated the presence in normal sera of antibodies that inhibit binding of a variety of autoantibodies. These inhibitory or blocking antibodies are generally considered to play a role in humoral self-tolerance. We examined sera from normal rabbits and from rabbits with experimental autoimmune encephalomyelitis (EAE), in search for antibodies capable to inhibit reactivity of autoantibodies directed to myelin basic protein (MBP). Rabbits injected with bovine myelin in complete Freund's adjuvant (EAE rabbits) or with adjuvant alone (control rabbits) were bled at various intervals post-injection. Sera were subjected to chomatography on a protein A-Sepharose column, retained and nonretained fractions were collected, and ability of these fractions to block reactivity of affinity-purified anti-MBP IgG-antibodies was analyzed by immunoblot technique. Protein A nonretained fraction from control rabbits inhibited anti-MBP IgG reactivity to the same degree at all intervals tested, whereas the same fraction from EAE animals showed an increase in inhibitory activity after induction of the disease. This inhibitory activity declined with the onset of clinical symptoms, and remained low in rabbits that did not recover from the disease. In contrast, the inhibitory activity remained at maximum value in EAE rabbits with spontaneous remission of clinical symptoms. We showed that the inhibitory activity is due to IgM-antibodies, and discussed the role of these antibodies in the development of EAE.

Passive transfer of experimental autoimmune encephalomyelitis in Wistar rats: dissociation of clinical symptoms and biochemical alterations.

We have used passive transfer of myelin-reactive lymphocytes in the Wistar rat model of experimental autoimmune encephalomyelitis (EAE) to investigate the nature of the central nervous system immunopathological alterations induced by these cells. Mononuclear cells from lymph nodes or spleen from sick myelin/complete Freund's adjuvant-immunized donors did not transfer clinical disease. However, depending on the previous treatment of the transferred cells, recipients develop central nervous system biochemical and histological alterations. Fresh cells from lymph nodes immediately transferred after procurement from the sick EAE donor rat were capable of inducing the most significant diminution in the content of myelin basic protein, sulfatides, and 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, with concomitant inflammatory infiltrations of white matter, principally in spinal cord and cerebellar lobules. Similar alterations were observed when animals were injected with spleen mononuclear cells activated in the presence of a nonspecific mitogen as concanavalin A. However, antigen-specific activated spleen cells generated by culturing in the presence of bovine myelin induced alterations to a lesser degree. Results point to a dissociation of the clinical disease from the central nervous system biochemical and histopathological lesions occurring in the EAE-transferred Wistar rats and indicate that these alterations in EAE are induced principally by T cells activated in vivo rather than by cells activated in vitro by myelin antigens. Therefore, these findings suggest a possible participation of lymphocytes unlike the encephalitogenic T cells in the induction of the described alterations and provide a useful model to explore further the subclinical responses to this experimental disease.

Humoral response against myelin antigens in two strains of rats with different susceptibility to experimental allergic encephalomyelitis (EAE).

Lewis (Lw) rats are susceptible and Wistar (Wr) rats are usually resistant to the induction of experimental allergic encephalomyelitis (EAE). In this study we analyze the humoral response to myelin antigens, providing evidence for different B cell response to myelin basic protein (MBP) and other myelin proteins between these two strains of rats with different susceptibility to EAE. In fact, IgG anti-MBP titers in Wr rats were markedly higher than in Lw ones. Moreover, an inverse relationship between the amount of antigen injected to induced EAE and the level of anti-MBP antibodies was observed in Wr rats, while IgG anti-MBP varied in a positive dose-depending manner in sera from Lw rats. Also, sera from Wr rats analyzed by immunoblotting showed a strong reactivity with MBP and other myelin proteins, but sera from Lw rats reacted only with MBP. Evaluation of IgA and IgM against MBP in Wr rats showed again higher titers of these isotypes when compared with the titers observed in Lw rats. The distribution of IgG subclasses in sera from both strains indicated that Wr produced low titers of specific IgG1, while Lw rats did not produce specific IgG1. However, Wr rats showed high levels of IgG2a, IgG2b and IgG2c subclasses while lesser titers of these isotypes were observed in Lw animals. These findings indicate that both strains have the capacity to develop antibodies against portions of the MBP molecule, but antibody production is greater in the resistant Wistar rats suggesting a B cell activation in these animals, that could be related to their lower susceptibility.

Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: relationship with ABH blood group determinants.

The capacity of cholera toxin (CT) and type I heat-labile enterotoxin produced by Escherichia coli isolated from human intestine (LTh) to interact with glycoconjugates bearing ABH blood group determinants from rabbit intestinal brush border membranes (BBM) was studied. On the basis of the type of intestinal compounds related to the human ABH blood group antigens, rabbits were classified as AB or H. Toxin binding to the intestinal glycolipids and glycoproteins depends on the blood group determinant borne by the glycoconjugate and on the analyzed toxin. LTh was capable of interacting preferentially with several blood group A- and B-active BBM glycolipids compared to those isolated from animals lacking these antigens (H rabbits). Also, LTh preferably bound to several BBM glycoproteins from AB rabbit intestines compared to those from H ones. One of these glycoproteins, the sucrase-isomaltase complex (EC 3.2.1.48-10) isolated from AB and H rabbits showed the same differential LTh binding. Conversely, CT practically did not recognize either blood group A-, B-, or H-active glycolipids and glycoproteins. These results may be relevant for carrying out in vivo experiments in rabbits in order to disclose the role of ABH active-glycoconjugates in the secretory response induced by LTh in rabbit intestine.