Effects of FR-91 on immune cells from healthy individuals and from patients with non-Hodgkin lymphoma.

The immune system is subject to destruction and dysfunction as a result of attacks by pathogenic and environmental agents. In addition, many clinical situations exist in which it is desirable to stimulate or suppress the immune system. The present study evaluated the screening efficacy of flow cytometric lymphocyte subset typing in peripheral blood mononuclear cells from healthy individuals (HI) and from patients with non-Hodgkin lymphoma (NHL) treated with different concentrations of FR-91, a standardized lysate of microbial cells belonging to the Bacillus genus, and in vitro cytokine production. Increased expression of subset markers (CD3, CD4, CD8) in NHL and CD3 in HI suggests an immunomodulating effect of FR-91. In addition the results of cytokine production also demonstrated a clear effect of FR-91 on both populations. A significant increase of IL-6, IL-12, IFN-gamma and TNF-alpha was observed in the HI group after treatment with FR-91. In a similar manner an increase of IL-2, IL-6, IL-12, IFN-gamma and TNF-alpha was also observed in the NHL group. In conclusion FR-91 seems to affect lymphocyte subpopulations, in vitro cytokine production, as well as mitogen-induced lymphocyte activation in a dose-dependent manner in both healthy individuals and NHL patients.

Effects of fish-derived lipoprotein extracts on activation markers, Fas expression and apoptosis in peripheral blood lymphocytes.

Several factors may influence numbers and function of peripheral blood lymphocytes (PBLs) by different processes. We conducted this study to evaluate the effect of E-CAB-94011 and E-JUR-94013, two marine fish extracts from S. scombrus and T. trachurus, respectively, on in vitro PBLs activation and on the expression and functionality of Fas, a cell surface molecule that plays a central role in immune homeostasis and cytotoxic activity. PBLs from 24 healthy volunteers were isolated and flow cytometry was performed to measure the state of activation, Fas expression and apoptosis of PBLs. Functionality of Fas was tested by assessing apoptosis after incubation of isolated lymphocytes with agonistic anti-Fas antibodies in blood samples treated with both E-CAB-94011 and E-JUR-94013. Studies on the lymphocyte cell marker suggest a clear immune activation as measured by the increased levels of CD25, CD8, CD38, CD19 and HLA-DR in vitro expression on lymphocytes treated with both extracts. In addition, a significant reduction in the percentages of apoptotic CD19(+)CD38(+) double positive lymphocytes could be demonstrated in the treated samples with respect to controls (p<0.05). Therefore the present results indicate that both E-CAB-94011 and E-JUR-94013 in vitro are powerful immunoregulatory, increasing immune surveillance.

A pharmacogenomic approach to Alzheimer's disease.

Single nucleotide polymorphisms (susceptibility genetics) and genomic point mutations (mendelian genetics) can be used in Alzheimer's disease (AD) for diagnostic, predictive and therapeutic purposes. Using a matrix genetic model, including APOE, PS1 and PS2 allelic variants, we have studied the distribution of 36 different genotypes in the AD population (N= 479) and the genotype-related cognitive response to a multifactorial therapy in AD patients with mild-to-moderate dementia. The 10 most frequent AD genotypes are the following: 1) E33P112P2 + (17.75%), 2) E33P112P2- (15.55%), 3) E33P111P2+ (10.85%), 4) E34P112P2+ (9.60%), 5) E34P112P2- (7.56%), 6) E33P111P2- (7.10%), 7) E34P111P2+ (4.80%), 8) E33P122P2+ (4.38%), 9) E34P111P2- (4.18%), and 10) E34P122P2+ (3.55%). APOE-4/4-related genotypes represent less than 3% in the following order: E44P112P2 + > E44P111P2+ = E44P111P2- > E44P112P2+ > E44P122P2+ = E44P122P2. Multifactorial therapy with CDP-choline (1,000 mg/day) + piracetam (2,400 mg/day) + anapsos (360 mg/day) did improve mental performance during the first 6-15 months in a genotype-specific fashion. The best responders in the APOE series were patients with APOE-3/4 genotype (r= +0.013), while the worst responders were APOE-4/4 patients (r= -0.93). PS1-related genotypes responded in a similar manner; and patients with a defective PS2 gene exon 5 (PS2+) always showed a poorer therapeutic response than PS2- patients. All these data suggest that the therapeutic outcome in AD exhibits a genotype-specific pattern, and that a pharmacogenomic approach to AD might be a valuable strategy for drug development and monitoring.

Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro apoptosis in healthy and Alzheimer's Disease (AD) individuals.

In order to investigate the possibility of whether or not the lymphocytes of patients with Alzheimer's Disease (AD) are in an activated state, blood mononuclear cells from 45 AD patients and 45 healthy age matched controls were immunophenotyped by measuring the expression of CD3, CD4, CD7, CD8, CD25, CD28, CD56 and HLA-DR by flow cytometry. Circulating and in-vitro-produced cytokines were also measured by ELISA tests. CD7 and CD8 were significantly decreased in AD patients (48.3% and 18.2%, respectively) when compared to healthy subjects (63.2% and 28.3%, respectively). A significant increase in the CD4, CD25 and CD28 antigen expression was also observed in the AD group (55.3% 24.8% and 65.1%) with respect to healthy subjects (44.5%, 10.3% and 54.3%). In addition there was a significant difference in the extent of apoptosis in lymphocyte culture, as measured by mean fluorescence intensity (MFI) of Fas antigen (CD95) expression on CD4+ T cells in 6 AD patients (MFI = 36% and 43%, by anti-CD3 and hyperthermia mediated-apoptosis, respectively) with respect to 6 healthy individuals (MFI = 24% and 31%, by anti-CD3 and hyperthermia mediated-apoptosis, respectively), as well as in T-cell proliferation assay. A decline of Fas antigen expression on CD8+ subset was observed in the AD group with both stimuli (19% and 28%) comparing to the control group (29% and 39%). No differences were observed on circulating cytokines and spontaneous in vitro production of proinflammatory interleukin 1beta (IL-1beta), Tumor Necrosis Factor-alpha (TNF-alpha), IL-6 and IL-10 cytokines. Lipopolysaccharide (LPS)-stimulated in vitro production of IL-1beta, TNF-alpha, IL-6 and IL-10 measured by a whole blood culture system was significantly higher in AD patients comparing to controls. Furthermore, the observed differences were more evident at late stages of disease. These findings suggest that immunological tests, based on lymphocyte immunophenotyping combined with pro-inflammatory cytokine determinations and measurement of apoptosis in peripheral blood might represent a useful tool to obtain more insight into the pathogenesis of AD and into the level of immune activation which could characterize the pathological state of lymphocytes from individual AD patients.

Epitope specificity of anti-HIV antibodies in human and murine autoimmune diseases.

This article reports the HIV epitope specificity of antibodies present in the sera of HIV-negative patients with autoimmune diseases. Recombinant gp120 and a panel of synthetic peptides derived from the amino acid consensus sequences of either related (gp120, gp41, and p24) or unrelated (Mage-1, necdin, heat shock protein [65 kDa], and amyloid) HIV proteins were tested by a specific ELISA. The first set of experiments performed on four patients with Sjögren's syndrome (SjS) and four patients with systemic lupus erythematosus (SLE) revealed a significant anti-gp120 antibody reactivity in autoimmune patients when compared to healthy HIV-negative controls. Moreover, such binding could be almost completely inhibited by preincubation with free gp120. A significant anti-p24 reactivity was observed in 18 of 29 sera from SjS patients and in 13 of 25 sera from SLE patients, while anti-gp41 was observed only in 3 of 14 SjS and in 2 of 20 SLE-affected patients. Similar analyses were performed in the murine model of autoimmunity, showing that sera from MRL/lpr mice were able to bind all HIV-related peptides in an age-dependent manner. The analysis of a panel of HIV-unrelated peptides showed that SLE as well as MRL/lpr sera bind both HIV-related and unrelated peptides, while SjS sera failed to do so, revealing the polyclonal nature of the SLE and MRL/lpr repertoire and the oligoclonal reactivity of SjS sera. This is also supported by inhibition experiments, which showed that SLE, but not SjS, sera competitively inhibited the binding to HIV gp120 peptide of sera from autoimmune MRL/lpr mice. These results indicate that an overlapping polyclonal repertoire is present in both SLE and MRL/lpr sera, while the oligoclonal specificity of SjS antibodies may be related to a specific, nonpolyclonal, activation against putative retroviral antigens.

19/20 kDa low molecular weight serum protein pattern: a novel potential biochemical prognostic marker for different types of dementia.

In recent years, several attempts have been made to identify biochemical and/or genetic markers which might have diagnostic and prognostic uses for Alzheimer's disease (AD). To look for possible new blood markers, a longitudinal study was carried out in our Central Nervous System Biomedical Research Center between October 1996 and July 1998. A total of 30 AD subjects were diagnosed as AD patients according to the DSM-IV and NINCDS-ADRDA criteria. Vascular dementia (VD, n= 17), mixed dementia (MXD, n = 18) and healthy age-matched control subjects (n = 15) have been included in the study. Serum samples have been analysed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the resulting protein patterns have been compared. The objectives of this study were to examine the presence of peripheral markers among patients with AD, VD and MXD and to explore the relationship between serum markers and APOE genotype. The findings suggest that two proteins of 19 and 20kDa molecular weight, respectively, might be associated with disease progression in different types of dementia.

Celastrol, a potent antioxidant and anti-inflammatory drug, as a possible treatment for Alzheimer's disease.

In the brains of patients with Alzheimer's disease (AD) signs of neuronal degeneration are accompanied by markers of microglial activation, inflammation, and oxidant damage. The presence of nitrotyrosine in the cell bodies of neurons in AD suggests that peroxynitrite contributes to the pathogenesis of the disease. A drug with antioxidant and anti-inflammatory activity may prevent neuronal degeneration in AD. Celastrol, a plant-derived triterpene, has these effects. In low nanomolar concentrations celastrol was found to suppress the production by human monocytes and macrophages of the pro-inflammatory cytokines TNF-alpha and IL-1beta. Celastrol also decreased the induced expression of class II MHC molecules by microglia. In macrophage lineage cells and endothelial cells celastrol decreased induced but not constitutive NO production. Celastrol suppressed adjuvant arthritis in the rat, demonstrating in vivo anti-inflammatory activity. Low doses of celastrol administered to rats significantly improved their performance in memory, learning and psychomotor activity tests. The potent antioxidant and anti-inflammatory activities of celastrol, and its effects on cognitive functions, suggest that the drug may be useful to treat neurodegenerative diseases accompanied by inflammation, such as AD.

Cerebrolysin reduces microglial activation in vivo and in vitro: a potential mechanism of neuroprotection.

Neurotrophins, such as NGF, BDNF and NT-3 play a regulatory role on the function of microglial cells in vivo and in vitro, and the identification of new compounds with neurotrophic properties is becoming a new strategy for the prevention and/or treatment of neurodegenerative disorders. In this study we describe the use of two different models to demonstrate the ability of Cerebrolysin to reduce microglial activation. The results of these in vitro and in vivo studies indicate that Cerebrolysin might exert a neuroimmunotrophic activity reducing the extent of inflammation and accelerated neuronal death under pathological conditions such as those observed in neurodegenerative diseases.

Oral Cerebrolysin enhances brain alpha activity and improves cognitive performance in elderly control subjects.

Cerebrolysin is a porcine brain derived peptide preparation with potential neurotrophic activity. The effects of a single oral dose of the Cerebrolysin solution (30 ml) on brain bioelectrical activity and on cognitive performance were investigated in healthy elderly people. A single oral dose of Cerebrolysin induced a progressive increase in relative alpha activity power from 1 to 6 hours after treatment in almost all the brain electrodes in elderly control subjects. As compared with baseline alpha power (45.8+/-9.5%), the increase in relative alpha activity in the left occipital electrode (O1) reached significant values at 1 hour (57.2+/-8.5%; p < 0.05), 3 hours (59.4+/-7.6%; p < 0.05) and 6 hours (63.4+/-9.8%; p < 0.05) after Cerebrolysin administration. Enhancement in relative alpha power was accompanied by a generalized decrease in slow delta activity that was maximum at 6 hours after Cerebrolysin intake. A significant improvement in memory performance, evaluated with items of the ADAS cog, was also found in elderly people taken a single dose of oral Cerebrolysin (6.9+/-1.0 errors at baseline versus 4.9+/-1.0 errors after treatment; p < 0.01). This memory improvement was more evident in recognition (2.8+/-0.6 errors vs. 1.5+/-0.7 errors; p < 0.05) than in recall tasks (4.1+/-0.5 errors versus 3.4+/-0.5 errors; ns). These data indicate that Cerebrolysin potentiates brain alpha activity, reduces slow EEG delta frequencies and improves memory performance in healthy elderly humans, suggesting that this compound activates cerebral mechanisms related to attention and memory processes. According to the present results, it seems that oral Cerebrolysin might be useful for the treatment of memory impairment and brain damage in eldely subjects with or without neurodegenerative disorders.

APOE-induced microglial activation: an in vitro assay to screen sera from Alzheimer's disease patients and novel therapeutic compounds.

Recent advances in molecular genetics have suggested that genetic predisposition can be considered one of the most important risk factors for Alzheimer's disease (AD) development. A significant increase in the number of amyloid plaques in AD patients with an apolipoprotein E4 (APOE) allele has been observed and the results of several genetic studies indicate that the etiology of this neurodegenerative disease is associated with the presence of the allele E4 of APOE gene. A potential source of damage in the AD brain is an altered response triggered by microglial activation, which is associated with senile plaque formation. In this study, in vitro cell cultures were established to investigate the effect of different concentrations of human sera (2.5% and 10%) with specific APOE genotypes from Alzheimer and non-Alzheimer subjects on ameboid and flat microglial cells obtained from adult rat hippocampus. Results show that this in vitro test can be applied as an in vitro model to test specific responses of microglia to human sera as well as a primary screening procedure to evaluate the effect of novel compounds for the treatment of AD and neuroimmune-associated disorders.

An experimental model to study the cytotoxic effects induced by beta-amyloid, histamine, LPS and serum from Alzheimer patients on cultured rat endothelial cells.

Lactate dehydrogenase (LDH) enzyme activity was measured in the supernatant of rat aortic endothelial cell cultures to evaluate the cytotoxic effects of two proinflammatory mediators such as LPS and histamine, as well as beta-amyloid protein (fragment 1-28) on endothelial cells. In the same culture we also studied the influence of different sera from patients with Alzheimer's disease (AD) or healthy elderly subjects. The results indicate that very low concentrations (1 microgram/ml) of beta-amyloid or histamine (1 microM) were able to produce cell damage after an incubation time of 4 h. Treatment with LPS induced cell damage at the highest concentration (2.5 micrograms/ml) after 24 h of incubation. When endothelial cells were treated with serum from AD and non-AD individuals, an inhibition of endothelial cell proliferation was observed only with activated AD serum. These results indicate that rat endothelial cell cultures represent an important model to study inflammatory mediators and to evaluate the therapeutic effect of antiinflammatory molecules in AD and other neurodegenerative disorders.

Effects of Cerebrolysin on in vitro primary microglial and astrocyte rat cell cultures.

In recent years the potential use of neurotrophic factors in the prevention and/or treatment of neurodegenerative diseases has received much attention. To determine whether Cerebrolysin, a porcine brain-derived peptide preparation, was able to modulate in vitro lipopolysaccharide (LPS)-induced microglial activation and to test the direct effect of Cerebrolysin on astrocyte morphology, survival and proliferation, rat glial and astrocyte cell culture experiments were carried out. The morphology of microglia, ameboid/activated and flat/resting, was examined under contrast microscopy and cell counts obtained. In addition, the release of interleukin (IL)-1 beta and brain-derived neurotrophic factor (BDNF) was measured from cell culture supernatant using an enzyme-linked-immunoassay (ELISA). The results obtained in this study clearly suggest a protective effect of Cerebrolysin as revealed by downregulation of microglial activation after LPS treatment as well as by the control of IL-1 beta expression. No significant differences were observed on astrocyte morphology, survival or the production and/or release of BDNF. In conclusion, these in vitro studies indicate that Cerebrolysin might exert a neuroimmunotrophic function which can in turn reduce the extent of inflammation and accelerate neuronal death under pathological conditions such as human neurodegenerative disorders.

Association between APOE epsilon4 allele and increased expression of CD95 on T cells from patients with Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, characterized by progressive impairment of cognitive functions. AD has a strong and complex genetic etiology, and multiple genes, acting independently and/or interacting, likely to influence the risk of developing AD. To test whether the expression of Fas receptor is upregulated in peripheral blood T lymphocytes and whether or not it correlates with APOE genotypes, 88 patients with AD and 24 normal individuals as controls were included in this study. T lymphocytes from patients as opposed to controls did undergo DNA fragmentation after in vitro exposure to IgM anti-Fas. In addition, several activation markers (CD25, HLA-DR, and CD45R0) were increased after 72 h in culture with respect to the controls, and Fas expression was also significantly different from the control group (p < 0.01). Reverse transcription PCR for Fas mRNA yielded the same results. T cells from both patients and controls showed upregulation of Fas receptor expression after in vitro anti-CD3 stimulation. Co-culture experiments with interleukin-4 downmodulated surface Fas receptor expression on T cells from patients and at a lesser extent in the control group. AD patients with the APOE allele 4 showed an increased expression of CD95 (53% +/- 6) with respect to APOE allele 3 (38% +/- 4). The control group showed a 22% +/- 3 (allele 4) and 31% +/- 5 (allele 3), respectively. Hyperexpression of Fas mRNA and surface Fas receptor on CD45RO(+) T lymphocytes may explain the occurrence of inflammatory cellular infiltrates in the CNS of AD patients.

Enhancement in immune function and growth using E-JUR-94013 supplementation.

Animal studies suggest that fish oils are capable of modulating the cell functions of immune system and there is some evidence that the effects of fish oils on immune function are due to fatty acids rather than trace elements or antioxidants. The major objectives of this study were: i) to identify a fish species with high nutritional value able to improve pig feeding conditions; ii) to utilize diets that modulate the immune system early in life in pigs and; iii) to enhance growth rate on a physiological basis. With the aim of maximizing feeding intake after weaning in order to reduce stress and increase growth rate, a study was carried out on 300 pigs supplemented with different fish extracts obtained by advanced biotechnological methods. The results of this work suggest that the lipoproteins obtained from the Trachurus trachurus (E-JUR-94013) species may have a great effect as both an immunomodulating compound (acting mainly on the regulation of IgA synthesis and/or release) and as a hypocholesterolemic compound, reducing the total cholesterol level in the serum of treated pigs. Both effects resulted in better pig growth, demonstrating that E-JUR-94013 can also be used as a natural growth promoter and an immune enhancer.

Microglial activation induced by factor(s) contained in sera from Alzheimer-related ApoE genotypes.

Several factors that increase the likelihood of developing Alzheimer's disease (AD) have already been identified. A correct evaluation of these may contribute to a better understanding of the etiology of the disease. The risk of developing AD definitely increases with (a) age, (b) head injuries, (c) family history of AD or Down syndrome, (d) sex (higher prevalence of AD in women), (e) vascular disease, (f) exposure to environmental toxins, (g) infectious processes, or (h) changes in immune function, and recent advances in molecular genetics have suggested that genetic predisposition (i) can be considered one of the most important risk factors in the development of AD. A significant increase in the number of amyloid plaques in AD patients with an apolipoprotein E4 (ApoE) allele has been observed and the results of several genetic studies indicate that the etiology of this neurodegenerative disease is associated with the presence of the allele E4 of ApoE. A potential source of damage in the AD brain is an altered response triggered by microglial activation, which is associated with amyloid plaques. It has become evident that a dysregulation of cytokine release appears within lesions of many types of brain disorders including infection, trauma, stroke, and neurodegenerative diseases. Many studies have shown that microglia secrete both cytokines and cytotoxins and since reactive microglia appears in nearly every type of brain damage, it is likely that their secreted products ultimately help to determine the rate of damaged brain tissue. In this study, in vitro cell cultures were established to investigate the effect of different concentrations of human sera (2.5% and 10%) with specific ApoE genotypes from Alzheimer's and non-Alzheimer's subjects on ameboid and flat microglial cells obtained from neonatal rat hippocampi. Results show that a modulation in the proliferation and activation of microglial cells was obtained and that AD sera, mainly in the ApoE 3/4 and 4/4 genotype contain factor(s) which are able to induce morphological changes, as measured by an increase in the ameboid cell type. In addition, major histocompatibility complex (MHC) class II antigen expression, as measured by flow cytometric analysis, and interleukin-1beta (IL-1beta) release as measured by enzyme linked immunoadsorbent assay (ELISA), in comparison with control groups and lipopolysaccharide (LPS)-treated cells, clearly demonstrate a direct effect of ApoE 3/4 and 4/4 and/or an indirect effect mediated by the release of IL-1beta on microglia activation. These results strongly suggest that primary in vitro microglial cell cultures can be used as a screening model to test human sera as well as the effect of new potential drugs aimed at down-regulating microglia activation.

Distinct appearance of differentiation markers in HL60 cell line treated with 1,25 dihydroxyvitamin D3 and phorbol esters (TPA).

Peripheral blood monocytes are comprised of a heterogeneous population of cells with respect to phenotype and functional activity. In this investigation we confirm the presence of two distinct subsets of peripheral blood adherent monocytes with respect to MHC class II antigens HLA DR and HLA DQ using two color FACS analysis. In the attempt to mimic the in vivo situation promyelocytic cells lines have been established which can be induced to undergo characteristic monocytic differentiation in response to a variety of agents. The HL 60 cell line can be induced in vitro by phorbol esters (TPA) to display membrane HLA DR and HLA DQ antigens along with the mature monocytic marker Leu M3. Physiological concentrations of 1,25 dihydroxy Vitamin D3 leads to the expression of HLA DR and Leu M3 antigens but not HLA DQ. These data suggest that these inducers may cause the differentiation of phenotypically distinct monocytic subpopulations.

Immunological studies in patients with central nervous system tumors.

Impairment of cell-mediated immunity in patients with primary central nervous system (CNS) tumors has repeatedly been reported but data to demonstrate the underlying immunological defect are not univocal. This report concerns a series of 31 patients harboring a glioma in which we studied: peripheral blood T-lymphocyte subpopulations by monoclonal antibody analysis; cellular responsiveness to mitogens; serum immunoglobulin values. The same parameters were also evaluated in 7 cases of intracranial meningioma and in 8 patients affected by non-proliferative, non-inflammatory CNS diseases. Thirty age-matched healthy volunteers formed the control group. Neither impairment of T-cells as regard to number, responsiveness and subsets, nor abnormal Ig values were found in these groups. However two patients, harboring respectively a third ventricle low grade astrocytoma and an anterior callosal glioblastoma, presented a striking T-cells impairment. These findings might suggest a correlation between hypothalamus and immune system, as already postulated by several previous experimental and clinical studies.