Statins and the brain: New perspective for old drugs.

Statins are one of the most popular lipid-lowering drugs (LLDs). Upon oral administration, these drugs are well absorbed by the intestine and effectively used for the treatment of dyslipidemias. Recently, statins are becoming also well-known for their cholesterol-independent effects and their potential use in brain diseases and different types of cancers. While still controversial, recent research has suggested that statin's cholesterol-independent activities work possibly through alterations on isoprenoid levels. This reduction of isoprenoids in the central nervous system might result in effective biochemical and behavioral improvements on certain neurological disorders. This manuscript aims to highlight current research describing the use of statin therapy in the brain and discuss whether statins might affect neuronal dynamics and function independently of their cholesterol regulatory role.

Impaired cognitive function and reduced anxiety-related behavior in a promyelocytic leukemia (PML) tumor suppressor protein-deficient mouse.

The promyelocytic leukemia (PML) protein is a tumor suppressor factor mostly known by its involvement in acute promyelocytic leukemia (APL). Interestingly, recent studies have provided evidence that, in the central nervous system, PML is involved in neurogenesis. However, prospective studies of PML in brain are lacking. To further understand the role of PML in the mammalian brain, we studied plasticity and behavioral changes in PML knockout mice. If PML is involved in neurogenesis, and neurogenesis is an important process for proper brain development as well as learning and memory functions, we hypothesized that PML might have a role in plasticity and cognition. Behavioral studies demonstrated that PML knockout mice present abnormalities in conditioned learning and spatial memory, as determined by fear conditioning and Morris water maze tasks. Experiments to determine normal exploratory behavior interestingly revealed that PML knockout mice present reduced anxiety-related responses as compared to control animals. This was confirmed when PML knockout mice spent more time in the open arms of an elevated plus-maze, which is an indication of decreased anxiety. Additionally, impairments in hippocampus-dependent learning were mirrored by altered long-term plasticity at Schaffer collateral-CA1 synapses. We now provide the first evidence for an important role of PML in the brain, indicating that PML might have a role in synaptic plasticity and associated behavioral processes.

Anti-viral properties of interferon beta treatment in patients with multiple sclerosis.

Viral infections are potentially associated with the etiology and pathogenesis of multiple sclerosis (MS). It has been speculated that the treatment efficacy of interferon beta (IFN beta) in MS may relate to its anti-viral properties. The study was undertaken to evaluate the in vivo anti-viral effects of IFN beta-1a in patients with MS. Human herpesvirus-6 (HHV-6) was studied as an example for being a latent neurotropic virus. IFN beta used at concentrations of approximately 0.5 microg/ml was shown to significantly reduce in vitro HHV-6 replication in a susceptible T-cell line. Sera derived from 23 MS patients treated with IFN beta-1a were examined for serum cell-free DNA of HHV-6 as an indicator for viral replication and the reactivity of IgM antibodies to a recombinant HHV-6 virion protein containing a known immunoreactive region. The results were compared with those of control sera obtained from untreated MS (n=29) and healthy individuals (n=21). The findings indicated that IFN beta treatment significantly reduced HHV-6 replication as evident by decreased cell-free DNA in treated MS specimens. The results correlated with decreased IgM reactivity to the HHV-6 antigen in treated MS patients compared to untreated controls, suggesting reduced exposure to HHV-6. The findings were confirmed in paired sera obtained from seven MS patients before and after the treatment The study provides new evidence indicating that IFN beta has potent in vivo anti-viral effects that may contribute to the treatment efficacy in MS.

Naturally processed HLA class II peptides reveal highly conserved immunogenic flanking region sequence preferences that reflect antigen processing rather than peptide-MHC interactions.

MHC class II heterodimers bind peptides 12-20 aa in length. The peptide flanking residues (PFRs) of these ligands extend from a central binding core consisting of nine amino acids. Increasing evidence suggests that the PFRs can alter the immunogenicity of T cell epitopes. We have previously noted that eluted peptide pool sequence data derived from an MHC class II Ag reflect patterns of enrichment not only in the core binding region but also in the PFRS: We sought to distinguish whether these enrichments reflect cellular processes or direct MHC-peptide interactions. Using the multiple sclerosis-associated allele HLA-DR2, pool sequence data from naturally processed ligands were compared with the patterns of enrichment obtained by binding semicombinatorial peptide libraries to empty HLA-DR2 molecules. Naturally processed ligands revealed patterns of enrichment reflecting both the binding motif of HLA-DR2 (position (P)1, aliphatic; P4, bulky hydrophobic; and P6, polar) as well as the nonbound flanking regions, including acidic residues at the N terminus and basic residues at the C terminus. These PFR enrichments were independent of MHC-peptide interactions. Further studies revealed similar patterns in nine other HLA alleles, with the C-terminal basic residues being as highly conserved as the previously described N-terminal prolines of MHC class II ligands. There is evidence that addition of C-terminal basic PFRs to known peptide epitopes is able to enhance both processing as well as T cell activation. Recognition of these allele-transcending patterns in the PFRs may prove useful in epitope identification and vaccine design.

Reactivity pattern and cytokine profile of T cells primed by myelin peptides in multiple sclerosis and healthy individuals.

Autoreactive T cells specific for candidate myelin antigens, including myelin basic protein (MBP) and proteolipid protein (PLP), are thought to play an important role in the pathogenesis of multiple sclerosis (MS). Myelin-reactive T cells primed in vivo by myelin breakdown products or microbial cross-reactive antigens during the disease processes may exhibit a reactivity pattern and cytokine profile different from those in the normal T cell repertoire. In this study, we examined the precursor frequency, the reactivity pattern and cytokine profile of myelin-reactive T cells that were primed in vitro with overlapping peptides of MBP and PLP in patients with MS and healthy individuals. The results revealed that T cells specific for peptides of MBP and PLP occurred at a relatively higher precursor frequency in patients with MS than that in healthy individuals. We identified a number of dominant T cell epitopes within MBP and PLP, some of which were not previously detected using whole myelin antigens as the primary stimuli. Some residues represented common immunodominant regions that were detected in both MS patients and healthy controls while others were associated only with MS. MBP-reactive T cell lines generally exhibited a Th0-like cytokine profile. There was significantly increased Th1 cytokine production (i. e. TNF and IFN-gamma) among MS-derived T cell lines. PLP-reactive T cell lines had a distinct cytokine profile, producing predominantly TNF-alpha and little or not IFN-gamma and IL-4. The findings have important implications in the understanding of the role of myelin-reactive T cells in MS.

Aberrant T cell responses to myelin antigens during clinical exacerbation in patients with multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of presumed T cell autoimmunity against self myelin. We hypothesized that if myelin-reactive T cells are associated with the disease processes, they may undergo activation and expansion during acute exacerbation. In this study, we examined the precursor frequency, epitope recognition and cytokine profile of myelin-reactive T cells in 14 relapsing/remitting MS patients during exacerbation and remission. The study revealed that T cells recognizing the immunodominant peptides of candidate myelin antigens, including myelin basic protein (MBP), proteolipid protein and myelin oligodendrocyte glycoprotein, occurred at increased precursor frequency during acute exacerbation. The T cell responses to MBP focused on the immunodominant regions (residues 83-99 and 151-170) during exacerbation and shifted toward other epitopes of MBP at the time of remission. Furthermore, there was a marked increase in the production of T(h)1 cytokines among T cell lines obtained during exacerbation compared to those obtained during remission. The study demonstrated that myelin-reactive T cells underwent selective activation and expansion during acute MS exacerbation. In contrast, myelin-reactive T cells found during remission in the same patients generally resembled those identified in healthy controls with some discrepancies. The findings suggest potential association of aberrant myelin-reactive T cell responses with acute exacerbation in MS, which may reflect transient activation of myelin-reactive T cell populations of pathogenic potential.

Reactivity and regulatory properties of human anti-idiotypic antibodies induced by T cell vaccination.

Immunization with irradiated autoreactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that preferentially recognize complementarity-determining region 3 sequences, contributing to clonal depletion of autoreactive T cells. However, it remains unknown whether T cell vaccination elicits anti-idiotypic humoral responses and whether the anti-idiotypic Abs play a similar role in the regulatory mechanism induced by T cell vaccination. In this study we examined the occurrence, the reactivity pattern, and the regulatory role of anti-idiotypic Abs elicited by T cell vaccination in patients with multiple sclerosis. We demonstrated for the first time that B cells producing anti-idiotypic Abs could be isolated from vaccinated patients. These EBV-transformed B cell lines were selected for specific reactivity to a 20-mer TCR peptide incorporating a common complementarity-determining region 3 sequence of the immunizing T cell clones. The resulting anti-idiotypic Abs were found to react with the original immunizing T cell clones and exhibit an inhibitory effect on their proliferation. The findings suggest that anti-idiotypic Ab responses can be induced by T cell vaccination in humans and that their regulatory properties are likely to contribute to the suppression of myelin basic protein-reactive T cells in vaccinated patients. The study has important implications in our understanding of the regulatory role of the anti-idiotypic humoral responses induced by T cell vaccination.

Immunoregulation and blocking antibodies induced by interferon beta treatment in MS.

OBJECTIVE: To examine the in vivo immunoregulatory properties of interferon beta-1a (IFN beta-1a) on the T cell responses to myelin basic protein (MBP) and to evaluate the occurrence of the blocking antibodies characterized by the ability to reverse the effects of IFN beta on T cells in MS patients treated with IFN beta. METHODS: The precursor frequency of T cells recognizing MBP and control antigens was estimated in a microwell culture system. The cytokine profile of T cell lines was measured in ELISA. The binding antibodies were determined in ELISA and Western blot. Cytopathic test and the T cell functional assays were used to determine the blocking effects of the binding antibodies. RESULTS: Treatment with IFN beta resulted in a substantial reduction in the precursor frequency of MBP-reactive T cells in MS patients. The cytokine profile of MBP-reactive T cells that sustained the treatment was altered toward an increased production of interleukin (IL)-10 and decreased production of tumor necrosis factor (TNF)alpha and IFN-gamma. The immunoregulatory properties of IFN beta on T cells could be blocked by the binding antibodies derived from a proportion of patients treated with IFN beta (4 of 64, 6.25%). The blocking antibodies also neutralized anti-viral activity of IFN beta in cytopathic assays, corresponding to previously described neutralizing antibodies. CONCLUSIONS: Treatment with IFN beta alters the cytokine profile by enhancing the production of IL-10 and downregulating Th1 cytokines, which may contribute to clinical benefit in MS. The treatment also induces blocking antibodies that impair the immunoregulatory properties of IFN beta in some individuals.

Aberrant T cell migration toward RANTES and MIP-1 alpha in patients with multiple sclerosis. Overexpression of chemokine receptor CCR5.

Trafficking of inflammatory T cells into the central nervous system (CNS) plays an important role in the pathogenesis of multiple sclerosis. The directional migratory ability of peripheral T cells is associated with interactions of chemokines with their receptors expressed on T cells. In this study, transmigration of peripheral T cells toward a panel of chemokines was examined in patients with multiple sclerosis and healthy individuals using Boyden chemotactic transwells. A significantly increased migratory rate preferentially toward RANTES and MIP-1alpha, but not other chemokines, was found in T cells obtained from multiple sclerosis patients as opposed to healthy individuals (P: < 0.001). The migratory T-cell populations represented predominantly Th1/Th0 cells while non-migratory T cells were enriched for Th2-like cells. The study demonstrated further that aberrant migration of multiple sclerosis-derived T cells toward RANTES and MIP-1 alpha resulted from overexpression of their receptors (CCR5) and could be blocked by anti-CCR5 antibodies. These findings have important implications for our understanding of the mechanism underlying aberrant T cell trafficking in multiple sclerosis.

Th2 immune regulation induced by T cell vaccination in patients with multiple sclerosis.

T cell responses to myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). In this study, we demonstrated that subcutaneous inoculations with irradiated autologous MBP-reactive T cell clones (T cell vaccination) elicited CD8+ anti-idiotypic T cell responses and CD4+ Th2 cell responses in patients with MS. Both regulatory cell types induced by T cell vaccination contributed to the inhibition of MBP-reactive T cells while they differed in the recognition pattern and functional properties. We describe for the first time that the Th2 regulatory cells reacted with activated but not resting T cells in the context of MHC class II molecules and inhibited the proliferation of MBP-reactive T cells through the secretion of IL-4 and IL-10. The T-T cell interaction mediated by Th2 regulatory cells was independent of the antigen specificity of activated T cells. The findings have important implications for our understanding of the regulatory mechanism induced by T cell vaccination.

Th1 and Th2 deviation of myelin-autoreactive T cells by altered peptide ligands is associated with reciprocal regulation of Lck, Fyn, and ZAP-70.

Th0 clones recognizing an immunodominant peptide of myelin basic protein (residues 83-99) were derived from patients with multiple sclerosis. We demonstrate that analogue peptides with alanine substitution at Val86 and His88 had a unique partial agonistic property in inducing Th0 -->Th1 and Th0 -->Th2 deviation of the myelin basic protein-reactive T cell clones, respectively. Th0 to Th1 deviation induced by peptide 86V-->A correlated with up-regulation of Fyn and ZAP-70 kinase activities. Conversely, Th0 to Th2 deviation induced by peptide 88H-->A was associated with complete failure to activate Fyn and ZAP-70 kinases. The observed Th1 and Th2 shift also correlated, to a lesser extent, with Lck kinase activity that was down-regulated with Th1 deviation and increased with Th2 deviation in some T cell clones. We demonstrated that the Th1 and Th2 shift induced by the analogue peptides was a reversible process, as the T cell clones previously exposed to either 86V-->A or 88H-->A peptide could revert to an opposite phenotype when rechallenged reciprocally with a different analogue peptide. The study has important implications in our understanding of regulation of TCR-associated tyrosine kinases by altered peptide ligands and its role in cytokine regulation of autoreactive T cells.

Proinflammatory cytokine and nitric oxide induction in murine macrophages by cell wall and cytoplasmic extracts of lactic acid bacteria.

Cells from a number of bacterial genera have been shown to possess mitogenic and polyclonal activating properties when cultured with cells of the immune system. Based on previously reported health immune-enhancing effects of fermented dairy products, we tested the potentiating effects of representative lactic acid bacteria and their extracts on leukocyte function. Specifically, the effects of in vitro exposure to heat-killed cells of Bifidobacterium, Lactobacillus acidophilus, L. bulgaricus, L. casei, L. gasseri, L. helveticus, L. reuteri, and Streptococcus thermophilus, their cell walls, and their cytoplasmic extracts on proliferation as well as cytokine and nitric oxide (NO) production were examined in the RAW 264.7 macrophage cell line. A similar strategy was applied to murine cultures composed of peritoneal, spleen, and Peyer's patch cells. Both the cell wall and cytoplasmic fractions of lactic acid bacteria were able to stimulate cloned macrophages to produce significant amounts of tumor necrosis factor-alpha, (interleukin) IL-6, and NO. Pronounced enhancement of IL-6 production by peritoneal cells was observed when cultured with those extracts, whereas, effects were not noted in spleen and Peyer's patch cell cultures from mice. Based on the results, it appears that, as a group, the lactic acid bacteria were capable of stimulating macrophages and possibly other immune cells to produce cytokines and NO, and both their cell walls and cytoplasm contributed to these capacities.

A common TCR V-D-J sequence in V beta 13.1 T cells recognizing an immunodominant peptide of myelin basic protein in multiple sclerosis.

T cell responses to the immunodominant peptide (residues 83-99) of myelin basic protein are potentially associated with multiple sclerosis (MS). This study was undertaken to examine whether a common sequence motif(s) exists within the TCR complementarity-determining region (CDR)-3 of T cells recognizing the MBP83-99 peptide. Twenty MBP83-99-reactive T cell clones derived from patients with MS were analyzed for CDR3 sequences, which revealed several shared motifs. Some V beta 13.1 T cell clones derived from different patients with MS were found to contain an identical CDR3 motif, V beta 13.1-LGRAGLTY. Oligonucleotides complementary to the shared CDR3 motifs were used as specific probes to detect identical target CDR3 sequences in a large panel of T cell lines reactive to MBP83-99 and unprimed PBMC. The results revealed that, in contrast to other CDR3 motifs examined, the LGRAGLTY motif was common to T cells recognizing the MBP83-99 peptide, as evident by its expression in the majority of MBP83-99-reactive T cell lines (36/44) and PBMC specimens (15/48) obtained from randomly selected MS patients. The motif was also detected in lower expression in some PBMC specimens from healthy individuals, suggesting the presence of low precursor frequency of T cells expressing this motif in healthy individuals. This study provides new evidence indicating that the identified LGRAGLTY motif is preferentially expressed in MBP83-99-reactive T cells. The findings have important implications in monitoring and targeting MBP83-99-reactive T cells in MS.

Ingestion of yogurt containing Lactobacillus acidophilus and Bifidobacterium to potentiate immunoglobulin A responses to cholera toxin in mice.

Lactic acid bacteria have been reported to have benefits for the prevention and treatment of some forms of diarrhea and related conditions. To determine whether these effects might involve direct stimulation of the gastrointestinal immune response, we administered yogurt to try to enhance mucosal and systemic antibodies against an orally presented immunogen, cholera toxin. Yogurts were manufactured with starter cultures containing different species and strains of lactic acid bacteria. Mice were fed these yogurts for 3 wk, during which they were also orally immunized twice with 10 micrograms of cholera toxin. Blood was collected on d 0 and 21, and fecal pellets were collected weekly. Mice that were immunized orally with cholera toxin responded by producing specific intestinal and serum immunoglobulin (Ig)A anti-cholera toxin. Antibody responses of the IgA isotype were significantly increased in mice fed yogurts made with starters containing the conventional yogurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus supplemented with Lactobacillus acidophilus, Bifidobacterium bifidum, and Bifidobacterium infantis. Yogurt that was manufactured with starters containing only conventional yogurt bacteria produced less IgA anti-cholera toxin than did the control group fed nonfat dry milk. Although strong responses were also observed for IgG anti-cholera toxin in serum, the responses did not differ among groups. Thus, administration of yogurt supplemented with L. acidophilus and Bifidobacterium spp. enhanced mucosal and systemic IgA responses to the cholera toxin immunogen.

Effects of lactic acid bacteria ingestion of basal cytokine mRNA and immunoglobulin levels in the mouse.

An increasing number of functional foods and pharmaceutical preparations containing lactic acid bacteria are being promoted with health claims based on the potential probiotic characteristics and on their capacity for stimulating the host immune system. However, the specific immune effects of oral administration of these microbes remain undefined. In this study, we tested the hypothesis that basal gastrointestinal immune status in mice is affected by orally administered lactic acid bacteria. The specific objective of this research was to evaluate the effects of repeated oral exposure to viable and nonviable lactic acid bacteria (Lactobacillus acidophilus, L. bulgaricus, L. casei, and Streptococcus thermophilus) in mice on basal cytokine mRNA expression in mucosal (Peyer's patches), systemic (spleen), and lymphoid tissue and on immunoglobulin levels. The results indicated that oral exposure to 10(9) CFU/day for up to 14 days did not significantly affect basal interferon-gamma, tumor necrosis factor-alpha, or interleukin-6 mRNA expression or total serum and intestinal immunoglobulins.

Ex vivo effects of lactobacilli, streptococci, and bifidobacteria ingestion on cytokine and nitric oxide production in a murine model.

Increasing numbers of functional foods and pharmaceutical preparations are being promoted with health claims based on the potential probiotic characteristics of lactic acid bacteria and on their capacity for stimulating the host immune system. However, the specific immune effects of oral administration of these microbes still remains undefined. In this study, we tested the hypothesis that production of immunologic mediators by leukocytes in mice is affected by orally administered lactic acid bacteria. The specific objectives of this study were to evaluate the effects of exposure to eight different lactic acid bacteria in mice on ex vivo cytokine and nitric oxide production in leukocyte cultures. Mice were gavaged with 1 X 10(9) viable bacteria and peritoneal, Peyer's patch and splenic leukocytes were isolated 8 h later. These were cultured for 2 or 5 days in the presence or absence of mitogens and then interleukin (IL)-6, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and nitric oxide production was measured. The results revealed that Lactobacillus acidophilus and L. casei potentiated IL-6 and IL-12 production by peritoneal cells whereas L. acidophilus upregulated IFN-gamma and nitric oxide. In contrast, L. helveticus, L. gasseri, L. reuteri, and Bifidobacterium attenuated the production of IL-6, IFN-gamma, and nitric oxide by peritoneal cells. TNF-alpha was not detectable in peritoneal cultures. None of the bacteria altered ex vivo production of cytokines or nitric oxide by Peyer's patch or spleen cell cultures. Taken together, the results suggest that prior oral exposure to lactic acid bacteria could differentially potentiate or attenuate subsequent cytokine and nitric oxide production by peritoneal cells.

Production of polyclonal antibody against ergosterol hemisuccinate using Freund's and Titermax adjuvants.

Ergosterol is the principal sterol in fungi and an important component of cell membranes. This sterol has been used previously to measure fungal growth in foods. In an attempt to develop antibodies for an immunoassay for ergosterol, we first derivatized the compound to its hemisuccinate (Erg-HS) and conjugated it to bovine serum albumin (Erg-HS-BSA). Six white female New Zealand rabbits were immunized by subcutaneous injections of Erg-HS-BSA conjugate followed by four booster injections. Freund's and Hunter's TiterMax adjuvants were used for stimulating strong and prolonged responses. An indirect enzyme-linked immunosorbent assay (ELISA) was used to assess the titers. Antibody titers to Erg-HS rose much earlier and were markedly higher when Freund's adjuvant was used than when TiterMax was used. Although titers as high as 6,400 were detectable by indirect ELISA, binding of these antibodies to an Erg-HS-BSA solid phase could not be inhibited by free ergosterol in a competitive indirect ELISA. The antibody was also evaluated for applicability in an enzyme-linked immunocytochemical assay for a variety of fungi but failed to detect the presence of ergosterol in the membranes of these organisms. Thus, although antibodies could be generated that showed high specificity for the Erg-HS-BSA conjugate, they could not detect free ergosterol.

Stimulation of cytokine production in clonal macrophage and T-cell models by Streptococcus thermophilus: comparison with Bifidobacterium sp. and Lactobacillus bulgaricus.

The effects of four commercial strains of Streptococcus thermophilus used in yogurt manufacturing on cytokine production were evaluated by using a macrophage model (RAW 264.7 cells) and a T-helper-cell model (EL4.IL-2 thymoma cells) and compared to immunologically active strains of Lactobacillus bulgaricus, Bifidobacterium adolescentis, and Bifidobacterium bifidum. All cytokines (TNF-alpha and IL-6 in RAW 264.7 cells and IL-2 and IL-5 in EL4.IL-2 cells) were affected by heat-killed S. thermophilus in a strain- and dose-dependent fashion. Organisms of all three genera induced significant increases in IL-6 production by the macrophage line ranging from 31- to 192-fold, with S. thermophilus St 133 showing the greatest activity. The four S. thermophilus strains also strongly induced TNF-alpha production (from 135- to 176-fold). IL-6 and, to a lesser extent, TNF-alpha production were also increased when the macrophages were costimulated with lipopolysaccharide and cells of the three groups of lactic acid bacteria. Upon concurrent stimulation of EL4.IL-2 cells with phorbol 12-myristate-13-acetate, seven of the eight strains displayed significant enhancement of IL-2 and IL-5 production, with S. thermophilus being most effective. Taken together, the S. thermophilus strains stimulated macrophage and T-cell cytokine production to a similar or greater extent than did the species of Bifidobacterium and Lactobacillus. These and previous results lend further support to the contention that lactic acid bacteria, in a concentration-dependent manner, can differentially induce cytokine production in macrophages, but that the effects on T cells required a costimulatory signal and were less remarkable.