Subcutaneous interferon ß-1a may protect against cognitive impairment in patients with relapsing-remitting multiple sclerosis: 5-year follow-up of the COGIMUS study.

OBJECTIVE: To assess the effects of subcutaneous (sc) interferon (IFN) -1a on cognition over 5 years in mildly disabled patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: Patients aged 18-50 years with RRMS (Expanded Disability Status Scale score ≤4.0) who had completed the 3-year COGIMUS study underwent standardized magnetic resonance imaging, neurological examination, and neuropsychological testing at years 4 and 5. Predictors of cognitive impairment at year 5 were identified using multivariate analysis. RESULTS: Of 331 patients who completed the 3-year COGIMUS study, 265 participated in the 2-year extension study, 201 of whom (75.8%; sc IFN ß-1a three times weekly: 44 µg, n = 108; 22 µg, n = 93) completed 5 years' follow-up. The proportion of patients with cognitive impairment in the study population overall remained stable between baseline (18.0%) and year 5 (22.6%). The proportion of patients with cognitive impairment also remained stable in both treatment groups between baseline and year 5, and between year 3 and year 5. However, a significantly higher proportion of men than women had cognitive impairment at year 5 (26.5% vs 14.4%, p = 0.046). Treatment with the 22 versus 44 µg dose was predictive of cognitive impairment at year 5 (hazard ratio 0.68; 95% confidence interval 0.48-0.97). CONCLUSIONS: This study suggests that sc IFN ß-1a dose-dependently stabilizes or delays cognitive impairment over a 5-year period in most patients with mild RRMS. Women seem to be more protected against developing cognitive impairment, which may indicate greater response to therapy or the inherently better prognosis associated with female sex in MS.

Fluoxetine inhibits corticotropin-releasing factor (CRF)-induced behavioural responses in rats.

Corticotropin-releasing factor (CRF) is a potent neuromodulator of stress-related behaviour but the neural mechanisms underlying these effects are not clear. Studies were designed to test the hypothesis that CRF-induced behavioural arousal involves interactions with brainstem serotonergic systems. To examine interactions between CRF and serotonergic systems in the regulation of behaviour, CRF (1 microg, intracerebroventricular (i.c.v.)) or vehicle was infused in the presence or absence of the selective serotonin re-uptake inhibitor fluoxetine (0, 0.1, 1 or 10 mg/kg, intravenous (i.v.)). Fluoxetine was used at these doses because it is known to decrease serotonin cell firing rates while increasing extracellular serotonin concentrations in select forebrain regions. We then measured behavioural, neurochemical and endocrine responses. CRF increased locomotion and spontaneous non-ambulatory motor activity (SNAMA) in the home cages. Fluoxetine decreased tissue 5-hydroxyindoleacetic acid concentrations, a measure of serotonin metabolism, in specific limbic brain regions of CRF-treated rats (nucleus accumbens shell region, entorhinal cortex, central nucleus of the amygdala). Furthermore, fluoxetine inhibited CRF-induced SNAMA. CRF and fluoxetine independently increased plasma corticosterone concentrations, but the responses had distinct temporal profiles. Overall, these data are consistent with the hypothesis that CRF-induced facilitation of behavioural activity is dependent on brainstem serotonergic systems. Therefore, fluoxetine may attenuate or alleviate some behavioural responses to stress by interfering with CRF-induced responses.

CD4+CD25+FOXP3+ regulatory T cells suppress anti-tumor immune responses in patients with colorectal cancer.

BACKGROUND: A wealth of evidence obtained using mouse models indicates that CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4(+) T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients. METHODOLOGY AND PRINCIPAL FINDINGS: Treg were identified and characterized as CD4(+)CD25(+)FOXP3(+) using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4(+) T cell responses, as observed by IFNgamma release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group. CONCLUSIONS/SIGNIFICANCE: Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy.

Modulation of the immune response by the cholera-like enterotoxins.

Cholera toxins and heat labile enterotoxin from E. coli differ from most soluble proteins in eliciting systemic immunity both against themselves and unrelated admixed antigens, rather than tolerance following administration to a mucosal surface. Several reports have also demonstrated preferential induction of Th2-type responses when these molecules are used as adjuvants. Conversely, these proteins and their non-toxic derivatives, including the B sub-units are also able prevent and alleviate autoimmune diseases in naïve and systemically immune hosts demonstrating wide-ranging effects on the immune system. The recent observation that amelioration of autoimmune disease is associated with the generation of regulatory T cells which inhibit pathogenic Th1 responses may also help to consolidate these two apparently contradictory outcomes of exposure to the cholera-like enterotoxins. Furthermore, the observation that EtxB is able to alleviate autoimmune disease in the absence of conjugation to autoantigen highlights its potential for use in the clinical setting where the target antigen is often unknown. Direct effects on T cells, B cells and APC have been demonstrated in vitro which have provided insights into how these molecules may elicit these diverse effects. Further investigation is required for elucidation of the mechanisms of action of adjuvanticity and tolerance induction by these molecules to realise their potential for use in vaccines and therapies for autoimmune disease in humans.

The B subunit of Escherichia coli heat labile enterotoxin abrogates oral tolerance, promoting predominantly Th2-type immune responses.

Mucosal antigen encounter usually results in a state of systemic non-responsiveness (tolerance). This failure to mount a protective response is a major hurdle to mucosal vaccine development. Hence, the identification of safe and effective mucosal adjuvants promoting protective immunity is of critical importance. The non-toxic B subunit of Escherichia coli heat labile enterotoxin(EtxB) is a potent nasal adjuvant; however, its usefulness following oral delivery is unconfirmed. We used DO11.10 chimeric mice to assess whether EtxB could abrogate tolerance to oral OVA. We show that admixing EtxB with OVA for oral immunization abrogates oral tolerance and results in a weak anti-OVA immune response. Importantly, EtxB profoundly modulated the nature of the response to subsequent parenteral challenge, promoting IgG1 in favor of IgG2a antibodies and depressing IFN-gamma production while elevating TGF-beta secretion. The addition of EtxB promoted T cell division, as assessed by loss of staining with carboxyfluorescein diacetate succinimidyl ester. Enhanced cell division promoted by EtxB was associated with T cell differentiation (increased numbers of CD45RBlow cells) in vivo, although dividing OVA-specific T cells were CD25-. These data suggest that although EtxB is a weak oral adjuvant, it can profoundly modulate the nature of the immune response to admixed antigen.

Increased bone morphogenetic protein-6 expression in mouse long bones after estrogen administration.

High-dose estrogen administration is known to induce new bone formation in mouse long bones. To study the role of regulatory proteins in this response, we examined associated changes in femoral messenger RNA (mRNA) for candidate factors. 17beta-estradiol (E2) 0.5 mg was administered to intact female mice by weekly injection, and Northern blot analysis was performed 1, 2, 4, 8, 12, and 16 days after the first injection. In contrast to other factors, an increase was observed in mRNA for bone morphogenetic protein-6 (BMP-6), which reached significance at day 8 and subsequent time-points. Estrogen-induced changes in BMP-6 protein expression were assessed by immunocytochemistry in longitudinal femoral sections. In untreated animals, BMP-6 was expressed by a significant proportion of growth plate chondrocytes and a subpopulation of bone marrow cells. In contrast, osteoblasts were consistently BMP-6 negative. From as early as 4 days after starting estrogen, clusters of slightly elongated BMP-6-positive cells were observed within the marrow cavity; the majority were close to active bone formation surfaces. Double immunolabeling studies revealed that only approximately 10% of BMP-6-positive bone marrow cells co-expressed the osteoblast transcription factor Cbfa1 suggesting that they are largely distinct from the osteoblast precursor population generated concurrently. BMP-6-positive cells expressed neither leukocyte nor erythroid markers (CD45 and TER-119, respectively), consistent with a stromal origin. We conclude that estrogen-induced osteogenesis in female mice is associated with increased levels of BMP-6 mRNA in mouse femurs, which seems to reflect the emergence of clusters of BMP-6 positive stromal cells adjacent to active bone formation surfaces. These findings raise the possibility that BMP-6 serves as a paracrine mediator of estrogen's osteogenic action in mice.

Estrogen-induced osteogenesis in mice is associated with the appearance of Cbfa1-expressing bone marrow cells.

Cbfa1 is a transcription factor recognised as being involved in early osteoblast differentiation during embryonic skeletogenesis. To determine whether Cbfa1 plays a similar role in bone formation in the adult, we analysed whether its expression is altered during estrogen-induced osteogenesis, following our recent studies which suggest that this response involves the generation of early osteoblast precursors within bone marrow. To facilitate identification of Cbfa1-expressing cells, these studies were performed in mice heterozygous for a cbfa1 gene deletion (cbfa1(+/-)) using beta-galactosidase (lacZ) as a genetic marker. Cbfa1-expressing cells were identified by lacZ staining of longitudinal sections of the proximal tibial metaphysis. Treatment of cbfa1(+/-) mice with 17beta-estradiol 0.5 mg/week for 24 days led to the appearance of new cancellous bone surfaces. This response was associated with a marked increase in number of Cbfa1-expressing cells within the metaphysis, consisting not only of osteoblasts on bone surfaces but also of cells within the adjacent bone marrow. We subsequently enumerated Cbfa1-expressing cells at earlier time-points following estrogen, in sections co-stained for ALP activity. After 4 days of estrogen treatment, a population of cells appeared within the marrow cavity which expressed Cbfa1, but were negative for ALP. At later time-points, large numbers of Cbfa1 + bone marrow cells were still present, but the majority of these were close to new trabecular bone surfaces at sites which showed high levels of ALP activity. An equivalent distribution of Cbfa1-expressing cells was observed in further studies where Cbfa1 expression was analysed in wild-type mice by immunohistochemistry. We conclude that estrogen-induced osteogenesis is associated with the appearance of a population of Cbfa1-expressing cells within bone marrow, which we hypothesize to represent the osteoblast precursor population responsible for subsequent new bone formation.