CX3CL1 (fractalkine) and CX3CR1 expression in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis: kinetics and cellular origin

Background  

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). It is associated with local activation of microglia and astroglia, infiltration of activated macrophages and T cells, active degradation of myelin and damage to axons and neurons. The proposed role for CX₃CL1 (fractalkine) in the control of microglia activation and leukocyte infiltration places this chemokine and its receptor CX₃CR1 in a potentially strategic position to control key aspects in the pathological events that are associated with development of brain lesions in MS. In this study, we examine this hypothesis by analyzing the distribution, kinetics, regulation and cellular origin of CX₃CL1 and CX₃CR1 mRNA expression in the CNS of rats with an experimentally induced MS-like disease, myelin oligodendrocyte glycoprotein (MOG)-induced autoimmune encephalomyelitis (EAE).     

A technique for preparing defined conjugates of horseradish peroxidase and immunoglobulin

Conjugates of horseradish peroxidase (HRP) and sheep anti-human immunoglobulin (anti-Ig) were prepared by means of a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The molar ratios of the conjugates could be changed by varying the degree of chemical substitution of anti-Ig and HRP. Gel filtration and affinity chromatography were used to separate conjugated anti-Ig from free HRP and unconjugated anti-Ig. The distribution of complex sizes and the presence of free HRP and anti-Ig in the final products were monitored by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. After final purification and characterisation conjugates were studied as reagents in enzyme immunoassays (EIA) with various antigens.Conjugates prepared by SPDP bridging were shown to yield more than 60% of the anti-Ig as conjugated small defined enzyme-protein complexes. A suitably labelled conjugate reacted in a standardised way resulting in a reproducible dose-response curve when a positive serum was assayed in different dilutions.     

Magnetophosphenes: a quantitative analysis of thresholds

Low-frequency and transient magnetic fields of moderate flux densities are known to generate visual phenomena, so-called magnetophosphenes. In the present study, time-variable very low frequency (10–50 Hz) electromagnetic fields of moderate flux density (0–40 mT) were used to induce magnetophosphenes. The threshold values for these phosphenes were determined as a function of the frequency of the magnetic field both in normal subjects and colour defective ones. Maximum sensitivity occurred at a frequency of approximately 20–30 Hz, and with broad-spectrum light the threshold flux density was 10–12 mT. The threshola values were found to be dependent upon the intensity and the spectral distribution of the background light. Sensitivity decreased during dark adaptation. In certain respects deutans differed from subjects with normal colour vision. Possible mechanisms for generation of magnetophosphenes are discussed. The present magnetic threshold curves show a close resemblance to corresponding curves obtained by electric stimulation at various frequencies provided the electric thresholds are divided by the a.c. frequency. These problems are under current investigation in our laboratory. This is in full agreement with the assumption that the fluctuating magnetic field affects retinal neurons by inducing currents which polarise synaptic terminals.     

Spatial learning and memory following fimbria-fornix transection and grafting of fetal septal neurons to the hippocampus

Summary The ability of intrahippocampal grafts of fetal septal-diagonal band tissue, rich in developing cholinergic neurons, to ameliorate cognitive impairments induced by bilateral fimbria-fornix transections in rats was examined in three experiments using the Morris water-maze to test different aspects of spatial memory. Experiment 1. Rats with fimbria-fornix lesions received either septal cell suspension grafts or solid septal grafts; normal rats and rats with lesions alone were used as controls. Sixteen weeks after surgery, the rats' spatial learning and memory were tested in the water-maze using a place test, designed to investigate place navigation performance, in which rats learned to escape from the water by swimming to a platform hidden beneath the water's surface. After 5 days of training, the rats were given a spatial probe test in which the platform was removed from the tank to test spatial reference memory. Experiment 2. The same rats used in Exp. 1 were tested in a delayed-match-to-sample, working memory version of the water-maze task. The platform was located in one of two possible locations during each trial, which was composed of 2 swims. If the rat remembered the location of the platform on the 2nd swim of a trial, it should find the platform more quickly on that swim, and thereby demonstrate working memory. Experiment 3. Prior to receiving fimbria-fornix lesions, normal rats were trained in a modification of the water-maze task using alternating cue navigation and place navigation trials (i.e., with visible or non-visible escape platforms). The retention and reacquisition of the place task and the spatial probe test were examined in repeated tests up to 6 months after the lesion and intrahippocampal grafting of septal cell suspensions. The effects of central muscarinic cholinergic receptor blockade with atropine were also tested. Normal rats performed well in both the place and spatial probe tests. In contrast, rats with fimbria-fornix lesions only were unable to acquire or retain spatial information in any test. Instead, these rats adopted a random, nonspatial search strategy, whereby their latencies to find the platform decreased in the place navigation tasks. Sixty to 80% of the rats with septal suspension or solid grafts had recovered place navigation, i.e., the ability to locate the platform site in the tank, in Exp. 1 and 3, and they showed a significantly improved performance in the working memory test in Exp. 2. Atropine abolished the recovered place navigation in the grafted rats, whereas normal rats were impaired to a lesser extent. In contrast, atropine had no effect on the non-spatial strategy adopted by rats with fimbria-fornix lesions only. The results show that: (1) fimbria-fornix lesions disrupt spatial learning and memory in both naive and pretrained rats; (2) with extended training the fimbria-fornix lesioned rats develop an efficient non-spatial strategy, which enables them to reduce their escape latency to levels close to those of intact controls; (3) intrahippocampal septal grafts can restore the ability of the lesioned rats to use spatial cues in the localization of the platform site; and (4) the behavioural recovery produced by grafts is dependent upon an atropine sensitive mechanism.     

Distinct T-cell subtypes induced with whole cell and acellular pertussis vaccines in children.

Recent clinical trials have demonstrated that new generation acellular pertussis vaccines can confer protection against whooping cough. However, the mechanism of protective immunity against Bordetella pertussis infection induced by vaccination remains to be defined. We have examined cellular immune responses in children immunized with a range of acellular and whole cell pertussis vaccines. Immunization of children with a potent whole-cell vaccine induced B. pertussis-specific T cells that secreted interferon-gamma (IFN-gamma), but not interleukin-5 (IL-5). In contrast, T cells from children immunized with acellular pertussis vaccines secreted IFN-gamma and/or IL-5 following stimulation with B. pertussis antigens in vitro. These observations suggest that protective immunity conferred by whole-cell vaccines, like natural immunity, is mediated by type 1 T cells, whereas the mechanism of immune protection generated with acellular vaccines may be more heterogeneous, involving T cells that secreted type 1 and type 2 cytokines.     

Messenger RNA processing sites in Trypanosoma brucei

In Kinetoplastids, protein-coding genes are transcribed polycistronically by RNA polymerase II. Individual mature mRNAs are generated from polycistronic precursors by 5' trans splicing of a 39-nt capped leader RNA and 3' polyadenylation. It was previously known that trans splicing generally occurs at an AG dinucleotide downstream of a polypyrimidine tract, and that polyadenylation is coupled to downstream trans splicing. The few polyadenylation sites that had been examined were 100-400 nt upstream of the polypyrimidine tract which marked the adjacent trans splice site. We wished to define the sequence requirements for trypanosome mRNA processing more tightly and to generate a predictive algorithm. By scanning all available Trypanosoma brucei cDNAs for splicing and polyadenylation sites, we found that trans splicing generally occurs at the first AG following a polypyrimidine tract of 8-25 nt, giving rise to 5'-UTRs of a median length of 68 nt. We also found that in general, polyadenylation occurs at a position with one or more A residues located between 80 and 140 nt from the downstream polypyrimidine tract. These data were used to calibrate free parameters in a grammar model with distance constraints, enabling prediction of polyadenylation and trans splice sites for most protein-coding genes in the trypanosome genome. The data from the genome analysis and the program are available from: .     

Temporal expression and cellular origin of CC chemokine receptors CCR1, CCR2 and CCR5 in the central nervous system: insight into mechanisms of MOG-induced EAE

Background  

The CC chemokine receptors CCR1, CCR2 and CCR5 are critical for the recruitment of mononuclear phagocytes to the central nervous system (CNS) in multiple sclerosis (MS) and other neuroinflammatory diseases. Mononuclear phagocytes are effector cells capable of phagocytosing myelin and damaging axons. In this study, we characterize the regional, temporal and cellular expression of CCR1, CCR2 and CCR5 mRNA in the spinal cord of rats with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE). While resembling human MS, this animal model allows unique access to CNS-tissue from various time-points of relapsing neuroinflammation and from various lesional stages: early active, late active, and inactive completely demyelinated lesions.     

Immunizing with mouse trophoblast to obtain mouse-human cross-reacting antibodies against normal and neoplastic human trophoblast

Monoclonal antibodies raised against living, early invasive mouse trophoblast cells were screened on paraffin sections from first- and third-trimester placentas and from hydatidiform moles and choriocarcinoma. Several mouse-human cross-reacting antibodies were recognized, which implies that mouse trophoblast cells can be used as immunogen for producing antibodies against human trophoblast. Among the new antibodies obtained, some were selected for further study. That panel includes a trophoblast specific antibody with capacity to differ between invasive and noninvasive molar tissues, and two antibodies, which detect antigen epitopes in the normal, but not in the neoplastic trophoblast.