Cloning of human hybridoma, myeloma and lymphoma cell lines using enriched human monocytes as feeder layer

Human monocytes were prepared from peripheral blood by buoyant density centrifugation and subsequent absorption-elution on a column of gelatin beads. The eluted fraction containing 60-80% monocytes was used as feeder layer in cloning of the human lymphoma line RH-L4, the human myeloma line SKO-007, and a human hybridoma cell derived from the latter line. Cloning efficiencies were high in both liquid and semisolid media with all 3 cell lines tested. Feeder monocytes could also be successfully used after having been stored in liquid nitrogen.     

Comparative study of tocainide and lidocaine in patients admitted for suspected acute myocardial infarction

The antiarrhythmic effects of tocainide, administered as a bolus injection of 750 mg followed by oral therapy, and conventional lidocaine therapy were evaluated in 40 patients admitted for suspected acute myocardial infarction (AMI) and showing high-grade premature ventricular complexes (PVCs). The mean hourly PVC rate before therapy was 928 and its reduction was equally significant in the tocainide group, 73%, and in the lidocaine group, 68%. The number of 5-minute periods with multiform, paired and R/T PVCs or ventricular tachycardia was also significantly reduced, by 78% in the tocainide group and by 71% in the lidocaine group. Ten patients in the tocainide group reported moderate side-effects, compared to 13 in the lidocaine group, where the infusion had to be discontinued in 5 patients and the rate had to be reduced in 4. Tocainide, an amine analogue of lidocaine, is considered just as effective as lidocaine in patients with high-grade PVCs and suspected AMI.     

Myelin antigen reactive T cells in cerebrovascular diseases.

T cell reactivities to the putative autoantigens myelin basic protein (MBP), MBP peptides with amino acid residues 110-128 and 148-165, and myelin proteolipid protein (PLP) were examined in patients with acute ischaemic cerebrovascular disease (CVD) and, for comparison, in patients with inflammatory neurological diseases and other neurological diseases. A quantitative measure of these T cell reactivities was obtained by assessing numbers of T cells among blood and cerebrospinal fluid (CSF) mononuclear cells that secreted IFN-gamma in response to antigen in vitro. Higher numbers of T cells reactive with each of these four antigens were detected in peripheral blood from patients with CVD compared with patients of the two control groups. Among blood cells from the CVD patients, their average number was 2.3-4.2/10(5) mononuclear cells. MBP reactive T cells were several-fold enriched in the CSF of CVD patients. The findings strongly suggest that brain damage in context with acute CVD leads to an in vivo expansion of myelin reactive T cells.     

Localization of evoked potentials in the digastric,masseteric, supra- and intertrigeminal subnuclei of the cat

Summary Extracellular focal potentials were evoked and mapped in the trigeminal motor nucleus and its surrounding borderzone in the cat. Graded electrical stimulation was used for orthodromic and antidromic excitation of the masseteric and digastric motoneurones and for orthodromic stimulation of the lingual and inferior alveolar nerves. The method of referring Horsley Clarke coordinates of microelectrode recording positions to their location of the actual histological section was studied and the total error affecting the method was calculated for the H, AP and L axes. The characteristics and the distribution of the evoked focal potentials were described and related to the histological section from the actual experiment. A phase reversal of the negative focal potential evoked by the lingual and inferior alveolar nerves in the main sensory nucleus and in the intertrigeminal nucleus was observed to indicate the dorso-lateral border of the motor nucleus. Other borders were given by the antidromic potentials evoked in the nucleus. Digastric motoneurones were found medially in the caudal third and ventro-medially in the middle third of the motor nucleus. The masseteric motoneurones were located laterally in the middle and rostral thirds of the nucleus. Potentials evoked in the supratrigeminal and intertrigeminal subnuclei, adjacent to the motor nucleus, were considered and discussed in relation to the available evidence of interneurones subserving trigeminal reflex arcs.     

Dipsogenic Effects of Intracarotid Infusions of Various Hyperosmolal Solutions

Thirst in response to intracarotid and intravenous infusions (1.5 ml/min) of various hypertonic, equi-osmolal solutions was studied in the goat. Intracarotid infusions of 1 M NaCl and of 2 M fructose induced conspicuous cumulative drinking. The amount of water drunk during intracarotid infusions of 2 M urea and glycerol was only about a third of that consumed during the corresponding infusions of NaCl and fructose. During intracarotid infusions of 2 M galactose and glucose drinking was inconsistent. Of the intravenous infusions only hypertonic NaCl had a consistent dipsogenic effect. However, the amount of water consumed was considerably smaller and the latency time for drinking much longer than during the intracarotid infusions of NaCI. It is concluded that intracarotid infusions of hypertonic solutions act as considerably stronger thirst stimuli than corresponding intravenous infusions, and that the most pronounced dipsogenic effect is obtained by intracarotid infusions of those hypertonic solutions which also most effectively release antidiuretic hormone in the hydrated goat. The possibility is discussed that intracarotid infusions may stimulate the thirst mechanism indirectly via a rise in the Na+ concentration of the cerebrospinal fluid.     

Analysis of nine autoantibodies associated with systemic autoimmune diseases using the Luminex technology. Results of a multicenter study

Luminex technology allows simultaneous detection of several analytes in a single well. Applications have been recently developed for the detection of autoantibodies. PURPOSE: To evaluate the performances and convenience of the Fidis analytical system (BioMedical Diagnostics, Marnes-la-Vallee, France) for the detection of nine autoantibodies associated with connective diseases: SS-A, SS-B, Sm, RNP, Scl-70, Jo-1, CENP-B, P ribosomal and double stranded DNA antibodies. MATERIALS AND METHODS: Three hospital laboratories analysed 366 samples taken from their serum banks and corresponding to the main systemic autoimmune diseases. Control samples included 120 sera from blood donors and 42 sera from patients with dysglobulinemia. RESULTS: In each laboratory, handling of this new analytical system was easy. Results are readily obtained: nine autoantibodies are quantitated in fourty-four sera in less than two hours. A clear-cut discrimination between negative and positive results was observed, due to very low backgrounds. Intra-assay and inter-assay variations were low: coefficients of variation were under 10% in 80 and 64% of the cases, respectively. Results obtained with Fidis correlated satisfactorily with those obtained with the numerous routine techniques used in each laboratory. The overall concordance exceeded 93%. CONCLUSION: Fidis is a reliable and time-saving analytical system for the detection of autoantibodies associated with systemic autoimmune diseases.     

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).     

Image analysis methods for assessing levels of image plane nonuniformity and stochastic noise in a magnetic resonance image of a homogeneous phantom

Magnetic response image plane nonuniformity and stochastic noise are properties that greatly influence the outcome of quantitative magnetic resonance imaging (MRI) evaluations such as gel dosimetry measurements using MRI. To study these properties, robust and accurate image analysis methods are required. New nonuniformity level assessment methods were designed, since previous methods were found to be insufficiently robust and accurate. The new and previously reported nonuniformity level assessment methods were analyzed with respect to, for example, insensitivity to stochastic noise; and previously reported stochastic noise level assessment methods with respect to insensitivity to nonuniformity. Using the same image data, different methods were found to assess significantly different levels of nonuniformity. Nonuniformity levels obtained using methods that count pixels in an intensity interval, and obtained using methods that use only intensity values, were found not to be comparable. The latter were found preferable, since they assess the quantity intrinsically sought. A new method which calculates a deviation image, with every pixel representing the deviation from a reference intensity, was least sensitive to stochastic noise. Furthermore, unlike any other analyzed method, it includes all intensity variations across the phantom area and allows for studies of nonuniformity shapes. This new method was designed for accurate studies of nonuniformities in gel dosimetry measurements, but could also be used with benefit in quality assurance and acceptance testing of MRI, scintillation camera, and computer tomography systems. The stochastic noise level was found to be greatly method dependent. Two methods were found to be insensitive to nonuniformity and also simple to use in practice. One method assesses the stochastic noise level as the average of the levels at five different positions within the phantom area, and the other assesses the stochastic noise in a region outside the phantom area.     

B cells expressing CD5 are increased in cerebrospinal fluid of patients with multiple sclerosis.

By two-colour flow cytometric analysis, we found increased numbers of B cells co-expressing the pan-T cell marker CD5 and the B cell marker CD19 in cerebrospinal fluid (CSF) of 21 patients with multiple sclerosis (MS), compared with 17 control subjects with muscular tension headache. Only one patient with MS, but nine controls lacked CD5+ B cells in CSF. This difference was not observed in peripheral blood. Numbers of CD5+19+ B cells were increased in CSF compared with blood in MS, but not in the controls. In both groups, CD5+19+ B cells were not restricted to small resting lymphocytes, but were also found among larger-sized lymphocytes. The relative density of CD5 molecules and of CD19 molecules was lower in CD5+19+ than in CD5-19+ B cells and CD5+19- T cells. CD5+ B cells are assumed to be responsible for autoantibody production, and our results suggest a pathogenetic role of such cells, predominantly within the central nervous system, in MS.     

Number of interleukin-4- and interferon-γ-secreting human T cells reactive with tetanus toxoid and the mycobacterial antigen PPD or phytohemagglutinin: distinct response profiles depending on the type of antigen used for activation

The enzyme-linked immunospot (ELISPOT) assay has been proven to be an efficient and sensitive method for the enumeration of single cells secreting antibodies or cytokines. Here we have used this method to determine the number of interleukin-4 (IL-4)- and interferon-γ (IFN-γ)-producing cells in in vitro secondary responses to tetanus toxoid (TT) and the mycobacterial antigen (purified protein derivative; PPD) or the mitogen phytohemagglutinin (PHA). PHA-induced IL-4 and IFN-γ secretion was well correlated suggesting polyclonal activation of cells. This was not the case with the specific antigens, where PPD preferentially induced IFN-γ- and very few IL-4-producing cells, while TT-induced both IL-4 and IFN-γ. These differences are probably a reflection of the types of immunity the two antigens induce, mycobacteria preferentially inducing a cell-mediated T helper type 1 (Th 1) type of immunity, while immunity to tetanus is an antibody-dependent, Th 2 type of response. In individuals recently boosted with TT, a significant increase in both IL-4- and IFN-γ-producing cells in response to TT was seen at day 7 after boost, followed by decline. This was in contrast to what was seen in response to PPD where an increase of IFN-γ-producing cells after the TT boost at day 7 persisted for at least 14 days. These results suggest that after an in vivo boost both antigen-specific and nonspecific T cells are activated and that antigen-specific cells home to other organs and therefore may be difficult to demonstrate in the circulation. Our data show that the ELISPOT assay is a powerful tool for determining the frequency of cells secreting cytokines. The assay has several advantages over other assays since it is sensitive, measures the number of actually secreting cells, and avoids the problems of binding of cytokines to their cell-bound or soluble receptors.