Detection of radiolabelled bone marrow cells bearing surface ace immunoglobulins by combined autoradiography and immunoperoxidase

A method is described for the simultaneous detection of radiolabelled bone marrow cells bearing surface immunoglobulins by combined autoradiography and immunoperoxidase. Bone marrow cells from normal CBA mice prelabelled in vivo with ¹²⁵IUDR or exposed in vitro to [³H]thymidine were incubated with rabbit anti-mouse immunoglobulins under capping conditions, washed, cytocentrifuged and treated with methanol and hydrogen peroxide to destroy endogenous peroxidase. Cells were then covered with peroxidase-conjugated goat anti-rabbit immunoglobulins, washed, treated with diaminobenzidine a and hydrogen peroxide and finally covered with autoradiographic stripping film and exposed for different times. Peroxidase-positive cells were typically capped and those radiolabelled had autoradiographic silver grains overlying the nucleus.     

Localization of [Met5]- and [Leu5]-enkephalin-like immunoreactivity in nerve terminals in human paravertebral sympathetic ganglia

Both [Leu⁵]- and [Met⁵]-enkephalin have been localized immunohistochemically in nerve fibres and in small, intensely fluorescent cells of adult human sympathetic ganglia. The nerve fibres showing enkephalin-like immunoreactivity formed a network varying in density around the sympathetic neurons, some being closely related to the perikarya. No labelled neuronal cell bodies were found. No structures within the ganglion were labelled after reaction with antibodies to vasoactive intestinal polypeptide, adrenocorticotrophin or substance P. No differences between the distributions of [Leu⁵]-and [Met⁵]-enkephalin-like immunoreactivities were found.The physiological roles of enkephalins are still unknown, but it is possible that they might act as neurotransmitters or neuromodulators in the human sympathetic nervous system.     

The blocking effect of antibodies against the products of the H-2 gene complex on lymphocyte complement (C3d) receptors. complement dependence and specificity

Antisera against the products of the major histocompatibility system (MHS), produced by immunization between congenic mouse strains differing only at that segment of the 17th chromosome rosettes which bears the H-2gene complex, exert a reproducible blocking effect on formation of with EACm (C3d rosettes). In many instances this effect is probably due to specific antibodies reactive with the lymphocytes, as indicated by immunochemical and adsorption experiments. Purified antibodies from the C3H anti-C3H.B10 immune ascites failed to exert this blocking effect. The blocking capacity can be restored, however, by addition of fresh normal mouse, rabbit or fetal calf serum, but not by heat inactivated serum. These experiments show that the presently defined H-2K, H-2D and H-2L antigens as well as some Ia antigens are themselves not the C3d receptors. The blocking effects observed in the presence of complement are possibly due to the proximity of H-2 or Ia molecules and C3d receptors, or to a rearrangement of membrane components after reaction with anti-MHC anti-MHC antibodies and complement.     

Molecular epidemiology of the RNA satellite of <Emphasis Type="Italic">Rice yellow mottle virus</Emphasis> in Africa

Summary. Satellite RNA was sought in 51 isolates of Rice yellow mottle virus (RYMV) representative of the geographical, molecular and pathogenic variability of the virus in Africa. Three-quarters of the isolates from cultivated rice and wild gramineaceous hosts supported a satellite RNA. The prevalence of RYMV isolates that were associated with a satellite differed among regions, being c. 100% in West and Central Africa and c. 36% in East Africa. The RYMV satellite showed a low diversity as only seven of the 220 sequenced positions were variable. One insertion also occurred after serial host passages of the satellite. Two forms of the satellite differed by six substitutions forming three base pairs in one branch of the predicted RNA secondary structure. There was no evidence of intermediates between these two forms, but double-infection occurred. Each form had a specific geographical distribution: one occurred in Central Africa, the other elsewhere in Africa. There was no relation between the occurrence or the forms of the satellite and the phylogeny of the helper virus. The satellite was not involved in symptom modulation or ability to break host-plant resistances to the disease.Received January 29, 2003; accepted April 12, 2003 Published online June 24, 2003     

Synergistic effect of azithromycin on the phagocytic killing ofStaphylococcus aureus by human polymorphonuclear leukocytes

Many macrolides have been shown to affect the interaction between bacteria and various immune defense mechanisms, such as chemotaxis, accumulation, and bioactivity within phagocytic cells. The interaction of azithromycin with human polymorphonuclear leukocytes (PMNs) was studied in vitro and compared with the interactions between other macrolides and PMNs. The opsonophagocytic killing ofStaphylococcus aureus was synergistically enhanced by azithromycin at concentrations below and above the minimal inhibitory concentration, with a reduction of up to 2.82 log₁₀ cfu/ml with 2 mg/ml of azithromycin. Other macrolides were effective only at subinhibitory concentrations. The beneficial azithromycin-leukocyte interaction may explain azithromycin's efficacy against intracellular pathogens.     

Interactive effects of the GABABergic modulation of calcium channels and calcium-dependent potassium channels in lamprey

The GABAB-mediated modulation of spinal neurons in the lamprey is investigated in this study. Activation of GABAB receptors reduces calcium currents through both low- (LVA) and high-voltage activated (HVA) calcium channels, which subsequently results in the reduction of the calcium-dependent potassium (KCa) current. This in turn will reduce the peak amplitude of the afterhyperpolarization (AHP). We used the modulatory effects of GABAB receptor activation on N-methyl-D-aspartate (NMDA)-induced, TTX-resistant membrane potential oscillations as an experimental model in which to separate the effects of GABAB receptor activation on LVA calcium channels from that on KCa channels. We show experimentally and by using simulations that a direct effect on LVA calcium channels can account for the effects of GABAB receptor activation on intrinsic membrane potential oscillations to a larger extent than indirect effects mediated via KCa channels. Furthermore, by conducting experiments and simulations on intrinsic membrane potential oscillations, we find that KCa channels may be activated by calcium entering through LVA calcium channels, providing that the decay kinetics of the calcium that enters through LVA calcium channels is not as slow as the calcium entering via NMDA receptors. A combined experimental and computational analysis revealed that the LVA calcium current also contributes to neuronal firing properties.     

Analysis of myocilin mutations in 1703 glaucoma patients from five different populations

A glaucoma locus, GLC1A, was identified previously on chromosome 1q. A gene within this locus (encoding the protein myocilin) subsequently was shown to harbor mutations in 2-4% of primary open angle glaucoma patients. A total of 1703 patients was screened from five different populations representing three racial groups. There were 1284 patients from primarily Caucasian populations in Iowa (727), Australia (390) and Canada (167). A group of 312 African American patients was from New York City and 107 Asian patients from Japan. Overall, 61 different myocilin sequence variations were identified. Of the 61 variations, 21 were judged to be probable disease-causing mutations. The number of probands found to harbor such mutations in each population was: Iowa 31/727 (4.3%), African Americans from New York City 8/312 (2.6%), Japan 3/107 (2.8%), Canada 5/167 (3.0%), Australia 11/390 (2.8%) and overall 58/1703 (3. 4%). Overall, 16 (76%) of 21 mutations were found in only one population. The most common mutation observed, Gln368Stop, was found in 27/1703 (1.6%) glaucoma probands and was found at least once in all groups except the Japanese. Studies of genetic markers flanking the myocilin gene suggest that most cases of the Gln368Stop mutations are descended from a common founder. Although the specific mutations found in each of the five populations were different, the overall frequency of myocilin mutations was similar ( approximately 2-4%) in all populations, suggesting that the increased rate of glaucoma in African Americans is not due to a higher prevalence of myocilin mutations.     

Cathepsin C gene: First compound heterozygous patient with Papillon-Lefèvre syndrome and a novel symptomless mutation

Papillon-Lefèvre syndrome (PLS) has recently been shown to be caused by mutations in the cathepsin C gene resulting in periodontal disease and palmoplantar keratosis. Thirteen different homozygous mutations have been characterised in PLS patients of different ethnic origin. In the present paper, a PLS patient is described who carries two novel mutations (706G>T and 872G>A) in the paternal and maternal chromosomes, respectively. This is the first compound patient described so far. In addition, a novel symptomless mutation (458C>T) in the cathepsin C gene is described in three homozygous individuals. Thus, not all mutations should be considered as a cause of disease, whether case studies or general population screening is performed. Another already described mutation that provoked the Haim-Munk syndrome (HMS) in Indian Jews has also been found to give rise to PLS in a Spanish family from Madrid. On the other hand, PLS patients are ameliorated by retinoids, which indicates that retinoids may be used as therapeutic agents in this immune system deficiency.     

Domaines d’application et enjeux de l’ingénierie et de la thérapie cellulaires et tissulaires: Quel choix stratégique pour l’EFS ?

A new medical field, known as regeneration medicine is developing and attracting more and more researchers and practitioners. Whereas hematopoietic cell-based therapies have already proven their efficacy in numerous – malignant or not – diseases, non-hematopoietic cell-based therapies have not. They can be useful to dozens, if not hundreds, of patients with various disorders, such as cardiopathy, diabetes, some types of cancer, osteoarticular and neurodegenerative disorders. In these fields, numerous clinical applications are possible for mesenchymal stem cells. Cell and tissue (corneas, bone, skin) therapy products require the definition of pharmaceutical standards with new European requirements in terms of quality and safety. The legitimacy of the Établissement Français du Sang (EFS) in cell and tissue engineering activities is established, it is recognized by most specialists and by regulatory authorities and has been asserted by the orientations of its “contrat d’objectifs et de moyens”. An independent committee has been set up by the EFS President to define an EFS-specific strategy. This committee made up of qualified specialists was required to draw up a rational organization plan for these activities, in order for EFS to be in a position to produce cells and tissues according to pharmaceutical standards. The committee proposals are based on economic data and an inventory of existing cell and tissue engineering activities. Public/private partnerships are required and efforts must focus towards the industrial valorization of EFS expertise in R&;D activities and staff know-how. Implementing such a new organization requires national management and the cooperation of institutional actors (university hospitals, cancer treatment centers, universities). For the success of this approach, EFS personnel must be convinced of its legitimacy and new skills must be encouraged. With its numerous assets, EFS can be ambitious and assert itself as a major actor in cell and tissue engineering in Europe.