Functional assay of multidrug resistant cells using JC-1, a carbocyanine fluorescent probe

Multidrug resistance (MDR) is characterized by a decrease in the efficiency of chemotherapeutic agents correlated with the expression and activity of a membrane protein: the permeability-glycoprotein (Pgp 170). Clinically, detection of MDR can be performed by functional tests based on the accumulation of fluorescent compounds such as rhodamine 123. With the aim of improving the sensitivity of such analysis, we have evaluated JC-1, a fluorescent lipophilic carbocyanine dye. Above a critical concentration, JC-1 aggregates in a 'liquid crystal' form. Aggregates display a specific red emission band centered at 597 nm whereas the monomers display a green emission band centered at 540 nm. JC-1 was avidly accumulated in sensitive K562 cells where it displayed both a green cytoplasmic and red mitochondrial fluorescence. In contrast, JC-1 was poorly accumulated in resistant K562 cells, which displayed only a slight green fluorescence. The level of JC-1 accumulation was correlated with the level of Pgp expression detected by MRK16 and UIC2 antibodies on a set of K562 subclones with increasing resistance levels. The specific fluorescence properties of JC-1 allow accurate discrimination between low-level resistant cells and sensitive cells. Chemosensitizers such as verapamil, cyclosporine A or S9788 restored JC-1 accumulation in resistant cells. The fluorescence properties of JC-1 could therefore be used for monitoring the effects of reversing agents.     

Preclinical evaluation of a novel group B meningococcal conjugate vaccine that elicits bactericidal activity in both mice and nonhuman primates

Group B meningococcal (GBM) conjugate vaccines were prepared using chemically modified N-propionylated polysialic acid, from Escherichia coli K1 polysaccharide capsule, coupled by reductive amination to tetanus toxoid and purified recombinant GBM porin (rPorB). All conjugates elicited high antibody levels in mice with good booster responses. However, only rPorB conjugates elicited bactericidal activity specific against a broad spectrum of five different GBM serotypes. Bactericial activity was completely inhibited by free N-propionylated polysaccharide. In baboons and rhesus monkeys, rPorB conjugates elicited high antibody titers, with IgG booster responses 9- to 15-fold higher than primary responses. Bactericial activity increased 19- to 39-fold over preimmune values, using rabbit complement; increased bactericial activity was also confirmed with human and monkey complement. IgG cross-reactivity for unmodified N-acetyl polysaccharide was <5% for 79% of mice and <10% for 80% of primates. These studies strongly suggest that the N-propionylated polysialic acid-rPorB conjugate is an excellent vaccine candidate for human use.     

Differential consequences of lateral and central fluid percussion brain injury on receptor coupling in rat hippocampus

We have identified alterations in the responses of muscarinic and metabotropic receptors in rat hippocampus that persist for at least 15 days after central fluid percussion injury. This study compares the effect of lateral fluid percussion and central fluid percussion on these responses. Moderate injury was obtained by displacement and deformation of the brain within the closed cranial cavity using a fluid percussion device positioned either centrally or laterally. Carbachol and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated polyphosphoinositide (PPI) hydrolysis was assayed in hippocampus from injured and sham-injured controls at 15 days following injury. At 15 days after central fluid percussion traumatic brain injury (TBI), the response to carbachol was enhanced by 30% and the response to trans-ACPD was enhanced by 75% compared to sham-injured animals. At 15 days after lateral fluid percussion TBI the response to trans-ACPD was enhanced by 40% both ipsilateral and contralateral to the side of injury. In contrast, the response to carbachol was enhanced by 29% contralateral to the side of injury but was diminished by 12% ipsilateral to the side of injury. Cresyl violet staining shows no hippocampal cell death after central fluid percussion injury or on the side contralateral to lateral fluid percussion injury but on the ipsilateral side cell death was identified in hippocampal area CA3. Thus, abnormal hippocampal cell signaling through the phosphoinositide pathway occurs in the absence of cell death and may contribute to cognitive impairment.     

Binding of anti-CD23 monoclonal antibody to the leucine zipper motif of FcepsilonRII/CD23 on B cell membrane promotes its proteolytic cleavage. Evidence for an effect on the oligomer/monomer equilibrium

In the present study we have compared the binding of two monoclonal antibodies to CD23, EBVCS1 and mAb25, which recognize the stalk and the lectin domain, respectively, on the CD23 molecule. At 4 degreesC, EBVCS1 binds to about 10% of the receptors recognized by mAb25 on the B cell surface. At 37 degreesC, whereas mAb25 reaches its maximal binding within a few seconds, EBVCS1 requires 60 min to bind to the same extent. Stabilization of the oligomeric structure of CD23 with IgE strongly affects in a dose-dependent fashion the number of binding sites seen by EBVCS1 but not the t1/2 to reach them, suggesting that EBVCS1 binds to the coiled coil region through an allosteric mechanism. EBVCS1 rapidly down-modulates the membrane CD23 expression with a coincident increase of CD23-soluble fragments in the culture medium, an effect that is inhibited by IgE. In contrast, mAb25, as well as IgE, protects CD23 from proteolytic cleavage and stimulates its endocytosis. These results suggest that EBVCS1 unravels the coiled coil structure of CD23, rendering it more susceptible to proteolytic attack. This supports the oligomeric model proposed previously (Gould, H., Sutton, B., Edmeades, R., and Beavil, A. (1991) Monogr. Allergy 29, 28-49). The biological significance of these observations is discussed.     

Antinuclear autoantibodies in human T-cell leukemia/lymphoma virus type I carriers in French Guiana

To compare the rate of antinuclear antibodies (ANA) in HTLV-I carriers and negative individuals in French Guiana, 350 sera (175 HTLV-I carriers, either symptomatic or not, and 175 controls) were screened for ANA, using an immunofluorescence assay. All positive sera were tested for autoantibodies against extractable nuclear antigens, histones and double stranded DNA. ANA were detected in 9.71% of the HTLV-I carriers and 3.43% of the control group (p < 0.05). There was no difference in ANA distribution by age, sex, or ethnic group. Neither was there any difference between asymptomatic and symptomatic HTLV-I individuals. However, ANA of medical interest were significantly higher (p < 0.04) in HTLV-I seropositive Creoles than in seropositive Noir-Marrons.     

Hypothalamic gamma-aminobutyric acid neurons project to the neocortex

Three groups of gamma-aminobutyric acid--containing neurons were found in the mammillary region of the posterior hypothalamus. The groups correspond to the tuberal, caudal, and postmammillary caudal magnocellular nuclei. Many cells in these nuclei were retrogradely labeled with fast blue after the injection of this fluorescent dye into the neocortex. Immunohistochemical experiments showed that these same neurons also contained the gamma-aminobutyric acid-synthesizing enzyme glutamate decarboxylase. These results provide morphological evidence for a gamma-aminobutyric acid pathway arising in magnocellular neurons of the posterior hypothalamus and innervating the neocortex.     

Surgery of the pudendal nerve in various types of perineal pain: course and results

In 1989, we reported our thoughts on the neurophysiological and anatomic aspects of pudendal nerve involvement in certain types of perinal pain. Since that time, the surgical approach has been modified. Here we report our follow-up of 40 patients with 48 operated nerves. Follow-up ranged from 6 months to 7 years and outcome revealed improvement in 67% and no change in 33%. Thus surgery had been useful in two-thirds of the cases; in 44% of the patients, there was either a frank improvement or no change. Early diagnosis appears to be the determining factor in improving results. Operating for the canal syndrome must be performed before lesions to the nervous trunk become too important.