Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig)

1. Cells were isolated by incubating chunks of tissue from the urinary bladder of the guinea-pig in a high potassium, low chloride medium containing 0.2 mM calcium plus the enzymes collagenase and pronase. After isolation, the cells were superfused with a physiological salt solution (PSS) containing 150 mM NaCl, 3.6 mM CaCl₂ and 5.4 mM KCl (35°C). Patch electrodes filled with an isotonic KCl-solution were used for whole cell recordings. With a single electrode voltage clamp we measured a capacitance of 50±5 pF per cell, an input resistance of 200±25 kOhm ·cm² and a series resistance of 44±4 Ohm·cm².
2. The cells had resting potentials of ?52±2 mV. They did not beat spontaneously but responded to stimuli with single action potentials (APs) which rose from the threshold (?38 mV) with a maximal rate of 6.5±1.8 V/s to an overshoot of 22±3 mV. The AP lasted for 36±4 ms (measured between threshold and ?40 mV). Continuous cathodal current produced repetitive activity, a pacemaker depolarization followed the AP and preceded the next upstroke.
3. Net membrane currents evoked by clamp steps to positive potentials were composed of an inward and an outward component. The inward component generating the upstroke of the AP was carried by Ca ions (i _Ca, Klöckner and Isenberg 1985). The repolarization resulted from a potassium outward currenti _K. Ca-channel blockers (5 mM NiCl₂) reducedi _K suggesting that (part of)i _K was Ca-activated.
4. i _K rose within about 100 ms to a peak of 40–200 μA/cm² from which it inactivated slowly and incompletely. The inactivatingi _K followed a bell-shaped voltage-dependence, the noninactivatingi _K an outwardly rectifying one. Both parts had similar steady state inactivation curves with a half maximal inactivation potential at ?36 mV and a slope of 9 mV.
5. Repolarization to ?50 mV induced outward tail currents which reversed polarity at ?85 mV (the calculated potassium equilibrium potential). The amplitude and the time course of the envelope of the tail currents varied in proportion toi _K during the prestep. Thus, the tail current is suggested to reflect the turning off of a potassium conductance which had been activated during the prepulse.
6. i _K was largely reduced but not blocked by 20 or 150 mM tetraethylammonium (TEA). TEA did not significantly change the resting potential, but it prolonged the AP and facilitated upstroke and overshoot.i _K could be blocked by loading the cells with Cs released from Cs-filled patch electrodes.
7. We compare the results with the data from multicellular tissue (Creed 1971). The more negative resting potential and the absence of spontaneous APs are mainly attributed to the absence of transmitter release from nerve terminals. The isolated cell is suggested as a model of the postsynaptic membrane properties.

     

Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain: a novel mechanism for acquisition of cross-reactivity by an autoantibody

We have recently shown that the anti-cardiolipin activity of human anti-phospholipid antibody UK4 (lambda) resides on its heavy chain. We now show that UK4 possesses strong reactivity to the plasma-protein beta2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 (kappa) with no reactivity to beta2-GPI, we now demonstrate that both the constructs possessing the auto-antibody-derived light chain exhibited significant binding to beta2-GPI. However, the construct possessing UK4 heavy chain in association with 4D5 light chain, exhibited no anti-beta2-GPI activity. Furthermore, there was a low increase (approximately 10%) in the binding of UK4 to cardiolipin in the presence of beta2-GPI. The results demonstrate that anti-beta2-GPI activity resides on UK4 light chain and, importantly, this activity could be transferred to a novel antibody construct via the light chain alone. Computer-generated models of the three-dimensional structures of UK4 and its hybrids, suggest predominant interaction of UK4 light chain with domain IV of beta2-GPI. Molecular docking experiments highlight a number of potential sites on beta2-GPI for interaction of UK4 and indicate as to how beta2-GPI recognition may occur primarily via the autoantibody light chain. The study provides first demonstration of the occurrence of anti-phospholipid and anti-beta2-GPI activities separately on heavy and light chains of an autoantibody. The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed.     

New therapies for systemic lupus erythematosus

In the past 40 years, prognosis for patients with systemic lupus erythematosus (SLE) has improved, with 10-year survival now approximately 90%. This is due probably to a combination of earlier disease diagnosis and diagnosis of milder disease, due in part to availability of multiple serological tests for SLE, use of steroids and other immunosuppressive agents, and availability of renal dialysis and transplantation. Despite this, however, the potential for significant morbidity and mortality remains in the group of patients with partially responsive or treatment resistant disease. More recently, advancements in the understanding of molecular mechanisms involved in the pathogenesis of SLE have translated to the development of novel therapies, offering possible alternatives to this patient cohort. Discussion of these pharmacological options and ongoing research forms the basis of this review.     

New therapies for rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease, which continues to cause significant morbidity in affected persons. In the past few years, a number of new exciting therapeutic options have become available. These reflect the application of knowledge obtained from advancements in understanding of disease pathogenesis and underlying molecular mechanisms. A number of these therapies are outlined in the following review, including the various biological modifiers, in particular, anti-tumour necrosis factor-alpha agents and interleukin-1 (IL-1) receptor antagonists, which have been developed in recognition of the role of pro-inflammatory cytokines in RA. Also notable, is the current interest centring on the development and trials with B cell depletion therapies, specifically rituximab, in patients with RA. This demonstrates acknowledgment for a more significant role for B cells in the aetiology of RA, in contrast to the long held view that RA was a predominantly T cell mediated disease. To evaluate this therapeutic option for RA, salient features from recent rituximab trials have been collated. Finally, a selection of other therapeutic alternatives, including anti-IL-6 receptor monoclonal antibody and tacrolimus, and newer anti-rheumatic therapies presently in development are summarized.     

Genotypic variation and slime production among blood and catheter isolates of Candida parapsilosis.

Candida parapsilosis is an important nosocomial pathogen that can proliferate in high concentrations of glucose and form biofilms on prosthetic materials. We investigated the genotypic diversity and slime production among 31 isolates of C. parapsilosis from individual patients with bloodstream or catheter infections. DNA subtyping was performed by using electrophoretic karyotyping plus restriction endonuclease analysis with BssHII followed by pulsed-field gel electrophoresis. Slime production was evaluated by growing organisms in Sabouraud broth with 8% glucose and examining the walls of the tubes for the presence of an adherent slime layer. Overall there were 14 DNA subtypes among the 31 isolates. Eighty percent of the isolates produced slime; 67% of the isolates were moderately to strongly positive, 13% were weakly positive, and 20% were not slime producers. The ability of isolates of a given DNA type to produce slime under these conditions was variable. The results of these studies indicate moderate genotypic variation among clinical isolates of C. parapsilosis. The propensity of these isolates to form slime in glucose-containing solutions suggests that this phenotypic characteristic may contribute to the ability of C. parapsilosis to adhere to plastic catheters and cause infections.     

Anti-SSA/Ro52 autoantibodies blocking the cardiac 5-HT4 serotoninergic receptor could explain neonatal lupus congenital heart block

The 52-kDa SSA/Ro (Ro52) ribonucleoprotein is an antigenic target strongly associated with the autoimmune response in mothers whose children develop neonatal lupus and congenital heart block. When sera from patients with systemic lupus erythematosus were used as autoimmune controls in an enzyme immunoassay to screen for antibodies against the human serotoninergic 5-HT4-receptor, a high correlation was found between the presence of anti-Ro52 protein antibodies in such sera and antibodies reacting with a synthetic peptide, corresponding to the second extracellular loop of the human 5-HT4 receptor (amino acid residues 165-185). Homology scanning between the 5-HT4 peptide and the sequence of the Ro52 protein indicated two potential common epitopes located between residues 365 and 396 of the Ro52 protein. Cross-reactivity was found between the peptide derived from the 5-HT4 receptor, and a peptide corresponding to residues 365-382 of the Ro52 protein. Autoantibodies, affinity-purified on the 5-HT4 receptor peptide, specifically recognized both the Ro52 protein and the 5-HT4 receptor protein in immunoblots. The affinity-purified antibodies antagonized the serotonin-induced L-type Ca channel activation on human atrial cells. This effect could explain the electrophysiological abnormalities in neonatal lupus.     

Use of disinfectants to reduce microbial contamination of hubs of vascular catheters.

The vascular catheter hub is a potential portal of entry for microorganisms that cause catheter-related sepsis. Thus, a reduction in catheter hub contamination might reduce the incidence of catheter-related sepsis. To develop a regimen suitable for reducing microbial contamination of the catheter hub, we experimentally contaminated catheter hubs and assessed the efficacies of disinfectant solutions. Catheter hubs were incubated overnight with suspensions of Staphylococcus epidermidis, Pseudomonas aeruginosa, or Candida parapsilosis. After removal of unattached microorganisms, the catheter hubs were swabbed by rotating cotton swabs dipped in 1% chlorhexidine, 1% chlorhexidine in 70% ethanol, 70% ethanol, 97% ethanol, or normal saline. Posttreatment swabs of the catheter hub were obtained and cultured quantitatively. The cleaning regimens containing ethanol were the most effective. Seventy percent ethanol was more effective than chlorhexidine and is likely to be the safest treatment. We conclude that cleaning of the catheter hub with disinfectant can dramatically reduce microbial contamination.