[Diagnosis and therapy of chlamydia infections].
Explore the source record for details and available documents.
Chlamydia Infections↗
Biomedical subjects
A Essig
Publications and source records attributed to A Essig.
Infection of Acanthamoeba castellanii by Chlamydia pneumoniae.
Chlamydia pneumoniae is an intracellular respiratory pathogen, which, similar to Legionella, might have developed mechanisms to escape the intracellular bactericidal activity of both human host cells and amoeba. We therefore investigated the intracellular growth and survival of C. pneumoniae in Acanthamoeba castellanii by using cell culture, immunofluorescence microscopy, and electron microscopy. A castellanii was incubated with purified elementary bodies of C. pneumoniae TW 183 at a concentration of 10(6) inclusion-forming units (IFU)/ml to give a ratio of approximately 1 IFU of C. pneumoniae per amoeba. Quantitative determination of chlamydial growth within A. castellanii revealed viable and infective C. pneumoniae in the range of 10(4) to 10(5) IFU/ml between days 7 and 14 postinfection. Immunofluorescence analysis and transmission electron microscopy with subsequent immunogold staining confirmed evidence of infection of the amoebae by C. Pneumoniae and additionally revealed that C. pneumoniae entered the typical growth cycle. Our results show that amoebae allow the survival of C. pneumoniae, suggesting that amoebae may serve as an additional reservoir for Chlamydia or Chlamydia-related organisms.
Acanthamoeba↗
Growth of Chlamydia pneumoniae induces cytokine production and expression of CD14 in a human monocytic cell line.
Chlamydia pneumoniae was able to survive and to multiply in the human monocytic cell line Mono Mac 6. Growth of C. pneumoniae induced production of tumor necrosis factor alpha, interleukin 1beta, and interleukin 6, as well as up-regulation of the CD14 molecule in a time-dependent manner. Infection of monocytic cells and a proinflammatory cytokine response may be important in C. pneumoniae pathogenesis.
Cell Line↗
A model of genital Chlamydia trachomatis infection using human xenografts in severe combined immunodeficiency mice.
We developed a new model of human genital Chlamydia trachomatis infection in order to characterize the pathogen-host relationship in a clinically relevant system using a human strain of C. trachomatis instead of the commonly employed mouse biovar (MoPn). Human endometrial tissue was xenografted into the skin of mice homozygous for the mutation severe combined immunodeficiency and inoculated with C. trachomatis serovar K. C. trachomatis efficiently infected the endometrium as shown by cell culture and immunofluorescence microscopy and persisted for more than 6 weeks. Chlamydial inclusions detected by direct immunofluorescence and electron microscopy appeared to be smaller than those produced by in vitro cell culture-grown chlamydiae. A pattern of localized mild infection prevailed, and infiltrative uncontrolled spread of chlamydiae was observed in only 1 of 10 infected grafts. This might correspond to the well-known tendency of the agent to cause asymptomatic infections. This model allows the study of a human genital infection resembling the clinical situation and offers the possibility to better characterize the host-parasite relationship with respect to pathogenicity and therapy.
Animals↗
Diagnosis of ornithosis by cell culture and polymerase chain reaction in a patient with chronic pneumonia.
We report the case of a woman who had pneumonia due to Chlamydia psittaci. A Chlamydia species was determined to be the causative agent of the pneumonia because it was isolated from bronchoalveolar lavage fluid, because it could be detected in lung biopsy specimens by the direct immunofluorescence technique, and because Chlamydia-specific antibodies could be detected by ELISA and microimmunofluorescence. The infectious agent could not be identified at the species level with use of serological techniques, but the isolate was determined to be C. psittaci by PCR with use of species- and genus-specific sequences within the chlamydial lipopolysaccharide biosynthesis gene gseA. The case reported herein exemplifies the problems encountered in diagnosing ornithosis and shows that isolation of the etiologic agent followed by identification of the species by PCR is helpful in diagnosing this rare disease. In addition, the findings in our case show that laboratory personnel who are conducting tests for Chlamydia pneumoniae should be aware of the risk of accidentally isolating highly infectious C. psittaci organisms.
Aged↗
Cleavage of tumor necrosis factor-alpha by Legionella exoprotease.
The role of the major secretory protein of Legionella pneumophila, a zinc protease, in Legionella infection is not known. Since an important step of the host reaction in Legionnaires' disease is the production of tumor necrosis factor-alpha (TNF-alpha) by alveolar macrophages, we studied the interaction of Legionella protease and U-937 cells with respect to TNF-alpha. The Legionella protease was purified by fractionated precipitation, gel filtration and hydrophobic interaction chromatography. The purified enzyme was added to U-937 cells, a promyelocytic cell line. In the supernatants of PMA-treated U-937 cells we found low concentrations of TNF-alpha after incubation with protease. Therefore we pursued the hypothesis of direct enzymatic degradation of TNF-alpha by Legionella protease. Enzymatic cleavage of TNF-alpha was proven by SDS-PAGE, ELISA and TNF-alpha bioassay with L-929 cells. The degradation of TNF-alpha by the Legionella protease was shown in all three systems. Enzymatic degradation of TNF-alpha might be important for the pathogenesis of Legionnaires' disease.
Chromatography, Gel↗
Growth in serum-free medium improves isolation of Chlamydia pneumoniae.
Infectivity titers were determined for eight Chlamydia pneumoniae strains simultaneously grown in serum-free and serum-supplemented cell culture media. Use of serum-free medium resulted in a 10- to 50-fold increase in the susceptibility of HL cells to chlamydial infection. Comparative primary isolation of a wild-type strain also produced higher inclusion counts in a serum-free environment. Serum-free cultivation is recommended to increase the efficiency of C. pneumoniae isolation from clinical material and to permit elementary body purification without interference caused by serum components.
Chlamydophila pneumoniae↗
Water movement: does thermodynamic interpretation distort reality?
In a recent theoretical analysis of water flow, Finkelstein (Water Movement Through Lipid Bilayers, Pores, and Plasma Membranes: Theory and Reality, 1987) has attacked the contributions of irreversible thermodynamics, stating that "the thermodynamic treatment of uphill water flow completely distorts reality." Instead he presents a mechanistic formulation. For a porous membrane, water flow is attributed to convection generated by a favorable hydrostatic pressure gradient within pores, even when in the presence of permeant solutes water moves against its chemical potential gradient; water flow may "drag", solute, to an extent determined by the solute partition coefficient, but the possibility that solute flow may drag water is excluded. We argue that this formulation violates the second law of thermodynamics. Water cannot move against its chemical potential gradient because of the influence of only part of the chemical potential gradient. Furthermore, the proposed mechanism requires that at one of the membrane-solution interfaces water must move against both its concentration gradient and the hydrostatic pressure gradient. Also considered by Finkelstein is the nature of the reflection coefficient sigma, a kinetic variable, which he concludes can be evaluated (in a porous membrane) by measurement of the (equilibrium) solute partition coefficient. We claim that in general it is not possible to evaluate a kinetic variable from measurements of equilibrium parameters alone. A valid kinetic analysis must incorporate the contribution of all coupled flows.
Body Water↗
Influence of serosal Cl on transport properties and cation activities in frog skin.
The effects of serosal substitution of isosmotic Na2SO4-Ringer solution for NaCl-Ringer solution were studied in the short-circuited frog skin (Rana pipiens, Northern variety). Despite prompt changes of transepithelial measurements, initial cellular effects were slight. After 30 to 45 min, however, the transcellular current had decreased and the cell electrical potential had depolarized, in association with decrease of the apical membrane fractional resistance and basolateral membrane conductance. Apical membrane slope conductance was unaffected. Similar effects were obtained with isolated epithelia. With the use of gluconate or NO3 in place of Cl, the effects on cellular current and conductance were minimal or insignificant, despite changes of the cell potential, fractional resistance, and basolateral conductance similar to those seen with sulfate. Following prolonged exposure to serosal SO4-Ringer, the extent of depolarization induced by raising the serosal K concentration decreased, indicating diminution of basolateral K conductance and the existence of other basolateral conductances. Equilibration in serosal gluconate-Ringer enhanced polarization on serosal restoration of Cl or removal of Na, again indicating a time-dependent change in the basolateral conductance pattern. Depolarization on removal of serosal Cl was not attributable to inhibition of the pump. Nor was it the result of decrease of the K equilibrium potential EK: exposure to serosal SO4-Ringer decreased cell K activity aKc from 104 +/- 6 to 58 +/- 4 mM (n = 5), but EK was reduced only slightly; exposure to serosal gluconate increased aKc and EK. Serosal sulfate lowered the cell Na activity aNac, but the electrochemical potential difference for Na across the apical surface was unaffected. The concurrent decrease of both aKc and aNac following serosal substitution of SO4 for Cl raises questions concerning mechanisms of osmoregulation.
Amiloride↗
Voltage dependence of cellular current and conductances in frog skin.
Knowledge of the voltage dependencies of apical and basolateral conductances is important in determining the factors that regulate transcellular transport. To gain this knowledge it is necessary to distinguish between cellular and paracellular currents and conductances. This is generally done by sequentially measuring transepithelial current/voltage (It/Vt) and conductance/voltage (gt/Vt) relationships before and after the abolition of cellular sodium transport with amiloride. Often, however, there are variable time-dependent and voltage-dependent responses to voltage perturbation both in the absence and presence of amiloride, pointing to effects on the paracellular pathway. We have here investigated these phenomena systematically and found that the difficulties were significantly lessened by the use of an intermittent technique, measuring It and gt before and after brief (less than 10 sec) exposure to amiloride at each setting of Vt. I/V relationships were characterized by these means in frog skins (Rana pipiens, Northern variety, and Rana temporaria). Cellular current, Ic, decreased with hyperpolarization (larger serosa positive clamps) of Vt. Derived Ic/Vt relationships between Vt = 0 and 175 mV (serosa positive) were slightly concave upwards. Because values of cell conductance, gc, remained finite, it was possible to demonstrate reversal of Ic. Values of the reversal potential Vr averaged 156 +/- 14 (SD, n = 18) mV. Simultaneous microelectrode measurements permitted also the calculation of apical and basolateral conductances, ga and gb. The apical conductance decreased monotonically with increasing positivity of Vt (and Va). In contrast, in the range in which the basolateral conductance could be evaluated adequately (Vt less than 125 mV), gb increased with more positive values of Vt (and Vb). That is, there was an inverse relation between gb and cellular current at the quasi-steady state, 10-30 sec after the transepithelial voltage step.
Amiloride↗
Cell sodium activity and sodium pump function in frog skin.
Cell Na activity, acNa, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and Na-selective microelectrodes. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular conductance, with NaNO3, Ringer on the apical surface. Under control conditions acNa averaged 8 +/- 2 mM (n = 9, SD). Apical addition of amiloride (20 microM) or Na replacement reduced acNa to 3 mM in 6-15 min. Sequential decreases in apical [Na] induced parallel reductions in acNa and cell current, Ic. On restoring Na after several minutes of exposure to apical Na-free solution Ic rose rapidly (approximately less than 30 sec) to a stable value while acNa increased exponentially, with a time constant of 1.8 +/- 0.7 min (n = 8). Analysis of the time course of acNa indicates that the pump Na flux is linearly related to acNa in the range 2-12 mM. These results indicate that acNa plays an important role in relating apical Na entry to basolateral active Na flux.
Animals↗
Basolateral membrane potential and conductance in frog skin exposed to high serosal potassium.
In studies of apical membrane current-voltage relationships, in order to avoid laborious intracellular microelectrode techniques, tight epithelia are commonly exposed to high serosal K concentrations. This approach depends on the assumptions that high serosal K reduces the basolateral membrane resistance and potential to insignificantly low levels, so that transepithelial values can be attributed to the apical membrane. We have here examined the validity of these assumptions in frog skins (Rana pipiens pipiens). The skins were equilibrated in NaCl Ringer's solutions, with transepithelial voltage Vt clamped (except for brief perturbations delta Vt) at zero. The skins were impaled from the outer surface with 1.5 M KCl-filled microelectrodes (Rel greater than 30 M omega). The transepithelial (short-circuit) current It and conductance gt = -delta It/delta Vt, the outer membrane voltage Vo (apical reference) and voltage-divider ratio (Fo = delta Vo/delta Vt), and the microelectrode resistance Rel were recorded continuously. Intermittent brief apical exposure to 20 microM amiloride permitted estimation of cellular (c) and paracellular (p) currents and conductances. The basolateral (inner) membrane conductance was estimated by two independent means: either from values of gt and Fo before and after amiloride or as the ratio of changes (-delta Ic/delta Vi) induced by amiloride. On serosal substitution of Na by K, within about 10 min, Ic declined and gt increased markedly, mainly as a consequence of increase in gp. The basolateral membrane voltage Vi (= -Vo) was depolarized from 75 +/- 4 to 2 +/- 1 mV [mean +/- SEM (n = 6)], and was partially repolarized following amiloride to 5 +/- 2 mV.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Time-dependent phenomena in voltage-clamped epithelia.
Explore the source record for details and available documents.
Animals↗
Cell K activity in frog skin in the presence and absence of cell current.
Cell K activity, acK, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30-60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO3 Ringer on the apical surface. Under control conditions acK was nearly constant among skins (mean +/- SD = 92 +/- 8 mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 microA/cm2). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase in acK after inhibition of cell current was observed, on the average the change was not significant (98 +/- 11 mM after amiloride, 101 +/- 12 mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from -42 to -77 mV, hyperpolarized after inhibition of cell current, initially to -109 +/- 5 mV, then depolarizing to a stable value (-88 +/- 5 mV) after 15-25 min. At this time K was above equilibrium (EK = 98 +/- 2 mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport. The measurement of acK permitted the calculation of the passive K current and pump current under control conditions, assuming a "constant current source" with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.
Amiloride↗
Capacitative transients in voltage-clamped epithelia.
In voltage-clamped epithelia the cell membrane potential transient during a + 10-mV transepithelial pulse conforms to the expected behavior for a series combination of two linear resistance-capacitance (RC) circuits. The evolution of the cell potential is characterized by a single time constant with values of 30-130 ms in frog skin and Necturus gallbladder. These observations have important consequences for the measurement of cell membrane resistance ratios and the interpretation of current-voltage relations.
Amiloride↗
Voltage-dependence of Ca2+ uptake and ATP hydrolysis of reconstituted Ca2+-ATPase vesicles.
Ca2+-ATPase from sarcoplasmic reticulum was reconstituted into phospholipid/cholesterol (9:1) vesicles (RO). Sucrose density gradient centrifugation of the RO vesicles separated a light layer (RL) with a high lipid/protein ratio and a heavy layer (RH). RH vesicles exhibited a high rate of Ca2+-dependent ATP hydrolysis but did not accumulate Ca2+. RL vesicles, on the other hand, showed an initial molar ratio of Ca2+ uptake to ATP hydrolysis of approximately 1.0. Internal trapping of transported Ca2+ facilitated studies over periods of several minutes. Ca2+ transport and ATP hydrolysis declined concomitantly, reaching levels near 0 with external Ca2+ concentrations less than or equal to 2 microM. Ca2+ uptake was inhibited by the Ca2+ ionophore A23187, the detergent Triton X-100, and the metabolic inhibitor quercetin. Ca2+ transport generated a transient electrical potential difference, inside positive. This finding is consistent with the hypothesis that the Ca2+ pump is electrogenic. Steady state electrical potentials across the membrane were clamped by using potassium gradients and valinomycin, and monitored with voltage-sensitive dyes. Over a range of +50 to -100 mV, there was an inverse relationship between the initial rate of Ca2+ uptake and voltage, but the rate of ATP hydrolysis was nearly constant. In contrast, lowering the external Ca2+ concentration depressed both transport and ATP hydrolysis. These findings suggest that the membrane voltage influences the coupling between Ca2+ transport and ATP hydrolysis.
Adenosine Triphosphate↗
Contribution of junctional conductance to the cellular voltage-divider ratio in frog skins.
It has been suggested that distribution of lateral interspace resistance in association with a highly conductive junction can significantly affect the measurement of outer membrane(o)/epithelial(t) voltage divider ratios (Fo = delta Vo/delta Vt), thereby leading to erroneous inferences regarding the outer membrane fractional resistance [fRo = Ro/Rc = Ro/(Ro + Ri)], where Ro and Ri are the outer and inner cell membrane resistance respectively and Rc is the total cell membrane resistance. We present here experimental evidence for this point of view. During seasons when frog skins were highly permeable to Cl, transepithelial conductance gt often exceeded 2 mS/cm2. High concentrations of external amiloride rapidly blocked cellular transport, but gt initially remained high and Fo remained appreciably less than 1.0. These values of Fo were found here to result from low junctional resistance Rj: increase of Rj, either gradually following the administration of amiloride, or abruptly with external replacement of Cl by other anions, was associated with increase of Fo to near unity, without effect on the membrane potential or significant change in the short-circuit current. Experimental results following amiloride validated a simple equivalent circuit model predicting near-linear increase in Fo with progressive decrease in gt and led to plausible values of Rj and lateral space resistance Rl. The possible influence of the paracellular resistance pattern on the evaluation of cell membrane resistances from voltage divider ratios is discussed.
Abdomen↗
Voltage-dependent K conductance at the apical membrane of Necturus gallbladder.
The epithelial and cellular effects of clamping the transepithelial potential (Vt, mucosa reference) have been investigated in the Necturus gallbladder. Following initial equilibration at short circuit, tissue conductance gt was 4.1 +/- 1.2 (SD) mS/cm2, the apical potential Va was -76 +/- 8 mV, and the apical fractional voltage on brief voltage perturbation (fa = delta Va/delta Vt, reflecting the ratio of apical membrane to transcellular resistance) was 0.72 +/- 0.11 (21 gallbladders, 34 impalements). On clamping Vt at positive values, Va depolarized and fa decreased; at the same time gt decreased. Clamping Vt at negative values produced converse effects. All of the above changes were related directly to the magnitude of the clamping potential Vt and were reversed on return to the short circuit state. Effects of Vt on fa are not due to changes in the extracellular pathway resistances (which, however, contribute to gt). Furthermore, the effects of Vt on fa were abolished by the mucosal application of TEA or Ba, or acidification of the mucosal solution. Thus, these experiments disclose the presence of a voltage-dependent apical K conductance that increases with apical membrane depolarization. The calculated dose-response curve of TEA inhibition of apical conductance and the values of the apparent dissociation constant were in good agreement with those found for K channels in excitable tissues. Mucosal application of the Ca ionophore A23187 shifted the voltage dependence curve of fa to more negative values of Va without altering its shape. The effect of A23187 suggests a possible role of intracellular Ca in the modulation of the apical K channels.
Animals↗