Conductance properties and voltage dependence of an anion channel in amphibian skeletal muscle

Single anion-selective channels were studied in excised membrane patches of adult frog toe muscle. The conductance and the probabilityp _o of the main open state were determined for various ionic compositions of the extra-and intracellular solutions. =280 pS in symmetrical 110 mM NaCl, pH 7.4 solutions at 15°C. Higher values were found at elevated internal or external NaCl concentrations, in 70 mM external CaCl₂ and at lower extracellular pH. Thep _o(E) curve declined steeply with hyperpolarization and was shifted towards more negative potentials at increased internal ionic strength and at higher external pH. Positive shifts were induced by extracellular Ca. The results show that the anion channel saturates at Cl concentrations >110 mM, that the potential profile of an open channel is almost symmetrical and that channel gating is affected by neighboring channels. It is suggested that the anion channel has a voltage sensor (effective gating charge 4.3) and a similar collection of local fixed charges on the extra- and intracellular sides as voltage-gated cation channels.     

Diminished neo-antigen response to keyhole limpet hemocyanin (KLH) vaccines in patients after treatment with chemotherapy or hematopoietic cell transplantation

Relapse is the most common cause of treatment failure for advanced cancer, even those treated with autologous hematopoietic cell transplantation (HCT). Effective tumor-specific immunotherapy may decrease relapse, however, this will fail if the immune system is unable to respond. We developed a strategy to test immune responses with a single injection of the bona fide neo-antigen KLH. The model was first tested in 37 normal volunteers using three KLH vaccines: Intracel KLH, Biosyn KLH, and Biosyn KLH + adjuvant. Despite finding the immunogenic epitope conserved in both products, intact Intracel KLH induced a better response compared to a purified 350/390 kDA subunit of KLH contained in the Biosyn KLH product. Addition of a synthetic oil adjuvant (Montanide ISA51) restored the response to a single injection of Biosyn KLH. A quantitative readout measured by a KLH-specific cellular and humoral response with isotype switching 1 month after KLH vaccination was established. To test the integrity of the adaptive immune response in cancer patients, we vaccinated 14 patients post-HCT and 19 patients with advanced cancer with KLH vaccines that elicited a 100% response rate in normal volunteers. In marked contrast to normal subjects, both responses were significantly impaired up to 16 months after autologous HCT with an intermediate response in advanced cancer patients. KLH vaccines are safe and require only a single injection to test neo-antigen responses providing an optimal platform for definitive testing of strategies to improve diminished immune recovery after chemotherapy or post-HCT.     

Saliva-mediated aggregation of Enterococcus faecalis transformed with a Streptococcus sanguis gene encoding the SSP-5 surface antigen.

The interaction of a high-molecular-weight salivary glycoprotein (agglutinin) with Streptococcus sanguis M5 leads to the formation of bacterial aggregates. We have previously shown that the SSP-5 surface antigen from S. sanguis M5 binds the salivary agglutinin and therefore may be involved in the aggregation process. Here we report the transformation of a nonaggregating Enterococcus faecalis strain with the SSP-5 gene and show that the protein is expressed on the cell surface and confers an aggregation-positive phenotype. E. faecalis S161 protoplasts were transformed with pAM401 EB-5, a shuttle vector containing the S. sanguis SSP-5 gene, resulting in the isolation of E. faecalis S161EB-5. Crude cell extracts from this transformant and from S. sanguis M5 were analyzed by Western blotting. Extracts from S. sanguis M5 possessed peptides of 190 and 205 kilodaltons that reacted strongly with polyclonal antibodies against the recombinant SSP-5 antigen. E. faecalis S161EB-5 contained only the 190-kilodalton immunoreactive protein, suggesting that the antigen may be processed differently in E. faecalis S161EB-5. The parent strain, E. faecalis S161, did not react with this antibody preparation. Immunogold labeling of intact E. faecalis S161EB-5 and S. sanguis M5 with anti-SSP-5 immunoglobulin G showed that both organisms expressed similar levels of the antigen. Both organisms formed visible aggregates upon incubation with salivary agglutinin. These results suggest that the SSP-5 antigen may mediate both the binding of agglutinin to S. sanguis M5 and the subsequent formation of bacterial aggregates.     

Improved immunoglobulin production in dialysis patients treated with recombinant erythropoietin.

Improvements in B lymphocyte function have been reported in hemodialysis patients receiving erythropoietin. The present investigation studied whether erythropoietin interferes with B cell function and the mechanisms of this effect. Antibody production by cultured peripheral blood mononuclear cells (PBMC) (7 days) from 15 dialysis patients before and during erythropoietin treatment and from 14 healthy controls was followed. IgG and IgA were formed less in the uremic group than in healthy subjects. After 8 weeks of erythropoietin (hematocrit rose from 19 to 31%) basal IgG formation by PBMC rose from 304 +/- 83 to 566 +/- 49 ng/ml (p less than 0.02), while IgA production rose from 380 +/- 121 to 563 +/- 362 ng/ml (p less than 0.01). IgM production, which appeared to be normal in uremia, remained unchanged during erythropoietin treatment. Production of IgG and IgA stimulated by pokeweed-mitogen was subnormal in uremia, but improved under erythropoietin therapy. To establish whether erythropoietin acted by itself or through correction of the renal anemia, healthy PBMC were directly incubated with 2 U/ml of erythropoietin. Under these conditions production of IgG (+19%), IgA (+28%), and IgM (+32%) was enhanced. Taken together these data indicate a direct stimulant effect of erythropoietin on B lymphocytes in end-stage renal failure.     

Effect of spleen exposure to ultrasound on cellular and antibody-mediated immune reactions in man

Spleen exposure to ultrasound has been reported to influence antibody response to sheep red blood cell injection in mice (decreased hemagglutination and hemolytic titers and IgM, IgG2a and IgG2b levels and elevated IgG1 levels). In a controlled clinical trial, we investigated the possible immunosuppressive side-effect of splenic exposure (2.0 mW/m², 3.5 MHz, 5 minutes) to ultrasound on the immune response to Rubella vaccination in 41 anti-Rubella antibody-negative volunteers. The measured parameters (blood cell count, IgA, IgM, IgG including subclasses IgG1-IgG4, isoagglutinins, anti-Rubella hemagglutinin and hemolysin titers, complement C3, skin tests to mumps and tuberculin, T, B and O lymphocytes, esterase-positive and negative T-cell subsets) suggest changes dependent on the time of vaccination, but provide no evidence of an immunosuppressive effect of ultrasound in man.     

Impaired sodium excretion, decreased glomerular filtration rate and elevated blood pressure in endothelin receptor type B deficient rats

The renal endothelin (ET) system, particularly the ET type B receptor, has been implicated in the regulation of sodium excretion and glomerular filtration rate (GFR). We analyzed kidney morphology and function in a rat strain characterized by complete absence of a functional ETB receptor. Due to Hirschsprung's disease limiting lifetime in these rats, studies were performed in 23-day-old rats. Kidney size and morphology (glomerular and interstitial matrix content, glomerular size and cell density and intrarenal vascular morphology) were normal in ETB-deficient rats. There were also no evidence of altered kidney cell cycle regulation in these rats. GFR was significantly lower, by 72% (P<0.001), in homozygous ETB-deficient rats than in wild-type rats. Fractional sodium excretion was likewise markedly reduced by 84% in homozygous ETB-deficient rats (P<0.001 versus wild-type rats). Treatment with the specific epithelial sodium channel blocker amiloride led to a much higher increase in fractional sodium excretion in ETB-deficient rats (934.2+/-73% in ETB-deficient rats versus 297+/-20% in wild-type rats, expressed as percentage of corresponding placebo treated control; P<0.001). Mean arterial blood pressure was elevated by 7.9 mmHg in homozygous ETB-deficient rats (P<0.05 versus wild-type rats). Our study demonstrates that ETB-deficiency causes early onset kidney dysfunction characterized by a markedly reduced sodium excretion, decreased GFR, and slightly elevated blood pressure. The complete absence of the ETB receptor causes in the kidney--in contrast to the colon--a functional rather than a developmental, neural crest cell dependent disease, since kidney morphology was normal in ETB-deficient rats. The much higher increase in the fractional sodium excretion in ETB-deficient rats after pharmacological blockade of the epithelial sodium channel indicates that the decreased fractional sodium excretion in ETB-deficient rats is most probably due to a lack of the inhibitory property of the ETB receptor on the epithelial sodium channel activity.     

Functional neuroanatomy of human sleep states after zolpidem and placebo: a H215O-PET study

Changes in the functional organization of the brain during the course of sleep and waking are reflected by different patterns of regional cerebral blood flow (rCBF). To investigate the effect of the hypnotic zolpidem, a benzodiazepine receptor agonist, drug or placebo were administered to eight young, healthy men prior to bedtime. The subjects were sleep-deprived to promote sleep during the 4-h recording period in the positron emission tomography scanner. Intravenous injections of labelled water were administered during pre-drug wakefulness, and during Stage 2, Stage 4 and rapid eye movement (REM) sleep, each injection being followed by an emission scan. Statistical parametric mapping was used to investigate the effects of treatment and sleep states. During sleep (combined Stages 2 and 4, and REM sleep) relative rCBF was lower after zolpidem than after placebo in the basal ganglia and insula, and higher in the parietal cortex. A 'multiple study' analysis of REM sleep revealed that rCBF in the anterior cingulum was lower after zolpidem than after placebo, whereas rCBF in the occipital and parietal cortex, parahippocampal gyrus and cerebellum was higher. When the pooled data (drug and placebo) of Stages 2 and 4 were compared with wakefulness, rCBF was lower in prefrontal cortex and insula, and higher in the occipital and parietal cortex. The results indicate that some differences in rCBF from wakefulness to non-REM sleep are further augmented by zolpidem.     

Comparison of pleural pressure and transcutaneous diaphragmatic electromyogram in obstructive sleep apnea syndrome

STUDY OBJECTIVES: Based on studies of the impact of esophageal pressure on cardiovascular variables during sleep, this signal can be used to refine the severity level in the clinical diagnosis of obstructive sleep apnea syndrome. We hypothesized that relative changes in diaphragmatic electromyogram (EMG) can reflect short-term changes in esophageal pressure durng obstructive apneas and hypopneas. DESIGN: Diaphragmatic EMG was sampled at 0.25 kHz; diaphragmatic EMG waveform was band-pass filtered and digitally converted; the electrocardiogram artifact was eliminated; using a gating procedure, the waveform was fast-Fourier transformed and digitally rectified; and a moving average of 200 milliseconds was calculated. For each inspiratory effort during apnea or hypopnea, we calculated maximum diaphragmatic EMG and esophageal pressure. Data were normalized calculating the percentage difference between the first obstructed and each subsequent inspiratory effort during the respiratory event. SETTING: Sleep disorders laboratory. PATIENTS: 9 patients with moderate obstructive sleep apnea syndrome presenting with apneas and hypopneas during sleep. INTERVENTION: None. MEASUREMENTS AND RESULTS: 861 respiratory events were scored, and the evolution between esophageal pressure and diaphragmatic EMG were compared. Normalized data showed a good correlation between the 2 measures during apneas and hypopneas. There was a significant difference between the percentage increase in esophageal pressure and diaphragmatic EMG for apneas and hypopneas (esophageal pressure, apnea: 118.1% +/- 118.5%, hypopnea: 76.1% +/- 74.3%, P = .000; diaphragmatic EMG, 123.5% +/- 131.7%, hypopnea: 73.3% +/- 74.2%, P = .000). No significant differences for apnea or hypopnea were noted between the 2 measures under investigation. CONCLUSION: Diaphragmatic EMG may be clinically useful to describe relative changes in respiratory effort under conditions of apnea and hypopnea during sleep and to reliably dissociate central from obstructive events where esophageal pressure monitoring is not readily available.