Regulatory volume decrease in a renal distal tubular cell line (A6) II. Effect of Na+ transport rate

A6 epithelia, a cell line originating from the distal tubular part of the kidney ofXenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (T _c), short-circuit current (I _sc) and transepithelial conductance (G _t) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na⁺ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease ofT _c described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na⁺ uptake by replacing apical Na⁺ byN-methyl-d-glucamine (NMDG⁺) did not affect cell volume under isotonic conditions. An increase ofT _c by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na⁺ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na⁺ uptake or Na⁺ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K⁺ channels by 5 mM Ba²⁺ elicited a significant increase inT _c of 16.3% above control. Quinine, a potent blocker of volume-activated K⁺ channels, did not changeT _c significantly. Basolateral hypotonicity elicited a rapid rise inT _c followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na⁺ uptake as well as after stimulating apical Na⁺ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K⁺ efflux at the basolateral side with Ba²⁺ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K⁺, whereas the effects of Ba²⁺ and quinine are most likely due to the blockage of the basolateral K⁺ pathway.     

Assessment of the antigenic response in humans to a recombinant mutant interferon beta

Cancer patients were given a recombinant mutant interferon by alternating IM and IV injections with weekly escalation of doses from 0.1 to 400 million U. Antibodies specific to the interferon of the IgG class were detected in 24 of 30 patients using an indirect enzyme-linked immunosorbent assay. Serum from only 1 of the 30 patients had detectable ability to neutralize interferon biological activity. Thein vivo interferon serum level, assayed as antiviral activity immediately after IV injection, was not lower than levels seen in the absence of antibodies. Antibodies did not alter the kinetics of clearance of interferon from the serum after IV administration. Antibody levels progressively decreased when interferon administration was discontinued. In most patients antibody levels decreased during a maintenance period when interferon was being administered only by the IV route. In a subsequent trial interferon was given IV, and antibody developed in only 2 of 36 patients. In contrast, in a trial in which interferon was given IM, 20 of 25 patients developed antibody. No antibody-related clinical sequelae could be detected in any of these patients.     

Somatic translocation and differential expression of Ig mu transgene copies implicate a role for the Igh locus in memory B cell development

Memory B cells of mice with Ig mu transgenes often carry transgene copies that have moved into the Igh locus via somatic translocation. This phenomenon has been attributed to a selection pressure for somatic hypermutations, which generally are observed at much higher frequencies in translocated copies than in ectopic copies. We tested this idea by immunizing Ig-mu transgenic mice in a manner designed to select B cells that required only one V(H) mutation for a switch in antigenic specificity and recruitment into the memory pool. Despite the minimal mutation requirement, hybridomas carrying somatic translocations to the Igh locus were obtained. Importantly, this occurred despite the fact that translocated and untranslocated mu-transgenes were mutated comparably. Evidently, a strong selection advantage was conferred upon B cells by the somatic translocations. Among the hybridomas, translocated mu-transgenes were active, while ectopic mu-transgenes were uniformly silent. The translocated copy that had conferred an affinity-based selection advantage was expressed at the highest level. Moreover, translocated copies were differentially expressed among hybridoma members, which belonged to a common post-mutational lineage. This suggests that adjustments in transgene expression levels had occurred during memory cell development. These results indicate that, apart from their potential influences on somatic hypermutagenesis and class switch recombination, elements in the Igh locus promote the selection of memory B cells in another way, possibly by regulating the level of Ig expression at various stages of antigen-driven differentiation.     

Mapping and ranking of potential cytotoxic T epitopes in the p53 protein: effect of mutations and polymorphism on peptide binding to purified and refolded HLA molecules

In many cancer cells, the p53 gene displays point mutations that result in stabilization and accumulation of the p53 protein. Therefore, p53 peptides could be presented to the immune system by tumor cells; thus, p53 might be a suitable target antigen for developing an immunotherapy against tumors using cytotoxic T lymphocytes (CTL). To map candidate CTL epitopes, we synthesized 150 peptides of 8–11 residues that contained putative anchor motifs required for binding to common HLA class I molecules. They were tested for their capacity to promote the assembly of purified and refolded HLA-A1, A2, B7 and B8 molecules. The following wild-type p53 peptides were found to be reactive with the HLA molecules tested: 196–205 and 226–234 bound moderately to HLA-A1; 25–35, 65–73, 129–137, 187–197, 263–272 and 264–272 bound strongly, and 187–195 and 256–264 moderately to HLA-A2; 26–35, 63–73, 189–197, 249–257 and 321–330 bound strongly to HLA-B7; and 135–143, 210–218 and 375–383 bound weakly to HLA-B8. We also analyzed the effects of p53 mutations occurring naturally in tumors on peptide/HLA assembly. We found substitutions that enhanced, diminished or had no effect on the peptide binding to HLA molecules. Polymorphism at position 72 mainly affected peptide/HLA-B7 binding, the proline allele P72 giving a less-reactive peptide (63–73) than the arginine allele R72. We have ranked potential p53 epitopes according to their reactivity for purified HLA molecules, allowing the selection of appropriate peptides and HLA molecules to attempt CTL induction in vitro.     

Youth Mental Health First Aid: Examining the Influence of Pre-Existing Attitudes and Knowledge on Training Effectiveness

Journal of Prevention - Youth Mental Health First Aid (YMHFA) is a gatekeeper training designed to assist professionals in their identification and response to youth mental illness. Despite...     

The effect of temazepam on melatonin secretion and light sensitivity

Seven healthy male volunteers had their light sensitivity (melatonin suppression) and melatonin phase measured before and after treatment with temazepam (20 mg) for 7 days. Temazepam did not alter the circadian phase of melatonin secretion (the Dim Light Melatonin Onset, the timing of the peak of secretion), the total melatonin secretion nor the sensitivity of melatonin secretion to suppression by full spectrum light. This is an important negative finding which suggests that the short half-life benzodiazepine hypnotic temazepam does not confound tests of light sensitivity or melatonin phase.     

A prospective comparison of central and peripheral vein access for parenteral nutrition in the newborn

Central venous parenteral nutrition (PN) is frequently used in preterm infants. Although central venous catheters (CVC) permit reliable delivery of hypertonic solution, they may be associated with more serious complications than when a peripheral venous infusion is used. The aim of this randomised prospective study was to compare complications of central versus peripheral venous access using Silastic catheters identical expect for intravascular length. Eighty such devices were inserted, 38 central (CVC), 42 peripheral (PVC). Catheter life was not significantly different between groups: median (range) CVC 10d (2-25); PVC 7d (1-22) with no difference in overall complication rate. Although peripherally sited catheters tended not to function for as long as CVCs, they offer a useful alternative to central venous catheterisation.     

Enhanced myocardial protection during global ischemia with 5'-nucleotidase inhibitors.

Depletion of adenosine triphosphate precursors, such as myocardial adenosine, during global ischemia results in poor postischemic adenosine triphosphate repletion and functional recovery. Neonatal hearts may be more resistant to this deleterious effect of ischemia, because they are characterized by low 5'-nucleotidase activity, which may result in higher sustained endogenous myocardial adenosine triphosphate precursor levels during ischemia. Adult hearts, however, have high levels of 5'-nucleotidase activity leading to depleted precursors during ischemia and poor postischemic functional recovery. Augmenting myocardial adenosine exogenously during ischemia in adult hearts has a beneficial effect on recovery. The present study tested if preservation of nucleotide precursors, better adenosine triphosphate repletion, and enhanced postischemic myocardial recovery in adult hearts could be achieved with a "neonatal" strategy. Therefore 5'-nucleotidase inhibitors were administered to isolated, perfused adult rabbit hearts subjected to 120 minutes of ischemia (at 34 degrees C) to determine if this improved functional recovery. Hearts received St. Thomas' Hospital cardioplegic solution (control hearts) or cardioplegic solution containing 5'-nucleotidase inhibitors: pentoxifylline, thioinosine, [s-(p-nitrophenyl)-4-thioinosine], or thioinosine's dimethyl sulfoxide vehicle alone. After ischemia and reperfusion, recovery of systolic function, diastolic function, and myocardial oxygen consumption was significantly better with 5'-nucleotidase inhibition. No changes in coronary flow were noted. We speculate and are pursuing the theory that the mechanism of 5'-nucleotidase inhibition's favorable action is due to preventing the catabolism, transport, and loss of nucleotide precursors during ischemia, maintaining adenosine triphosphate precursor availability.     

Early predicted time to normalization of tumor markers predicts outcome in poor-prognosis nonseminomatous germ cell tumors.

PURPOSE: The prognostic relevance of the rate of decline of serum alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) during the first 3 weeks of chemotherapy for nonseminomatous germ cell tumors (NSGCT) was studied in the context of the International Germ Cell Cancer Collaborative Group (IGCCCG) classification. PATIENTS AND METHODS: Data from 653 patients prospectively recruited in clinical trials were studied. Tumor markers were obtained before chemotherapy and 3 weeks later. Decline rates were calculated using a logarithmic formula and expressed as a predicted time to normalization (TTN). A favorable TTN was defined when both AFP and HCG had a favorable decline rate, including cases with normal values. RESULTS: The median follow-up was 50 months (range, 2 to 151 months). Tumor decline rate expressed as a predicted TTN was associated with both progression-free survival (PFS; P <.0001) and overall survival (OS; P <.0001). The 4-year PFS rates were 64% and 38% in patients from the poor-prognosis group who had a favorable and an unfavorable TTN, respectively. The 4-year OS rates were 83% and 58%, respectively. This effect was independent from the initial tumor marker values, the primary tumor site, and the presence of nonpulmonary visceral metastases: tumor marker decline rate remained a strong predictor for both PFS (hazard ratio = 2.5; P =.01) and OS (hazard ratio = 4.6; P =.002) in patients from the IGCCCG poor-prognosis group in multivariate analysis. CONCLUSION: Early predicted time to tumor marker normalization is an independent prognostic factor in patients with poor-prognosis NSGCT and may be a useful tool in the therapeutic management of these patients.