Pregnancy in women with Hereditary Angioedema due to C1-inhibitor deficiency: Results from the ITACA cohort study on outcome of mothers and children with in utero exposure to plasma-derived C1-inhibitor.

Background: In women with Hereditary Angioedema (HAE) due to C1-inhibitor (C1INH) deficiency (C1INH-HAE), pregnancy counseling and treatment can be challenging. Despite the evidence of the immediate favorable outcome and safety of plasma-derived (pd)C1INH concentrate, there are no data regarding any difference among women who underwent or not pdC1INH during pregnancy or on children with in utero exposure to pdC1INH. The present interview study aimed at analyzing outcome of C1INH-HAE mothers and children according to pdC1INH-exposure during pregnancies. Methods: C1INH-HAE women who experienced at least 1 pregnancy were included from seven centers of the Italian Network for Hereditary and Acquired Angioedema (ITACA). The interview study retrospectively analyzed pregnancies who underwent (group 1) or not (group 2) pdC1INH. The overall goals of the study included immediate and long-term outcomes, in terms of outcomes in the time interval between pregnancy and survey. Results: A total of 168 pregnancies from 87 included women were analyzed. At term delivery (>37 gestation-week, GW) has been registered in 73.8% of cases, while spontaneous abortion (SA) occurred in 14.2% of cases with a mean GW 7 ± 2. The group 1 including pdC1INH-treated pregnancies comprised a third of the cohort (51/168, time interval 1.5 ± 10.4 yrs), while the group 2 represented 69.6% (117/168, time interval 32.8 ± 14 yrs). The same prevalence of SA occurred when comparing group 1 (11.7%) with group 2 (15.4%) with a similar GW at SA. The group 1 was older at the pregnancy time and younger at the interview than the group 2 (P < 0.01 for both); moreover, the group 1 showed a higher prevalence of cesarean delivery (P < 0.0001). The overall prevalence of obstetrical syndromes was similar between two groups: however, gestational diabetes was described only in pdC1INH-untreated pregnancies. In utero pdC1INH-exposed children (n = 45) did not show differences compared with unexposed ones (n = 99) in neonatal short-term outcomes. Conclusion: Through appropriate management and counseling, most of C1INH-HAE women undergo successful pregnancy and delivery. For pregnant C1INH-HAE women being treated with pdC1INH, our findings are reassuring and might lead to an improvement of both the knowledge about treatments and the experience of HAE itself.

Effect of the erbium dopant architecture on the femtosecond relaxation dynamics of silicon nanocrystals.

Femtosecond pump-probe absorption spectroscopy is used to investigate the role of Er(3+) dopants in the early relaxation pathways of photoexcited Si nanocrystals. The fate of photoexcited electrons in three different Si nanostructures was studied and correlated with the effect of Er-doping and the nature of the dopant architecture. In Si nanocrystals without Er(3+) dopant, a trapping component was identified to be a major electron relaxation mechanism. Addition of Er(3+) ions into the core or surface shell of the nanocrystals was found to open up additional nonradiative relaxation pathways, which is attributed to Er-induced trap states in the Si host. Analysis of the photodynamics of the Si nanocrystal samples reveals an electron trapping mechanism involving trap-to-trap hopping in the doped nanocrystals, whereby the density of deep traps seem to increase with the presence of erbium. To gain additional insights on the relative depths of the trapping sites on the investigated nanostructures, benzoquinone was used as a surface adsorbed electron acceptor to facilitate photoinduced electron transfer across the nanocrystal surface and subsequently assist in back electron transfer. The established reduction potential (-0.45 V versus SCE) of the electron acceptor helped reveal that the erbium-doped nanocrystal samples have deeper trapping sites than the undoped Si. Furthermore, the measurements indicate that internally Er-doped Si have relatively deeper trapping sites than the erbium surface-enriched nanocrystals. The electron-shuttling experiment also reveals that the back electron transfer seems not to recover completely to the ground state in the doped Si nanocrystals, which is explained by a mechanism whereby the electrons are captured by deep trapping sites induced by erbium addition in the Si lattice.

Efficacy of tacrolimus in rheumatoid arthritis patients who have been treated unsuccessfully with methotrexate: a six-month, double-blind, randomized, dose-ranging study.

OBJECTIVE: To assess the efficacy, safety, and optimal dose of tacrolimus monotherapy in patients with rheumatoid arthritis (RA). METHODS: This phase II, randomized, double-blind, placebo-controlled monotherapy study was set in 12 community sites and 9 university-based sites. Two hundred sixty-eight patients with RA who were resistant to or intolerant of methotrexate (mean dose 15.2 mg/week) and had active disease for at least 6 months (mean tender joint count 28.2, mean erythrocyte sedimentation rate 46.5 mm/hour) were randomized to receive treatment after discontinuation of methotrexate. Those who received at least 1 dose of tacrolimus were analyzed; 141 completed the study. Stable dosages of nonsteroidal antiinflammatory drugs and low-dose prednisone were allowed during treatment. All patients were given 1, 3, or 5 mg of tacrolimus or placebo once daily for 24 weeks. The American College of Rheumatology definition of 20% improvement (ACR20) and the tender and swollen joint counts at the end of treatment were the primary outcomes. RESULTS: ACR20 response rates demonstrated a clear dose response. The ACR20 response was observed in 15.5% of patients receiving placebo (95% confidence interval [95% CI] 7.1-23.9%), 29% of the 1 mg tacrolimus group (95% CI 18.3-39.7%) (P < 0.058); 34.4% of the 3 mg group (95% CI 22.7-46.0%) (P < 0.013), and 50% of the 5 mg group (95% CI 37.8-62.3%) (P < or = 0.001). The tender joint count improved statistically significantly in all tacrolimus groups. The swollen joint count, physical function, and patient-assessed pain improved statistically significantly in the 3 mg and 5 mg groups. The incidence of creatinine elevation > or =40% above baseline levels increased in a dose-dependent manner. Dropout rates were high (41-59%) and were more common for inefficacy in the placebo patients (71.4%), whereas they were more common for toxicity in the high-dose tacrolimus groups (31-33%). Discontinuation for creatinine elevation occurred in the 3 mg (3.1%) and 5 mg (10.9%) tacrolimus groups. CONCLUSION: Tacrolimus improved disease activity in methotrexate-resistant or -intolerant patients with RA. A dose response was observed when efficacy and toxicity were assessed at different doses. The optimal dose of tacrolimus appears to be >1 mg but < or=3 mg daily.

Energetic determinants of tyrosine phosphorylation of focal adhesion proteins during hypoxia/reoxygenation of kidney proximal tubules.

Anaerobic mitochondrial metabolism of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules.(34) The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130(cas) migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to approximately 5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of beta1 and alpha6 integrin subunits, pY, and paxillin was lost or markedly decreased. During reoxygenation without supplemental substrates, recovery of pY and basal localization of the focal adhesion proteins was poor. alpha-Ketoglutarate and aspartate, which maintained slightly higher levels of ATP during hypoxia, also maintained 2.5-fold higher levels of pY during this period, and promoted full recovery of pY content and basal localization of focal adhesion proteins during subsequent reoxygenation. Similarly complete recovery was made possible by provision of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate only during reoxygenation. These data emphasize the importance of very low energy thresholds for maintaining the integrity of key structural and biochemical components required for cellular survival and reaffirm the value of approaches aimed at conserving or generating energy in cells injured by hypoxia or ischemia.

Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.

We have further examined the mechanisms for a severe mitochondrial energetic deficit, deenergization, and impaired respiration in complex I that develop in kidney proximal tubules during hypoxia-reoxygenation, and their prevention and reversal by supplementation with alpha-ketoglutarate (alpha-KG) + aspartate. The abnormalities preceded the mitochondrial permeability transition and cytochrome c loss. Anaerobic metabolism of alpha-KG + aspartate generated ATP and maintained mitochondrial membrane potential. Other citric-acid cycle intermediates that can promote anaerobic metabolism (malate and fumarate) were also effective singly or in combination with alpha-KG. Succinate, the end product of these anaerobic pathways that can bypass complex I, was not protective when provided only during hypoxia. However, during reoxygenation, succinate also rescued the tubules, and its benefit, like that of alpha-KG + malate, persisted after the extra substrate was withdrawn. Thus proximal tubules can be salvaged from hypoxia-reoxygenation mitochondrial injury by both anaerobic metabolism of citric-acid cycle intermediates and aerobic metabolism of succinate. These results bear on the understanding of a fundamental mode of mitochondrial dysfunction during tubule injury and on strategies to prevent and reverse it.

Evidence for the involvement of Rab3A in Ca(2+)-dependent exocytosis from adrenal chromaffin cells.

Bovine chromaffin cells are nondividing primary secretory cells that store and secrete catecholamine and a variety of proteins including chromogranins, opiate peptides, and opiate precursors. A transient transfection technique based upon the expression of human growth hormone as a reporter for the regulated secretory pathway was used to study the role of a Ras-like, GTP-binding protein, Rab3a, in Ca(2+)-dependent exocytosis. Immunocytochemistry and flow cytometry revealed that growth hormone and Rab proteins were coexpressed in the same cells. Overexpression of the wild type protein and expression of a mutant protein Rab3aQ81L both inhibited nicotinic agonist-stimulated exocytosis in intact cells. Expression of Rab3aQ81L also inhibited Ca(2+)-dependent secretion from permeabilized cells. Two other mutants, Rab3aN135I and Rab3aT36N, which correspond to dominant acting mutants of Ras, caused limited and no inhibition, respectively, of agonist-stimulated exocytosis. These data provide direct evidence that Rab3a plays an important role in Ca(2+)-triggered exocytosis. We suggest that Rab3a is an inhibitor of secretion, perhaps as part of a pre-fusion complex with secretory vesicles. Elevated Ca2+ may trigger exocytosis by overcoming the inhibition by Rab3a.

Transient transfection studies of secretion in bovine chromaffin cells and PC12 cells. Generation of kainate-sensitive chromaffin cells.

We have developed a transient transfection method to measure protein secretion from non-dividing, primary bovine chromaffin cells and from the continuous cell line, PC12. A plasmid coding human growth hormone (GH) was expressed in sufficient amounts in bovine chromaffin and PC12 cells to allow precise measurements of secretion from the small fraction (less than 1%) of transfected cells in a dish. GH was secreted in a similar proportion to endogenous catecholamine upon nicotinic stimulation, depolarization with elevated K+, and upon permeabilization with digitonin and subsequent stimulation with micromolar Ca2+. GH in homogenates from GH-transfected chromaffin cells cosedimented with catecholamine on discontinuous sucrose gradients. The data indicate that transiently expressed human GH in chromaffin and PC12 cells is localized predominantly in secretory vesicles in the regulated secretory pathway. With transient transfection there is a high probability of coexpression in the same cell of two plasmids which are cotransfected. Coexpression of a plasmid for GH and a plasmid for the non-N-methyl-D-aspartate glutamate receptor, GluR1, created chromaffin cells in which Ca(2+)-dependent GH secretion could be stimulated by the glutamatergic agonist kainate. The ability to coexpress a plasmid of interest with a plasmid for GH will allow the investigation of the role of other cloned proteins in the regulated secretory pathway in differentiated, non-dividing cells.

MgATP-independent and MgATP-dependent exocytosis. Evidence that MgATP primes adrenal chromaffin cells to undergo exocytosis.

The MgATP dependency of secretion was investigated in digitonin-permeabilized adrenal chromaffin cells. Shortly after permeabilization there is a component of Ca2+-dependent secretion that occurs in the absence of MgATP in the medium. This secretion occurs from cells which are permeable to Ca2+/[ethylene-bis(oxyethylenenitrilo)]tetraacetic acid buffers, to nucleotides, and to proteins. It is prevented by treatment of cells with metabolic inhibitors to reduce cellular ATP prior to permeabilization. The rate of MgATP-independent secretion is rapid and terminates by approximately 2 min after introduction of Ca2+. MgATP-independent secretion is labile and is lost unless Ca2+ is introduced within 8 min of permeabilization. MgATP-dependent secretion occurs at a slower rate than MgATP-independent secretion and continues at a constant rate for 12 min. Preincubation of permeabilized cells with MgATP enhances Ca2+-dependent secretion during a subsequent incubation in the absence of MgATP. Similar MgATP sensitivities are observed when MgATP is present only prior to or only during stimulation with Ca2+ with half-maximal stimulation occurring at 0.4-0.5 and 0.6 mM MgATP, respectively. The data indicate that intact cells are primed by intracellular ATP so that immediately upon permeabilization, there is a component of secretion which is independent of medium MgATP. MgATP partially maintains the primed state after permeabilization by acting before Ca2+ in the secretory pathway.

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells.

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.

Evaluation of nabumetone in the treatment of active adult rheumatoid arthritis.

The safety and efficacy of nabumetone and placebo were compared in a three-week, multicenter, double-blind, randomized, parallel evaluation involving patients with class II or III definite or classical rheumatoid arthritis. No patient received concomitant treatment with other nonsteroidal anti-inflammatory agents; however, disease-modifying agents (gold, steroids) were permitted. Of the 139 patients who entered the double-blind phase of the study, all were evaluable for safety, and 113 were evaluable for efficacy. Sixty-one patients received 1,000 mg of nabumetone per day at bedtime, and 50 were given placebo tablets; patients in both groups were permitted up to 3,250 mg of acetaminophen per day as needed for pain. After three weeks, nabumetone-treated patients exhibited a greater degree of improvement from baseline than did the placebo-treated patients, and the degree of improvement was statistically significant for four of seven variables.

Effects of osmolality and ionic strength on secretion from adrenal chromaffin cells permeabilized with digitonin.

Hyperosmotic solutions inhibit exocytosis of catecholamine from adrenal chromaffin cells at a step after Ca2+ entry into the cells. The possibility that the inhibition resulted from an inability of shrunken secretory granules to undergo exocytosis was investigated in cells with plasma membranes permeabilized by digitonin. The osmoticants and salts used in this study rapidly equilibrated across the plasma membrane and bathed the intracellular organelles. When sucrose was the osmoticant, secretion was not significantly inhibited unless the osmolality was raised above 1,000 mOs. When the osmolality was raised with the tetrasaccharide stachyose or a low-molecular-weight maltodextrin fraction (average size a tetrasaccharide), one-half maximal inhibition occurred at 900-1,000 mOs. Prior treatment of permeabilized cells with Ca2+ in hyperosmotic solution did not result in enhanced secretion when cells were restored to normal osmolality. Increased concentrations of potassium glutamate or sodium isethionate were more potent than carbohydrate in inhibiting secretion. Half-maximal inhibition occurred at 600-700 mOs or when the ionic strength was approximately doubled. The inhibition by elevated potassium glutamate also occurred when the osmolality was kept constant with sucrose. Increasing the ionic strength did not alter the Ca2+ sensitivity of the secretory response. Reducing the ionic strength by substituting sucrose for salt reduced the Ca2+ concentration required for half-maximal stimulated secretion from approximately 1.2 microM to 0.5 microM. Chromaffin granules, the secretory granules, are known to shrink in hyperosmotic solution. The experiments indicate that shrunken chromaffin granules can undergo exocytosis and suggest that in intact cells elevated ionic strength rather than chromaffin granule shrinkage contributes to the inhibition of secretion by hyperosmotic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma membrane and chromaffin granule characteristics in digitonin-treated chromaffin cells.

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.

Effects of phorbol ester on catecholamine secretion and protein phosphorylation in adrenal medullary cell cultures.

The effects of phorbol 12-myristate 13-acetate (PMA) on catecholamine secretion and protein phosphorylation from intact and digitonin-treated chromaffin cells were investigated. PMA (10-300 nM), an activator of protein kinase C, caused a slow Ca2+-dependent release of catecholamine from intact chromaffin cells that was potentiated by the Ca2+ ionophore ionomycin. PMA also enhanced secretion induced by Ba2+. In cells with plasma membranes rendered permeable by digitonin to Ca2+, ATP, and protein, PMA (100 nM) enhanced Ca2+-dependent secretion approximately 70% at 0.5 microM Ca2+ and 30% at 10 microM Ca2+. PMA enhanced the maximal response to Ca2+ approximately 25% and decreased the Ca2+ concentration required for half-maximal secretion approximately 30%. The effects of PMA on chromaffin cells were associated with a 2- to 3-fold increase in the phosphorylation of a 56-kDa protein that may be tyrosine hydroxylase. Other proteins were phosphorylated to a lesser extent. The experiments suggest that PMA increases protein kinase activity and secretion in chromaffin cells and raise the possibility that protein kinase C modulates catecholamine secretion in chromaffin cells.

Figure reversals and creativity: a research note.

In a series of studies, Bergum and Bergum (1979a, 1979b) noted a positive relationship between college students' self-perceptions of creativity and their passive rates of ambiguous figure reversal. While these authors suggest that a relationship may also exist between figure-reversal rate and creativity, as assessed by external measures, their research does not support this claim. Indeed, other research has not substantiated a relationship between rate of figure reversal and objective tests of creativity (Bloomberg, 1971; Bergum & Flamm, 1975). It may also be the case that students' perceptions of their own creative ability differ markedly from externally-derived measures of such ability. As part of a larger study relating figure-reversal rate, creativity, and handedness, the present authors attempted to replicate and extend the work of Bergum and Bergum through the use of professors' judgments of students' creativity. The subjects were 48 senior students of architecture (40 males, 8 females). Each student initially read a description of six factors commonly associated with creativity in the psychological literature and then rated himself in creative ability in comparison to his classmates. In accordance with Bergum and Bergum (1979a, 1979b), the students passively viewed (and recorded) figure reversals of six ambiguous figures. The six figures were presented for 60 sec. each, with 10-sec. intervals, in two random orders. Students' creative ability was also determined from rankings by two architecture professors who were familiar with the students' work. To guide their rankings, the professors used the same description of creativity as was given to the students.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that the H+ electrochemical gradient across membranes of chromaffin granules is not involved in exocytosis.

The possibility that the large H+ electrochemical potential of chromaffin granules, the secretory granules of adrenal medullary chromaffin cells, plays an important role in exocytosis was investigated in cultures of chromaffin cells from bovine adrenal medulla. Methylamine uptake into the cells, [gamma-31P]phosphate nmr of ATP within intracellular chromaffin granules, O2 consumption of intracellular mitochondria, and MgATP-stimulated catecholamine uptake into chromaffin granules isolated from cultured chromaffin cells were assessed to determine whether various manipulations altered the H+ electrochemical gradients of intracellular chromaffin granules or mitochondria. Catecholamine secretion was not significantly altered by ammonium, methylamine, nigericin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or dicyclohexylcarbodiimide under conditions when the pH of intracellular chromaffin granules was reduced or when granular or mitochondrial processes were uncoupled from H+ electrochemical gradients. The data indicate that the H+ electrochemical gradient across the chromaffin granule membrane does not play a role in exocytosis.

Relationship between Ca2+ uptake and catecholamine secretion in primary dissociated cultures of adrenal medulla.

Carbachol or elevated K+ stimulated 45Ca2+ uptake into chromaffin cells two- to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca2+ uptake and catecholamine secretion induced by the mixed nicotinic-muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca2+ uptake occurred within 15 s of stimulation by carbachol or elevated K+ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K+ was similar to that of Ca2+ uptake. There was a close correlation between Ca2+ uptake and catecholamine secretion at various concentrations of Ca2+. The concentration dependencies for inhibition of both processes by Mg2+ or Cd2+ were similar. Ca2+ uptake saturated with increasing Ca2+ concentrations, with an apparent Km for both carbachol-induced and elevated K+-induced Ca2+ uptake of approximately 2 mM. The Ca2+ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca2+ entry and a presumed increase in cytosolic Ca2+ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca2+-dependent processes associated with catecholamine secretion. Ca2+ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage-sensitive Ca2+ channels.