Isolated lymphocytic infiltration of pituitary stalk preceding the diagnosis of germinoma in 2 prepubertal children treated with growth hormone.

We report the clinical course of 2 patients with central diabetes insipidus and evolving to panyhypopituitarism which prompted the diagnosis of an isolated pituitary stalk thickening (PST). In both patients, all etiological investigations were normal and the first biopsy revealed an isolated lymphocytic infiltrate with no sign of malignancy. Close clinical follow-up accompanied by serial brain MRIs was proposed to determine a precise diagnosis and for early detection and treatment of neoplastic disease. In our first case, the diagnosis of germinoma was made 9 months after the PST diagnosis owing to tumor progression. In the second case, the time course was even longer with the diagnosis of germinoma 6 years following initial presentation. In these cases, it is speculated that the lymphocytic infiltrates represent the first sign of a host reaction to an occult germinoma. To our knowledge, this is the third case reported of lymphocytic infiltrates preceding a germinoma in a prepubertal girl, and the only case reported in a prepubertal boy. These cases underline the difficulties in establishing the diagnosis of germinoma in a patient with isolated PST.

Coexistence of hERG current block and disruption of protein trafficking in ketoconazole-induced long QT syndrome.

BACKGROUND AND PURPOSE: Many drugs associated with acquired long QT syndrome (LQTS) directly block human ether-a-go-go-related gene (hERG) K(+) channels. Recently, disrupted trafficking of the hERG channel protein was proposed as a new mechanism underlying LQTS, but whether this defect coexists with the hERG current block remains unclear. This study investigated how ketoconazole, a direct hERG current inhibitor, affects the trafficking of hERG channel protein. EXPERIMENTAL APPROACH: Wild-type hERG and SCN5A/hNa(v) 1.5 Na(+) channels or the Y652A and F656C mutated forms of the hERG were stably expressed in HEK293 cells. The K(+) and Na(+) currents were recorded in these cells by using the whole-cell patch-clamp technique (23 degrees C). Protein trafficking of the hERG was evaluated by Western blot analysis and flow cytometry. KEY RESULTS: Ketoconazole directly blocked the hERG channel current and reduced the amount of hERG channel protein trafficked to the cell surface in a concentration-dependent manner. Current density of the hERG channels but not of the hNa(v) 1.5 channels was reduced after 48 h of incubation with ketoconazole, with preservation of the acute direct effect on hERG current. Mutations in drug-binding sites (F656C or Y652A) of the hERG channel significantly attenuated the hERG current blockade by ketoconazole, but did not affect the disruption of trafficking. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that ketoconazole might cause acquired LQTS via a direct inhibition of current through the hERG channel and by disrupting hERG protein trafficking within therapeutic concentrations. These findings should be considered when evaluating new drugs.

Drug-induced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine.

BACKGROUND AND PURPOSE: Fluoxetine (Prozac) is a widely prescribed drug in adults and children, and it has an active metabolite, norfluoxetine, with a prolonged elimination time. Although uncommon, Prozac causes QT interval prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms underlying this clinical finding by analysing the effects of fluoxetine and norfluoxetine on ion channels in vitro. EXPERIMENTAL APPROACH: We studied the effects of fluoxetine and norfluoxetine on the electrophysiology and cellular trafficking of hERG K+ and SCN5A Na+ channels heterologously expressed in HEK293 cells. KEY RESULTS: Voltage clamp analyses employing square pulse or ventricular action potential waveform protocols showed that fluoxetine and norfluoxetine caused direct, concentration-dependent, block of hERG current (IhERG). Biochemical studies showed that both compounds also caused concentration-dependent reductions in the trafficking of hERG channel protein into the cell surface membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous current. Mutations in the hERG channel drug binding domain reduced fluoxetine block of IhERG but did not alter fluoxetine's effect on hERG channel protein trafficking. CONCLUSIONS AND IMPLICATIONS: Our findings show that both fluoxetine and norfluoxetine at similar concentrations selectively reduce IhERG by two mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel protein trafficking. These two effects are not mediated by a single drug binding site. Our findings add complexity to understanding the mechanisms that cause drug-induced long QT syndrome.

Identification of the t-type calcium channel (Ca(v)3.1d) in developing mouse heart.

During cardiac development, there is a reciprocal relationship between cardiac morphogenesis and force production (contractility). In the early embryonic myocardium, the sarcoplasmic reticulum is poorly developed, and plasma membrane calcium (Ca(2+)) channels are critical for maintaining both contractility and excitability. In the present study, we identified the Ca(V)3.1d mRNA expressed in embryonic day 14 (E14) mouse heart. Ca(V)3.1d is a splice variant of the alpha1G, T-type Ca(2+) channel. Immunohistochemical localization showed expression of alpha1G Ca(2+) channels in E14 myocardium, and staining of isolated ventricular myocytes revealed membrane localization of the alpha1G channels. Dihydropyridine-resistant inward Ba(2+) or Ca(2+) currents were present in all fetal ventricular myocytes tested. Regardless of charge carrier, inward current inactivated with sustained depolarization and mirrored steady-state inactivation voltage dependence of the alpha1G channel expressed in human embryonic kidney-293 cells. Ni(2+) blockade discriminates among T-type Ca(2+) channel isoforms and is a relatively selective blocker of T-type channels over other cardiac plasma membrane Ca(2+) handling proteins. We demonstrate that 100 micromol/L Ni(2+) partially blocked alpha1G currents under physiological external Ca(2+). We conclude that alpha1G T-type Ca(2+) channels are functional in midgestational fetal myocardium.

Monovalent cations contribute to T-type calcium channel (Cav3.1 and Cav3.2) selectivity.

Low voltage-activated (LVA) Ca2+ channels regulate chemical signaling by their ability to select for Ca2+. Whereas Ca2+ is the main permeating species through Ca2+ channels, Ca2+ permeation may be modified by abundant intra- and extracellular monovalent cations. Therefore, we explored monovalent cation regulation of LVA Ca2+ permeation in the cloned T-type Ca2+ channels alpha1G (Cav3.1) and alpha1H (Cav3.2). In physiological [Ca2+], the reversal potential in symmetrical Li+ was 19 mV in alpha1G and 18 mV in alpha1H, in symmetrical Cs+ the reversal potential was 36 mV in alpha1G and 37 mV in alpha1H, and in the bi-ionic condition with Li+ in the bath and Cs+ in the pipette, the reversal potential was 46 mV in both alpha1G and alpha1H. When Cs+ was used in the pipette, replacement of external Cs+ with Li+ (or Na+) shifted the reversal potential positive by 5-6 mV and increased the net inward current in alpha1G. Taken together the data indicate that in physiological [Ca2+], external Li+ (or Na+) permeates more readily than external Cs+, resulting in a positive shift of the reversal potential. We conclude that external monovalent cations dictate T-type Ca2+ channel selectivity by permeating through the channel. Similar to Li+, we previously reported that external [H+] can regulate T-type Ca2+ channel selectivity. Alpha1H's selectivity was more sensitive to external pH changes compared to alpha1G. When Cs+ was used in the pipette and Li+ was used in the bath external acidification from pHo 7.4 to 6.0 caused a negative shift of the reversal by 8 mV in alpha1H. Replacement of internal Cs+ with Li+ reduced the pH-induced shift of the reversal potential to 2 mV. We conclude that, similar to other external monovalent cations, H+ can modify T-type Ca2+ channel selectivity. However, in contrast to external monovalent ions that readily permeate, H+ regulate T-type Ca2+ channel selectivity by increasing the relative permeability of the internal monovalent cation.

pH modification of human T-type calcium channel gating.

External pH (pH(o)) modifies T-type calcium channel gating and permeation properties. The mechanisms of T-type channel modulation by pH remain unclear because native currents are small and are contaminated with L-type calcium currents. Heterologous expression of the human cloned T-type channel, alpha1H, enables us to determine the effect of changing pH on isolated T-type calcium currents. External acidification from pH(o) 8.2 to pH(o) 5.5 shifts the midpoint potential (V(1/2)) for steady-state inactivation by 11 mV, shifts the V(1/2) for maximal activation by 40 mV, and reduces the voltage dependence of channel activation. The alpha1H reversal potential (E(rev)) shifts from +49 mV at pH(o) 8.2 to +36 mV at pH(o) 5.5. The maximal macroscopic conductance (G(max)) of alpha1H increases at pH(o) 5.5 compared to pH(o) 8.2. The E(rev) and G(max) data taken together suggest that external protons decrease calcium/monovalent ion relative permeability. In response to a sustained depolarization alpha1H currents inactivate with a single exponential function. The macroscopic inactivation time constant is a steep function of voltage for potentials < -30 mV at pH(o) 8.2. At pH(o) 5.5 the voltage dependence of tau(inact) shifts more depolarized, and is also a more gradual function of voltage. The macroscopic deactivation time constant (tau(deact)) is a function of voltage at the potentials tested. At pH(o) 5.5 the voltage dependence of tau(deact) is simply transposed by approximately 40 mV, without a concomitant change in the voltage dependence. Similarly, the delay in recovery from inactivation at V(rec) of -80 mV in pH(o) 5.5 is similar to that with a V(rec) of -120 mV at pH(o) 8.2. We conclude that alpha1H is uniquely modified by pH(o) compared to other calcium channels. Protons do not block alpha1H current. Rather, a proton-induced change in activation gating accounts for most of the change in current magnitude with acidification.

Freeze-drying of Streptococcus thermophilus: a comparison between the vacuum and the atmospheric method.

Frozen suspensions of Streptococcus thermophilus were freeze-dried in a vacuum or a fluidized adsorbent bed at atmospheric pressure. Optimum operating conditions for each process were defined. For the duration of processing and survival rate of bacteria, in each case vacuum freeze-drying seemed more satisfactory than atmospheric pressure freeze-drying. The use of reconstituted skimmed milk as a suspension medium provides good protection for S. thermophilus.

Effect of cyclosporine on mycophenolic acid area under the concentration-time curve in pediatric kidney transplant recipients.

Mycophenolic acid (MPA), the active compound of mycophenolate mofetil (MMF), shows substantial interindividual and intraindividual variability. It was recently shown that in vitro calcineurin inhibitors alter the bioavailability of MPA by dose-dependent inhibition of MPA glucuronidation. The authors retrospectively analyzed full 10-point profiles for both MPA and cyclosporine (CyA) in 23 pediatric patients receiving MMF and cyclosporine microemulsion (Neoral; Novartis Pharmaceuticals Canada; Dorval, Quebec, Canada). Mycophenolic acid was measured using a commercially available EMIT (Novartis Pharmaceuticals, Canada) assay. As the majority of patients were treated with low doses of cyclosporine after adding MMF, the area under the concentration-time curve (AUC) for cyclosporine showed a wide scatter ranging from 296 to 6400 ng x h/mL. The mean cyclosporine dose was 100 +/- 76 mg/m2 per day (range: 28 to 331). There was no correlation between MPA AUC and MPA dose, and there was substantial interindividual variation. However, there was a significant negative correlation between dose-normalized MPA AUC and cyclosporine AUC ( r2 = 0.23, p < 0.0220). When dividing the MPA profiles into two groups (11 and 12 patients) with a CyA AUC less than or greater than 1600 ng x h/mL, there was a significantly higher 8-hour concentration in the patients with the lower CyA AUC, secondary to a higher second peak. The data demonstrate that the cyclosporine AUC is a determining factor for the MPA AUC and that MPA dose should be reduced when cyclosporine dose is reduced to achieve the same MPA AUC. The significantly higher peak in the group with a lower CyA profile supports the concept of a dose-dependent cyclosporine-induced inhibition of MPA glucuronidation.

Detection rates of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and swine influenza virus in porcine proliferative and necrotizing pneumonia.

A retrospective study on pig lung tissues from 60 cases of proliferative and necrotizing pneumonia (PNP) was performed to determine the presence of porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and porcine circovirus type 2 (PCV2) in these lesions. Cases selected included 30 cases diagnosed between 1988 and 1992 and 30 cases diagnosed between 1997 and 2001. In each group of 30 cases, 10 were from suckling piglets, whereas the other 20 were from postweaned animals representing either nursery or grower-finisher pigs. Immunohistochemistry using a monoclonal antibody to influenza virus type A was used to determine the presence of SIV, and in situ hybridization was used for the detection of PRRSV and PCV2 nucleic acids. PRRSV was detected in 55 of the 60 cases examined (92%), PCV2 in 25 cases (42%), and SIV in only 1 case (2%). In 30 cases (50%), PRRSV was the only virus detected, whereas in 25 other cases (42%), a combination of PRRSV and PCV2 could be detected in the lungs with PNP lesions. PCV2 could not be detected in the lungs of suckling pigs with PNP. All PCV2-positive cases were found in postweaned pigs and were always in combination with PRRSV. In this latter age group, PCV2 was detected in 63% of the cases (25/40). Data from our study indicate that SIV is rarely identified in PNP and that PCV2 infection is not essential for the development of PNP lesions. The results of the present study demonstrate that PRRSV is consistently and predominantly associated with PNP and should be considered the key etiologic agent for the condition.