Intracellular Ca2+ release‐dependent inactivation of Ca2+ currents in thalamocortical relay neurons

Neuronal Ca²⁺ channels are rapidly inactivated by a mechanism that is termed Ca²⁺‐dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca²⁺ release on CDI of high‐voltage‐activated Ca²⁺ channels in rat thalamocortical relay neurons by combining voltage‐clamp, Ca²⁺ imaging and immunological techniques. Double‐pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration‐dependent increase in CDI that was accompanied by an increase in the duration of Ca²⁺ transients. Inhibition of ryanodine receptors and endoplasmic Ca²⁺ pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5‐tris‐phosphate receptors by intracellular application of 2‐aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2‐aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L‐type Ca²⁺ channels was emphasized by the near‐complete block of CDI by nifedipine, an effect only surpassed when Ca²⁺ was replaced by Ba²⁺ and chelated by 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′,‐tetraacetic acid (BAPTA). Trains of action potential‐like stimuli induced a strong reduction in high‐voltage‐activated Ca²⁺ current amplitude, which was significantly reduced when intracellular Ca²⁺ stores were made inoperative by thapsigargin or Ba²⁺/BAPTA. Western blotting revealed expression of L‐type Ca²⁺ channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross‐signalling between L‐type Ca²⁺ channels and ryanodine receptors that controls the amount of Ca²⁺ influx during neuronal activity.     

Combined procoagulant activity and proteolytic activity of acute promyelocytic leukemic cells: reversal of the bleeding disorder by cell differentiation

In the human promyelocytic cell line HL60, we observed both a strong procoagulant activity (PCA) on the cell membrane and proteolytic activity in the lysate of these cells. Because these cell-line cells are susceptible to differentiation to either a more mature granulocytic or monocytic form, we were able to study the hypothesis that the combination of PCA and proteolytic activity is confined to the promyelocyte. This may explain the severe coagulopathy seen in patients with acute promyelocytic leukemia. Cell differentiation in a myeloid direction induced by retinoic acid or DMSO led to a diminished PCA, while not affecting the fibrinolytic activity. On the other hand, monocytic differentiation obtained by culturing the cells in the presence of 1; 25 dihydroxy vitamin D3 led to the complete disappearance of the proteolytic activity of the cell lysate, although the procoagulant activity was still present. Furthermore, we found that the elastase activity almost disappeared after monocytic differentiation. We also studied the PCA, proteolytic activity, and elastase activity of blast cells of patients with acute myeloid leukemia. Only in patients with acute promyelocytic leukemia did we observe both a strong PCA and fibrinolytic activity. This supports our hypothesis that the combination of these activities is unique to the promyelocyte and may explain the observed bleeding complications in patients with acute promyelocytic leukemia.     

Long-term follow-up of endurance and safety outcomes during enzyme replacement therapy for mucopolysaccharidosis VI: Final results of three clinical studies of recombinant human N-acetylgalactosamine 4-sulfatase

The objective of this study was to evaluate the long-term clinical benefits and safety of recombinant human arylsulfatase B (rhASB) treatment of mucopolysaccharidosis type VI (MPS VI: Maroteaux-Lamy syndrome), a lysosomal storage disease. Fifty-six patients derived from 3 clinical studies were followed in open-label extension studies for a total period of 97–260 Weeks. All patients received weekly infusions of rhASB at 1 mg/kg. Efficacy was evaluated by (1) distance walked in a 12-minute walk test (12MWT) or 6-minute walk test (6MWT), (2) stairs climbed in the 3-minute stair climb (3MSC), and (3) reduction in urinary glycosaminoglycans (GAG). Safety was evaluated by compliance, adverse event (AE) reporting and adherence to treatment. Results: A significant reduction in urinary GAG (71–79%) was sustained. For the 12MWT, subjects in Phase 2 showed improvement of 255 ± 191 m (mean ± SD) at Week 144; those in Phase 3 Extension demonstrated improvement from study baseline of 183 ± 26 m (mean ±  SE) in the rhASB/rhASB group at Week 96 and from treatment baseline (Week 24) of 117 ± 25 m in the placebo/rhASB group. The Phase 1/2 6MWT and the 3MSC from Phase 2 and 3 also showed sustained improvements through the final study measurements. Compliance was 98% overall. Only 560 of 4121 reported AEs (14%) were related to treatment with only 10 of 560 (2%) described as severe. Conclusion: rhASB treatment up to 5 years results in sustained improvements in endurance and has an acceptable safety profile.     

Characterization of novel Mycobacterium tuberculosis pncA gene mutations in clinical isolates from the Ukraine

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis cases in the Ukraine are increasing. Pyrazinamide (PZA) is critically important for first- and second-line tuberculosis (TB) treatment regimes. However, PZA drug susceptibility testing is time consuming and technically challenging. The present study utilized Next-generation sequencing (NGS) to identify mutations in the pncA gene from clinical isolates and to assess the prevalence of pncA gene mutations in MDR/XDR-TB patients. Clinical isolates were inactivated in molecular transport media and shipped from Kharkiv, Ukraine, to San Antonio, TX. Whole-genome and targeted pncA gene sequencing was carried out using Illumina MiSeq instrumentation. Mutations were noted in 67 of 91 (74%) clinical isolates comprising substitutions, insertions, and deletions in the pncA coding and upstream promoter region. Of 45 mutation types, there were 11 novel, i.e., to date unknown, pncA mutations identified of which 3 were confirmed PZA resistant. Seven isolates contained mixed base mutations, whereas 4 harbored doubled mutations. Data reported here further support use of NGS for pncA gene characterization and may contribute in significant fashion to PZA therapy, especially in MDR- and XDR-TB patients.     

Pleiotropic effects of intravascular haemolysis on vascular homeostasis

The breakdown of senescent or defective red blood cells releases red cell contents, especially haemoglobin, which scavenges nitric oxide (NO) and decomposes to haem and free iron. These are potent oxidants, all of which have promoted the evolution of inducible and vasculoprotective compensatory pathways to rapidly clear and detoxify haemoglobin, haem and iron. Chronic haemolytic red cell disorders as diverse as sickle cell disease, thalassaemia, unstable haemoglobinopathy, cytoskeletal defects and enzymopathies have been linked to a clinical constellation of pulmonary hypertension, priapism, leg ulceration and possibly cerebrovascular disease and thrombosis. Besides free haemoglobin, haemolysis has been associated with extracellular arginase that limits substrate availability to NO synthase, endogenous inhibitors of NO synthase activity, and inappropriate activation of haemostatic pathways. This article reviews the haemolytic disorders that have been reported to manifest vascular complications, and explores the speculative possibility that haemolysis mediates some of the vascular complications of inflammation and diabetes.     

Spirituality and medicine

INTRODUCTION: Governing bodies for medical education recommend that spirituality and medicine be incorporated into training. AIM: To pilot a workshop on spirituality and medicine on a convenience sample of preclinical medical students and internal medicine residents and determine whether content was relevant to learners at different levels, whether preliminary evaluation was promising, and to generate hypotheses for future research. SETTING: Private medical school and university primary care internal medicine residency program, both in the Northeast. CURRICULUM DESCRIPTION: The authors designed and implemented a required 2-hour workshop for all second-year medical students and a separate required 1.5-hour workshop for all primary care internal medicine house staff. The workshops used multiple educational strategies including lecture, discussion, and role-play to address educational objectives. PROGRAM EVALUATION: Learners completed optional, anonymous pre and postworkshop surveys with six 5-point Likert-rated statements and space to cite the most useful part of the curriculum and their remaining questions. One hundred and thirty-seven learners participated and 100 completed both surveys. Medical students and residents had increased (all P< or =.002): agreement regarding the appropriateness of inquiring about spiritual and religious beliefs in the medical encounter, their perceived competence in taking a spiritual history, and their perceived knowledge of available pastoral care resources. Medical students, but not residents, had an increase in their perceived comfort in working with hospital chaplains. DISCUSSION: A brief pilot workshop on spirituality and medicine had a modest effect in improving attitudes and perceived competence of both medical students and residents.