Coupling an HCN2-expressing cell to a myocyte creates a two-cell pacing unit

We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell–cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell–cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of ∼0.6 to 1.7 Hz (1.09 ± 0.05 Hz). Addition of carbenoxolone (200 μ m ), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 μ m 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.     

Rearrangement of the T cell receptor gamma-chain gene in childhood acute lymphoblastic leukemia

We analyzed rearrangements of the T cell receptor gamma-chain (T gamma) gene as well as rearrangements of the T cell receptor beta-chain (T beta) gene and immunoglobulin heavy-chain (IgH) gene in 68 children with acute lymphoblastic leukemia (ALL). All 15 patients with T cell ALL showed rearrangements of both T beta and T gamma genes. Twenty-four of 53 non-T, non-B ALL patients (45%) showed T gamma gene rearrangements and only 14 of these also showed T beta gene rearrangements. Only a single patient rearranged the T beta gene in the absence of T gamma gene rearrangement. The rearrangement patterns of the T gamma gene in non-T, non-B ALL were quite different from those observed in T cell ALL, as 20 of 23 patients retained at least one germline band of the T gamma gene. In contrast, all T cell ALL patients showed no retention of germline bands. These data indicate that rearrangement of the T gamma gene is not specific for T cell ALL. Further, the results also suggest that T gamma gene rearrangement precedes T beta gene rearrangement. The combined analysis of rearrangement patterns of IgH, T beta, and T gamma genes provides new criteria for defining the cellular origin of leukemic cells and for further delineation of leukemia cell heterogeneity.     

Safety and tolerability of as-needed nalmefene in the treatment of alcohol dependence: results from the Phase III clinical programme

Objective: To investigate safety and tolerability of nalmefene for reduction of alcohol consumption in alcohol-dependent patients.

Methods: Pooled data from three randomized, placebo-controlled studies (two 6-month; one 12-month) of 18 mg nalmefene (as-needed use) in alcohol-dependent patients looking at the total population (placebo n = 824, nalmefene n = 1123) and patients with high/very high drinking risk levels at screening and randomization (target population: placebo n = 374, nalmefene n = 450).

Results: In the study, 62.7% of patients on placebo and 74.7% on nalmefene in the total population had treatment-emergent adverse events (TEAEs). Fourty-seven (5.9%) on placebo and 149 (13.0%) on nalmefene dropped out due to TEAEs. Thirty-five (4.4%) on placebo and 57 (5.0%) on nalmefene had serious adverse events. Tolerability and safety were similar in the target population and total population. Most frequent TEAEs were transient, mainly occurring at treatment initiation. There was no difference in tolerability and safety if nalmefene was taken daily or intermittently; no signal of increased risk of suicide-related behavior with nalmefene. The higher incidence of psychiatric events in the nalmefene group was mainly due to the TEAE of confusional state.

Conclusions: Although there was a higher incidence of TEAEs and TEAEs leading to dropout, nalmefene was well-tolerated and no major safety issues were identified.     

Direct evidence for new T-cell generation by patients after either T-cell-depleted or unmodified allogeneic hematopoietic stem cell transplantations

Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution of normal T-cell immunity. Recipient thymic activity, biologic features of the allograft, and preparative regimens all contribute to immune reconstitution. We evaluated circulating T-cell phenotypes and T-cell receptor rearrangement excision circles (TRECs) in 331 blood samples from 158 patients who had undergone allogeneic HSCTs. All patients had received myeloablative conditioning regimens and were full donor chimeras in remission. Younger patients exhibited more rapid recovery and higher TRECs (P =.02). Recipients of T-cell-depleted allografts initially had lower TRECs than unmodified allograft recipients (P <.01), but the difference abated beyond 9 months. TREC level disparities did not achieve significance among adults with respect to type of allograft. Measurable, albeit low, TREC values correlated strongly with severe opportunistic infections (P <.01). This finding was most notable during the first 6 months after transplantation, when patients are at greatest risk but before cytofluorography can detect circulating CD45RA(+) T cells. Low TRECs also correlated strongly with extensive chronic graft-versus-host disease (P <.01). Recipients of all ages of either unmodified or T-cell-depleted allografts therefore actively generate new T cells. This generation is most notable among adult recipients of T-cell-depleted allografts, most of whom had also received antithymocyte globulin for rejection prophylaxis. Low TREC values are significantly associated with morbidity and mortality after transplantation. T-cell neogenesis, appropriate to age but delayed in adult recipients of T-cell-depleted allografts, justifies interventions to hasten this process and to stimulate desirable cellular immune responses.     

Purified membrane and soluble folate binding proteins from cultured KB cells have similar amino acid compositions and molecular weights but differ in fatty acid acylation.

A membrane-associated folate binding protein (FBP) and a soluble FBP, which is released into the culture medium, have been purified from human KB cells using affinity chromatography. By NaDodSO4/PAGE, both proteins have an apparent Mr of approximately 42,000. However, in the presence of Triton X-100, the soluble FBP eluted from a Sephadex G-150 column with an apparent Mr of approximately 40,000 (similar to NaDodSO4/PAGE) but the membrane-associated FBP eluted with an apparent Mr of approximately 160,000, indicating that this species contains a hydrophobic domain that interacts with the detergent micelles. The amino acid compositions of both forms of FBP were similar, especially with respect to the apolar amino acids. In addition, the 18 amino acids at the amino termini of both proteins were identical. The membrane FBP, following delipidation with chloroform/methanol, contained 7.1 mol of fatty acid per mol of protein, of which 4.7 mol was amide-linked and 2.4 mol was ester-linked. The soluble FBP contained only 0.05 mol of fatty acid per mol of protein. These studies indicate that the membrane FBP of KB cells contains covalently bound fatty acids that may serve to anchor the protein in the cell membrane.     

How physical therapists instruct patients with stroke: an observational study on attentional focus during gait rehabilitation after stroke

Purpose: People without neurological impairments show superior motor learning when they focus on movement effects (external focus) rather than on movement execution itself (internal focus). Despite its potential for neurorehabilitation, it remains unclear to what extent external focus strategies are currently incorporated in rehabilitation post-stroke. Therefore, we observed how physical therapists use attentional focus when treating gait of rehabilitating patients with stroke.

Methods: Twenty physical therapist-patient couples from six rehabilitation centers participated. Per couple, one regular gait-training session was video-recorded. Therapists’ statements were classified using a standardized scoring method to determine the relative proportion of internally and externally focused instructions/feedback. Also, we explored associations between therapists’ use of external/internal focus strategies and patients’ focus preference, length of stay, mobility, and cognition.

Results: Therapists’ instructions were generally more external while feedback was more internal. Therapists used relatively more externally focused statements for patients with a longer length of stay (B?=??0.239, p?=?0.013) and for patients who had a stronger internal focus preference (B?=??0.930, p?=?0.035).

Conclusions: Physical therapists used more external focus instructions, but more internally focused feedback. Also, they seem to adapt their attentional focus use to patients’ focus preference and rehabilitation phase. Future research may determine how these factors influence the effectiveness of different attentional foci for motor learning post-stroke.

• IMPLICATIONS FOR REHABILITATION

• Physical therapists use a balanced mix of internal focus and external focus instructions and feedback when treating gait of stroke patients.

• Therapists predominantly used an external focus for patients in later rehabilitation phases, and for patients with stronger internal focus preferences, possibly in an attempt to stimulate more automatic control of movement in these patients.

• Future research should further explore how a patients’ focus preference and rehabilitation phase influence the effectiveness of different focus strategies.

• Awaiting further research, we recommend that therapists use both attentional focus strategies, and explore per patient which focus works best on a trial-and-error basis.