HL-60 cells degrade alpha-actinin to produce a fragment that promotes monocyte/macrophage maturation

An amino-terminal fragment of alpha-actinin can promote monocyte/macrophage maturation. This fragment was initially isolated from media of HL-60 myeloid leukemia cells cultured on extracellular bone marrow matrix. To determine the source of this fragment in this culture system, we investigated whether HL-60 cells grown on bone marrow stroma have increased intracellular levels of alpha-actinin that may be released into the media during cell apoptosis. HL-60 cells grown on matrix showed no evidence of increased cellular alpha-actinin compared to cells grown on plastic substrata as measured by flow cytometry. In addition, there was no evidence of increased apoptosis as determined by DNA fragmentation assays or flow cytometry. However, 100 kD alpha-actinin was found in the extracellular matrix of bone marrow stroma by Western blot analysis and immunofluorescence microscopy. The alpha-actinin content in the stroma was markedly decreased after exposure to HL-60 cells. Furthermore, lysates of HL-60 cells or of peripheral blood monocytes can degrade exogenous alpha-actinin to produce a 31 kD fragment, which promotes monocyte/macrophage maturation. We conclude that when alpha-actinin is present in the extracellular matrix, it can be modified by HL-60 cells to produce a maturation promoting 31 kD fragment.     

Detection of genomic deletions of PKP2 in arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease that predominantly affects the right ventricle and is associated with ventricular arrhythmias that may lead to sudden cardiac death. Mutations within at least seven separate genes have been identified to cause ARVC, however a genetic culprit remains elusive in approximately 50% of cases. Although negative genetic testing may be secondary to pathogenic mutations within undiscovered genes, an alternative explanation may be the presence of large deletions or duplications involving known genes. These large copy number variants may not be detected with standard clinical genetic testing which is presently limited to direct DNA sequencing. We describe two cases of ARVC possessing large deletions involving plakophilin‐2 (PKP2) identified with microarray analysis and/or multiplex ligation‐dependent probe amplification (MLPA) that would have been classified as genotype negative with standard clinical genetic testing. A deletion of the entire coding region of PKP2 excluding exon 1 was identified in patient 1 and his son. In patient 2, MLPA analysis of PKP2 revealed deletion of the entire gene with subsequent microarray analysis demonstrating a de novo 7.9 Mb deletion of chromosome 12p12.1p11.1. These findings support screening for large copy number variants in clinically suspected ARVC cases without clear disease causing mutations following initial sequencing analysis.     

Age-dependent neurological phenotypes in a mouse model of PRRT2-related diseases

Paroxysmal kinesigenic dyskinesia is an episodic movement disorder caused by dominant mutations in the proline-rich transmembrane protein PRRT2, with onset in childhood and typically with improvement or resolution by middle age. Mutations in the same gene may also cause benign infantile seizures, which begin in the first year of life and typically remit by the age of 2 years. Many details of PRRT2 function at the synapse, and the effects of mutations on neuronal excitability in the pathophysiology of epilepsy and dyskinesia, have emerged through the work of several groups over the last decade. However, the age dependence of the phenotypes has not been explored in detail in transgenic models. Here, we report our findings in heterozygous and homozygous Prrt2 knockout mice that recapitulate the age dependence of dyskinesia seen in the human disease. We show that Prrt2 deletion reduces the levels of synaptic proteins in a dose-dependent manner that is most pronounced at postnatal day 5 (P5), attenuates at P60, and disappears by P180. In a test for foot slippage while crossing a balance beam, transient loss of coordination was most pronounced at P60 and less prominent at age extremes. Slower traverse time was noted in homozygous knockout mice only, consistent with the ataxia seen in rare individuals with biallelic loss of function mutations in Prrt2. We thus identify three age-dependent phenotypic windows in the mouse model, which recapitulate the pattern seen in humans with PRRT2-related diseases.

     

Processes of knowing in the translation of a health communication intervention for dialysis patients awaiting kidney transplantation

ObjectiveTo strengthen patients’ health literacy and their role as active knowledge actors, we developed a health communication intervention including a film-viewing and counselling session for patients awaiting kidney transplantation. We aimed to explore processes of knowing in the translation of the intervention.MethodsWe applied an ethnographic research approach, observing nine intervention sessions with patients and dialysis nurses. Afterwards, the patients and the nurses were interviewed in-depth. Data were analysed using Engebretsen’s modified version of Lonergans’ four-step model of knowing.ResultsThe following knowing processes were identified: i) Knowing as meaning-making; ii) Knowing as acquiring confidence; and iii) Accessing professionals’ and peer experts’ knowledge. Divergent considerations were taken by the different knowledge actors, which had a direct influence on the knowing processes and knowledge translation.ConclusionsThe findings support active interactions between patients and healthcare providers in processes of knowing. These include self-conscious approaches and critical questioning in both parties.Practice implicationsFor transplant professionals, this study demonstrates knowing processes in a real-life context. It also spotlights professional skills and attitudes regarding the importance of self-conscious questioning and a critical interrogating position (for both patients and providers).     

Concept of variable angle locking—evolution and mechanical evaluation of a recent technology

Applications for fracture‐adapted screw positioning offered by variable angle locking screws are increasing. The locking strength of the variable angle locking mechanism at different insertion angles was compared to conventional fixed angle locking screws. Stainless steel (S) and titanium (Ti) variable and fixed angle 2.4 mm locking screws, inserted at different inclinations (0°?15°), and locked at 0.8 Nm were subjected to a load‐to‐failure test. Ultimate failure moment at the screw‐head interface and failure mode of the screws were determined. Significant differences were detected by one‐way ANOVA (p < 0.05). Stainless steel and titanium variable angle locking screws inserted at 2°?10° inclination exhibited a failure moment comparable to the 0° position (S 1.67± 0.04 Nm; Ti 1.67 ± 0.14 Nm) and failed predominantly at the screw neck, with the head remaining fixed to the plate. Inserted at 15° inclination, screws revealed a lower failure moment (S 1.33 ± 0.06 Nm, Ti 1.58 ± 0.05Nm), and failed predominantly by breakout of the head thread. Fixed angle locking screws inserted at an inclination >2° did not lock properly in the plate hole, providing insufficient locking strength. Variable angle locking screws offer a stable head‐locking mechanism at different inclinations, comparable to the locking strength of orthogonal inserted fixed angle locking screws. Marginal inclinations >15° should be used with care. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:988–992, 2015.

     

Differential regulation of colony-stimulating factors and interleukin 2 production by cyclosporin A.

Stimulation of T lymphocytes with mitogens or antigens is followed by proliferation and lymphokine production. Although cyclosporin A (CsA), an immunosuppressive drug, has been shown to inhibit the production of certain lymphokines, including interleukin 2 (IL-2), interleukin 3 (IL-3), and gamma-interferon, its effect on the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) has not been evaluated. In the current study, concanavalin A (Con A)-stimulated murine spleen cells secreted GM-CSF, IL-3, and IL-2, and in the presence of CsA (0.1-1.0 micrograms/ml), IL-2 and IL-3 activities were inhibited. In contrast, significant activity was detected when the CsA-treated culture supernatants were assayed on a cell line that is dependent on GM-CSF and/or IL-3. Similar CsA-resistant activity was observed when the EL-4 thymoma cells were stimulated with a phorbol ester [phorbol 12-myristate 13-acetate (PMA)] in the presence of CsA. The activity resistant to CsA was identified as GM-CSF by the ability of specific antibodies against murine recombinant GM-CSF to neutralize its activity. These findings indicate that GM-CSF, in contrast to IL-2 and IL-3, was not inhibited by CsA. In additional experiments, transfer blot of poly(A)+ RNA isolated from PMA-induced EL-4 cells in the presence or the absence of CsA was hybridized with GM-CSF and IL-2 cDNA probes. Expression of the GM-CSF gene in EL-4 cells was detected independent of CsA, whereas CsA inhibited the expression of the IL-2 gene. The present data show that production of IL-2 and IL-3, but not that of GM-CSF, is inhibited by CsA and suggest a differential control mechanism for lymphokine synthesis in T lymphocytes.     

Reliability and validity of the sideways step test and its correlation with motor function after stroke

[Purpose] This study investigated the intra-rater, inter-rater and test-retest reliability of the sideways step test (SST), its correlation with other indicators of stroke-specific impairment, and the cut-off count best discriminating subjects with stroke from their healthy counterparts. [Subjects and Methods] Forty-three subjects with chronic stroke and 41 healthy subjects older than 50 years participated in this study. The SST was administered along with the Fugl-Meyer motor assessment for the lower extremities (FMA-LE), the five-times sit to stand (5TSTS) test, the Berg Balance Scale (BBS), the movement velocity (MVL) by the limits of stability (LOS) test, the ten-metre walk (10mW) test, the timed “Up and Go” (TUG) test and the Activities-specific Balance Confidence (ABC) scale. [Results] The SST showed good to excellent intra-rater, inter-rater and test-retest reliability. The SST counts correlated with 5TSTS times, 10mW times, TUG times, and the FMA-LE and BBS scores. SST counts of 11 for the paretic leg and 14 for the non-paretic leg were found to distinguish the healthy adults from subjects with stroke. [Conclusion] The sideways step test is a reliable clinical test, which correlates with the functional strength, gait speed, and functional balance of people with chronic stroke.Key words: Balance, Stroke, Rehabilitation     

Assessment of aldehyde dehydrogenase in viable cells

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.     

Protection of murine bone marrow by dexamethasone during cytotoxic chemotherapy

Corticosteroids have the ability to suppress the production of growth factors and cytokines and are thus implicated in the negative regulation of hematopoiesis. We have shown that the corticosteroids, prednisolone and dexamethasone, were able to effectively protect progenitor cells in four strains of mice against cell-cycle-specific antimetabolic chemotherapy agents. The highest levels of protection against 5-fluorouracil (FU; 200 mg/kg) were achieved when two or three intraperitoneal injections of dexamethasone were administered between - 7 and +3 hours at a dose of 7.5 mg/kg/injection (optimal dose) or by continuous infusion between -4 and +20 hours. This protective effect is manifested as an increase in the number of high proliferative potential colony-forming cells that survive in the bone marrow 3 days after treatment with FU from between 0.5% and 11% to between 10% and 34% of normal. The bone marrow progenitors and blood cell numbers return to normal from 3 to 5 days and 1 to 2 days earlier, respectively. Less dexamethasone than prednisolone is required to give an equivalent protective effect, which is consistent with their anti-inflammatory potency. These findings are further evidence of the negative regulatory role played by corticosteroids, and indicate that the treatment schedules of corticosteroids during cancer therapy need to be reexamined to obtain the maximum benefit from their use.