The Role of the Calcium Channel α2δ-1 Subunit in Skeletal Muscle

Journal of Muscle Research and Cell Motility -     

Evaluation of a vectored equine herpesvirus type 1 (EHV-1) vaccine expressing H3 haemagglutinin in the protection of dogs against canine influenza.

In 2004, canine influenza virus (CIV) was identified as a respiratory pathogen of dogs for the first time and found to be closely related to H3N8 equine influenza virus (EIV). We generated a recombinant vectored vaccine that expresses H3 of a recent isolate of EIV using equine herpesvirus type 1 (EHV-1) as the delivery vehicle. This EHV-1 vectored vaccine exhibited robust and stable EIV H3 expression and induced a strong influenza virus-specific response in both mice and dogs upon intranasal or subcutaneous administration. Furthermore, upon challenge with the recent CIV isolate A/canine/PA/10915-07, protection of vaccinated dogs could be demonstrated by a significant reduction in clinical sings, and, more importantly, by a significant reduction in virus shedding. We concluded that the EHV-1/H3 recombinant vector can be a valuable alternative for protection of dogs against clinical disease induced by CIV and can significantly reduce virus spread.     

Effects of the depth of anesthesia on distortion product otoacoustic emissions

To analyze the effects of the depth of anesthesia on inner ear function measured with distortion product otoacoustic emissions (DPOAEs) at 2f ₁ ? f ₂. Thirty patients who underwent tonsillectomy under general anesthesia (GA) were included. Patients were assigned randomly to one of two groups: group 1 (n = 15) received propofol, group 2 (n = 15) sevoflurane as anesthetic agent. The sedation level was assessed by the bispectral index system. DPOAE measurements were performed before premedication (T ₁), 5 min after premedication (T ₂), 3 min after induction of general anesthesia (T ₃) and every 10 min (T ₄, T ₅) thereafter until the end of surgery at about 23 min post-anesthetic induction, while sedation levels were obtained starting at the beginning until the end of anesthesia. After premedication, both blood oxygen saturation and heart rate decreased. Following induction of anesthesia systolic and diastolic blood pressure decreased, while, as expected, the level of sedation increased. Analyzing the propofol and sevoflurane group separately, both groups showed comparable overall courses of DPOAE levels at higher frequencies (2.8 kHz p = 0.310, 4 kHz p = 0.193, 6 kHz p = 0.269, 8 kHz p = 0.223) and no changes of DPOAE levels compared with baseline values were observed. At T₅ the 1 kHz DPOAE level increased in the propofol group and slightly decreased in the sevoflurane group (p < 0.001). While the 1.4 kHz DPOAE level in the propofol group did not change over time the 1.4 kHz DPOAE level decreased in the sevoflurane group (baseline to T ₄ p = 0.045; Baseline to T ₅ p = 0.004). While overall there were different courses between these two groups in the 2 kHz DPOAE level, in the post hoc analysis only a tendency in the change from baseline to T ₄ could be observed (p = 0.082). These results indicate that while the amplitudes of certain DPOAEs were influenced by GA, the depth of anesthesia had no effect on this measure of cochlear function in clinical routine. Therefore, DPOAE measurements in sedation and during GA are useful but the effect of anesthetic agents on DPOAE levels needs to be taken into account when analyzing the test.     

CD133-enriched CD34(-) (CD33/CD38/CD71)(-) cord blood cells acquire CD34 prior to cell division and hematopoietic activity is exclusively associated with CD34 expression

We compared the cell division behavior of CD34(-) and CD34(+) (CD33/CD38/CD71)-negative (Lin(-)) CD133(+) cord blood cells stimulated with the cytokines Flt3-ligand, stem cell factor, and thrombopoietin. Within a 4-day time frame, Lin(-)CD34(-) CD133(+) (CD34(-)) cells underwent more cell divisions in serum-free culture than their Lin(-)CD34(+) CD133(+) (CD34(+)) counterparts. The majority of CD34(-) cells acquired expression of CD34 in vitro, including most undivided cells. Moreover, hematopoietic activity from both CD34(-) and CD34(+) cells was exclusively retained within the cell fraction expressing CD34 after 4 days in culture. Most strikingly, in cultures from Lin(-)CD34(-) cells hematopoietic activity was associated with the fraction of divided cells, whereas in cultures of CD34(+) cells, hematopoietic activity associated with the undivided cell fraction. Therefore, clonogenic CD34(+) cells either do not divide or lose their clonogenic capacity upon cell division in vitro, while CD34(-) cells divide and retain this capacity under the same specific conditions. In conclusion, we demonstrate that CD133-enriched Lin(-)CD34(-) cord blood cells acquire CD34 prior to cell division and that long-term hematopoietic activity is associated exclusively with expression of CD34.     

Proliferative activity of leukaemic blasts and cytosine arabinoside pharmacodynamics are associated with cytogenetically defined prognostic subgroups in acute myeloid leukaemia

The biological mechanisms responsible for the association of specific karyotypes with prognosis in acute myeloid leukaemia (AML) remain largely unclear. A prospective study was performed to evaluate how far cytogenetically defined prognostic subgroups of AML differ in their proliferative activity as a potential mechanism for differential sensitivities to S-phase-specific induction chemotherapy comprising cytosine arabinoside (AraC). One hundred and eighty-seven patients with de novo AML were included in the study; 25 patients with a favourable [inv(16), t(8;21), t(15;17)] karyotype, 99 with a normal karyotype, 29 with an unfavourable karyotype (-5, 5q-, -7, 7q-, complex aberrations) and 34 with cytogenetic aberrations of unknown prognostic significance (all others). The favourable group demonstrated the highest ex vivo proliferative activity (PA) (3.41 pmol/105 cells), significantly (P = 0.02) exceeding the unfavourable group with the lowest PA (0.72) and the group with a normal karyotype (1.06) or with karyotype of unknown significance (1.05) that both demonstrated an intermediate PA. Samples with a high PA (> median of the whole group) were more likely to produce interleukin 3, granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF) (56%, 43% and 50%) than cells with a low PA (33%, 36% and 36%; n.s.). The effect of priming by exogenous GM-CSF or G-CSF was significantly more pronounced in samples with a low PA than in rapidly proliferating samples (P < 0.01). For the whole group, a high PA was closely associated with an increased incorporation of AraC triphosphate (AraCTP) into the DNA (P < 0.0001). Clinically, a high PA was associated with a better complete remission (CR) rate in the normal (95% versus 62%) and the unfavourable group (75% versus 33%). The significant differences in proliferative activity between cytogenetic subgroups of AML are associated with increased cytosine arabinoside pharmacodynamics and constitute one potential mechanism for the different response of cytogenetic subgroups to AraC-based induction therapy.     

The vast majority of bone-marrow-derived cells integrated into mdx muscle fibers are silent despite long-term engraftment

Bone-marrow-derived cells can contribute nuclei to skeletal muscle fibers. However, serial sectioning of muscle in mdx mice implanted with GFP-labeled bone marrow reveals that only 20% of the donor nuclei chronically incorporated in muscle fibers show dystrophin (or GFP) expression, which is still higher than the expected frequency of "revertant" fibers, but there is no overall increase above controls over time. Obviously, the vast majority of incorporated nuclei either never or only temporarily turn on myogenic genes; also, incorporated nuclei eventually loose the activation of the beta-actin::GFP transgene. Consequently, we attempted to enhance the expression of dystrophin. In vivo application of the chromatin-modifying agents 5-azadeoxycytidine and phenylbutyrate as well as local damage by cardiotoxin injections caused a small increase in dystrophin-positive fibers without abolishing the appearance of "silent" nuclei. The results thus confirm that endogenous repair processes and epigenetic modifications on a small-scale lead to dystrophin expression from donor nuclei. Both effects, however, remain below functionally significant levels.     

Association between plasmin activation system and intravascular ultrasound signs of plaque instability in patients with unstable angina and non-st-segment elevation myocardial infarction

Background

The association between intravascular ultrasound (IVUS) signs of plaque instability and plasma levels of biomarkers was determined in patients with unstable angina and non-ST-segment elevation myocardial infarction (UA/NSTEMI).

Methods

Fifty-two patients underwent coronary angiography and IVUS 8 ± 5 hours after the onset of chest pain. IVUS analysis included plaque morphology, disruption, thrombi and eccentricity, lumen, external elastic membrane, and plaque plus media areas of culprit lesion and reference segments and arterial remodeling. Plasma levels of the thrombin activation system (thrombin-antithrombin complex [TAT], tissue factor pathway inhibitor [TFPI], and prothrombin fragments 1+2 [F1+2]) and plasmin activation system (tissue and urokinase-type plasminogen activator [t-PA and u-PA], plasminogen activator inhibitor-1 [PAI-1], and D-dimer) were measured with enzyme-linked immunosorbent assay kits before angiography.

Results

Elevated levels of TAT (7.2 ± 6.0 μg/L), F1+2 (1.8 ± 1.0 nmol/L), TFPI (179.1 ± 131.0 ng/mL), PAI-1 (95.4 ± 54.6 ng/mL), t-PA (10.6 ± 8.8 ng/mL), and u-PA (2.6 ± 0.9 ng/mL) were found in patients with UA/NSTEMI. The serum levels of D-dimer (40.0 ± 39.5 ng/mL) remained in reference range. Expansive and constrictive remodeling were found in 18 (35%) and 12 (23%) patients, respectively. Expansive remodeling of the culprit lesion was associated with significantly higher plasma levels of PAI-1 (121.6 ± 55.0 vs 87.7 ± 61.5 and 77.4 ± 42.8 ng/ml, P = .039), and u-PA (3.0 ± 1.2 vs 2.2 ± 0.5 and 2.5 ± 0.7 ng/mL, P = .026) as compared with constrictive and neutral remodeling. Increased plasma levels of u-PA were associated with plaque rupture (3.0 ± 0.7 vs 2.5 ± 0.9 ng/mL, P = .062). Plasma levels of PAI-1 and u-PA correlated positively with plaque plus media (P = .0297 and P = .0093) and external elastic membrane areas (P = .010 and P = .0002).

Conclusions

Elevated levels of biomarkers of plasmin activation system are associated with signs of plaque instability of culprit lesion in UA/NSTEMI and might therefore serve as non-invasive determinants of the population that is at high risk for subsequent adverse events.     

The Jak2V617F oncogene associated with myeloproliferative diseases requires a functional FERM domain for transformation and for expression of the Myc and Pim proto-oncogenes

The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5.     

Chlorhexidine and silver-sulfadiazine coated central venous catheters in haematological patients—a double-blind,randomised, prospective,controlled trial

Background Central venous catheters (CVCs) are essential for the intensive care of patients with haematological illness. Catheter-related infections (CRI) are an important problem in modern medicine, which may lead to life-threatening situations, to prolonged hospitalisation and increased cost. In immunocompromised patients suffering from haemato-oncological diseases, CRI is a significant factor for adverse outcome. Several clinical studies have shown that CVCs coated with antiseptics such as chlorhexidine and silver-sulfadiazine (CHSS) reduce the risk of catheter-related bacteraemia. Most studies, however, were performed on intensive care patients not suffering from chemotherapy-induced immunosuppression.Patients and methods A prospective double-blind, randomised, controlled trial was performed to investigate the effectiveness of CHSS-coated catheters in haemato-oncological patients. A total number of 184 catheters (median duration of placement, 11 days) were inserted into 184 patients (male 115, female 69), of which 90 were antiseptically coated. After removal, all catheters were investigated for bacterial growth.Main results Catheters coated with CHSS were effective in reducing the rate of significant bacterial growth on either the tip or subcutaneous segment (26%) compared to control catheters (49%). The incidence of catheter colonisation was also significantly reduced (12% coated vs 33% uncoated). Data obtained show a significant reduction of catheter colonisation in CHSS catheters. There was no significant difference in the incidence of catheter-related bacteraemia (3% coated vs 7% uncoated). However, due to the overall low rate of CRI, we could not observe a significant reduction in the incidence of catheter-related bacteraemia.Conclusion Our data show that the use of CHSS catheters in patients with haematological malignancy reduces the overall risk of catheter colonisation and CRI, although the incidence of catheter-related bacteremia was similar in both groups.