Granulocyte-macrophage progenitor cell preservation at 4°C

Eighteen bone marrows collected from patients without haematological diseases and from normal subjects were tested for the effects of 4 d storage at 4 degrees C on CFU-C growth. Results indicate that unfractionated bone marrow cells may be stored at 4 degrees C for 4 d with 97% +/- 8 SEM recovery of the CFU-C evaluated by the agar culture assay. On the other hand, the same preservation procedure on peripheral blood CFU-C of 13 normal subjects yielded only 5% +/- 2 SEM recovery of in vitro growth capacity. The present results have practical implications. They might be exploited to preserve bone marrow CFU-C for transplantation therapy or laboratory investigation. In contrast this single preservation procedure seems not appropriate for preserving blood CFU-C.     

Sternomastoid muscle function and fatigue in breathless patients with severe respiratory disease

In patients with severe respiratory disease, the work of breathing is increased and the respiratory muscles, particularly those of inspiration, may become fatigued. Hitherto, there has been little information on the incidence of respiratory muscle fatigue in acutely breathless patients. We studied 34 patients with severe respiratory disease on admission to hospital when they were most breathless, and then, if possible, 7 to 14 days later after recovery for evidence of sternomastoid muscle fatigue or increased fatigability. Frequency/force curves, numerically expressed as the 20:50 ratio, were carried out in all patients on admission. Three of the 34 patients had evidence of low frequency fatigue (i.e., greater than 15% reduction in 20:50 ratio) in the sternomastoid muscle on admission when first studied (mean +/- SEM 20:50 ratio, 56.3 +/- 1.2%; n = 3). The mean 20:50 ratio in the remaining 31 patients on admission was 75.7 +/- 1.6% (n = 31) compared with 77.8 +/- 1.4% (n = 25) when symptomatically better (p less than 0.05). The mean 20:50 ratio on admission was also significantly lower than the mean 20:50 ratio in a group of age- and sex-matched normal control subjects (i.e., 78.5 +/- 1.4%, n = 25; p less than 0.05). Twenty-five patients were studied completely both on admission and recovery, including a fatigability test that involved the performance of 50 fatiguing head lifts with measurements of the 20:50 ratio 10 and 60 min later. Sternomastoid muscle fatigability was significantly increased on admission when the patients were most breathless, compared with recovery when they were less breathless (p less than 0.001 at both 10 and 60 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

FIH-1 Disrupts an LRRK1/EGFR Complex to Positively Regulate Keratinocyte Migration

Factor inhibiting hypoxia-inducible factor 1 (FIH-1; official symbol HIF1AN) is a hydroxylase that negatively regulates hypoxia-inducible factor 1α but also targets other ankyrin repeat domain–containing proteins such as Notch receptor to limit epithelial differentiation. We show that FIH-1 null mutant mice exhibit delayed wound healing. Importantly, in vitro scratch wound assays demonstrate that the positive role of FIH-1 in migration is independent of Notch signaling, suggesting that this hydroxylase targets another ankyrin repeat domain–containing protein to positively regulate motogenic signaling pathways. Accordingly, FIH-1 increases epidermal growth factor receptor (EGFR) signaling, which in turn enhances keratinocyte migration via mitogen-activated protein kinase pathway, leading to extracellular signal–regulated kinase 1/2 activation. Our studies identify leucine-rich repeat kinase 1 (LRRK1), a key regulator of the EGFR endosomal trafficking and signaling, as an FIH-1 binding partner. Such an interaction prevents the formation of an EGFR/LRRK1 complex, necessary for proper EGFR turnover. The identification of LRRK1 as a novel target for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration.CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2014 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.The asparaginyl hydroxylase factor-inhibiting hypoxia-inducible factor 1α (FIH-1; official symbol HIF1AN) was originally identified as a protein that interacts with and inhibits the activity of hypoxia-inducible factor 1α (HIF-1α) in the C-terminal transactivation domain^1,2 by coupling the oxidative decarboxylation of 2-oxoglutarate to the hydroxylation of HIF-1α.³ Significantly, proteins containing the ankyrin repeat domain, such as Notch, are other substrates for FIH-1.³ Only recently has FIH-1 been recognized to have pleiotropic roles in maintaining epithelial homeostasis.^4,5 For example, FIH-1 negatively regulates glycogen metabolism in corneal epithelium in a HIF-1α–independent manner via the direct involvement of the Akt/glycogen synthase kinase 3β signaling pathway.⁴ Furthermore, in epidermal and corneal epithelial keratinocytes, FIH-1 was shown to act as a negative regulator of differentiation via a coordinate decrease in Notch signaling.⁵ What is not clear in these studies is whether FIH-1 affects other signaling pathways known to influence keratinocyte growth, differentiation, and migration. For example, the regulation of Notch 1 activity by FIH-1⁵ raises the possibility of cross-talk with the epidermal growth factor receptor (EGFR)-signaling pathway, since EGFR signaling has been shown to be a negative regulator of Notch 1 gene expression and activity in keratinocytes.⁶Once EGF binds to the EGFR, numerous signaling pathways are activated that impact on cell proliferation, migration, differentiation, and survival.^7–9 With respect to the skin, EGFR impacts on epidermal and hair follicle development, keratinocyte proliferation, survival, cancer, and immune homeostasis.¹⁰ EGFR signaling also plays a prominent role in epidermal and corneal epithelial migration and wound repair. For example, in the epidermis, EGFR signaling has been shown to promote keratinocyte migration and wound repair.¹¹ Likewise, corneal perturbations activate the EGFR and downstream Ras-Raf-Mek-Erk1/2 (Ras, Raf, mitogen-activated protein kinase kinase, extracellular signal–regulated kinase 1/2) and phosphoinositide 3 kinase–Akt signaling cascades, which are required for efficient wound healing and are attenuated in patients with diabetic keratopathies.^12–14The activation of EGFR also commences endocytic trafficking, whereby the receptor is either packaged in lysosomes for degradation or recycled to the cell surface.^15,16 Endosomal trafficking is essential for establishing the extensiveness of the EGF-mediated signal, and thus much attention has been directed toward understanding the steps involved in the movement of the EGFR from the cell surface to cytoplasmic vesicles, such as the endosome, multivesicular body, and lysosome.^16–18 Recently, leucine-rich repeat kinase 1 (LRRK1) was recognized as a key regulator of EGFR endosomal trafficking.^19,20 Specifically, it is believed that LRRK1 forms a complex with activated EGFR through an interaction with growth factor receptor–bound protein 2 and that this complex is internalized in early endosomes.¹⁹ The mechanism by which LRRK1 regulates EGFR transport is from early to late endosomes.¹⁹LRRK1 protein kinase is one of the ROCO proteins, which contain a GTPase-like domain [Ras of complex proteins (Roc)] and a C-terminal of Roc (COR) domain.²¹ ROCO proteins have a series of leucine-rich repeats and/or ankyrin repeats, with LRRK1 containing six N-terminal ankyrin repeats.²² This latter aspect of LRRK1 is noteworthy since, as mentioned above, proteins with ankyrin repeat domains are potential substrates for FIH-1.³ Thus, FIH-1 has the potential to directly interact with LRRK1, which could impact on EGFR signaling.Here we show that ectopic expression of FIH-1 in keratinocytes increases phosphorylation of the EGFR, which positively affects keratinocyte migration via stimulation of the mitogen-activated protein kinase pathway, resulting in an increase in phosphorylated ERK1/2. Such enhanced migration is independent of Notch signaling. Moreover, our studies reveal that FIH-1 interacts with LRRK1 and prevents the formation of an EGFR/LRRK1 complex necessary for proper EGFR turnover.¹⁹ The identification of LRRK1 as a substrate for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration. Thus, the breadth of FIH-1 epithelial biology is considerably larger than previously realized.     

Long-term effects of various cleaning methods on polypropylene/ethylene copolymer retainer material

Objectives:To evaluate long-term light transmittance, surface roughness, and flexural modulus of polypropylene/ethylene copolymer retainer material after exposure to different cleaning methods.Materials and Methods:Standardized polypropylene/ethylene copolymer retainer specimens (n = 70, 50.8 mm × 12.7 mm × 1.0 mm) were subjected to seven chemical cleaning solutions: Invisalign cleaning crystals, Retainer Brite, Polident, Listerine mouthwash, 2.5% acetic acid, 0.6% NaClO, and 3% H₂O₂ for 6 months. The specimens were exposed to the different solutions twice a week for 15 minutes or according to manufacturer''s instructions, then stored in artificial saliva at 37°C. Another group of specimens (n = 10) were brushed with a standardized toothbrushing machine for 2 minutes twice a week. At baseline and 6 months, light transmittance, surface roughness, and flexural modulus of the specimens were quantified using spectrophotometry, profilometry and three-point bend testing, respectively. Qualitative analysis was performed using a scanning electron microscope (SEM). Statistical analyses were performed at a significance level of .05.Results:The results showed that light transmittance decreased significantly from baseline for all cleaning methods at 6 months. For an individual method, no significant differences were observed between specimens at baseline and 6 months in surface roughness and flexural modulus. No discernible differences in surface features were observed on SEM images.Conclusions:The results indicate that different cleaning methods affect the long-term light transmittance of the studied polypropylene/ethylene copolymer retainer material. However, for an individual cleaning method, no significant differences were shown for surface roughness or flexural modulus values at 6-months compared to baseline.     

Cell lineage analysis of acute leukemia relapse uncovers the role of replication-rate heterogeneity and microsatellite instability

Human cancers display substantial intratumoral genetic heterogeneity, which facilitates tumor survival under changing microenvironmental conditions. Tumor substructure and its effect on disease progression and relapse are incompletely understood. In the present study, a high-throughput method that uses neutral somatic mutations accumulated in individual cells to reconstruct cell lineage trees was applied to hundreds of cells of human acute leukemia harvested from multiple patients at diagnosis and at relapse. The reconstructed cell lineage trees of patients with acute myeloid leukemia showed that leukemia cells at relapse were shallow (divide rarely) compared with cells at diagnosis and were closely related to their stem cell subpopulation, implying that in these instances relapse might have originated from rarely dividing stem cells. In contrast, among patients with acute lymphoid leukemia, no differences in cell depth were observed between diagnosis and relapse. In one case of chronic myeloid leukemia, at blast crisis, most of the cells at relapse were mismatch-repair deficient. In almost all leukemia cases, > 1 lineage was observed at relapse, indicating that diverse mechanisms can promote relapse in the same patient. In conclusion, diverse relapse mechanisms can be observed by systematic reconstruction of cell lineage trees of patients with leukemia.     

Delayed left main stem obstruction following successful TAVI with an Edwards SAPIEN XT valve: successful resuscitation and percutaneous coronary intervention using a non-invasive automated chest compression device (AutoPulse)

Acute coronary artery obstruction at the time of device implantation is a recognized, albeit rare, complication of TAVI and is most frequently managed by emergency percutaneous intervention. This complication usually manifests with circulatory collapse due to compromising left ventricular ischemia and is most often observed immediately following valve deployment in the catheter laboratory or in theater. Immediate circulatory support is often necessary. We describe the first report of delayed left main stem obstruction 3.5 hours after successful deployment of a 26 mm Edwards SAPIEN XT valve via transfemoral implantation, with sudden development of circulatory collapse on the ward. Circulatory support was rapidly and effectively instituted with an automated non-invasive cardiac massage device, AutoPulse, that delivers continuous chest compressions. Successful emergency percutaneous intervention was then undertaken to the left main stem to displace a calcified nodule during automated external cardiac massage with the AutoPulse.     

Calcium homeostasis influences radiological fracture healing in postmenopausal women

Introduction

Recent studies suggest that calcium and 25-[OH]-cholecalciferol represent substantial co-factors in fracture healing. However, there still seems to be no sustainable consensus regarding the influence on fracture healing patterns. In this study, the influence of calcium and vitamin D levels on fracture callus formation was prospectively analysed using pQCT scan.

Methods

94 postmenopausal females with distal radius fractures and consecutive surgery were included. Calcium, 25-[OH]-cholecalciferol, parathyroid hormone and bone-specific alkaline phosphatase levels were obtained prior surgical treatment and after 6 weeks. A pQCT scan was performed on both sites. Bone mineral density and fracture callus area were determined after detecting the outer border contour at a threshold of 280 mg/ccm. Patients received daily supplements of 1000 mg calcium and 880 IU 25-[OH]-cholecalciferol.

Results

Mean 25-[OH]-cholecalciferol level was 19.61 ± 21.87 ng/ml, mean parathyroid hormone level was 52.6 ± 58.9 ng/l and mean Ca level was 2.23 ± 0.35 mmol/l. After 6 weeks of supplementation a significant increase of calcium (p < 0.001) and 25-[OH]-cholecalciferol (p < 0.001), and a significant decrease of parathyroid hormone (p < 0.001) levels were observed. Sixth week follow-up fracture callus area correlated significantly with postoperative normal range calcium levels on the fractured site (p = 0.006). Bone mineral density correlated with age (p < 0.001), but not with calcium and 25-[OH]-cholecalciferol levels after 6 weeks. All fractures presented timely adequate callus formation.

Conclusion

Calcium and parathyroid hormone serum levels influence fracture callus area interpreted as fracture callus formation patterns. Calcium levels within physiological range accounted for highest fracture callus area. Therefore, a balanced calcium homeostasis is required for appropriate callus formation.