Proteomic analysis reveals a potential role for extracellular vesicles within the erythroblastic island niche.

Introduction: Erythroblastic island (EBI) macrophages play an essential role in the production and maturation of the vast numbers of red blood cells (RBCs) that are produced throughout life. Their location within the bone marrow makes it difficult to study the cellular and molecular interactions associated with their action so we have used an in vitro model of the EBI niche using macrophages derived from human induced pluripotent stem cells (hiPSCs). We previously demonstrated that the activation of the transcription factor KLF1 enhanced the activity of hiPSC-derived EBI macrophages. Methods: To elucidate the mechanisms associated with EBI-like activity we carried out a quantitative proteomic analysis and assessed the role of extracellular vesicles using Nanosight Tracking analyses and media filtration. Results and Discussion: Gene ontology analysis showed that many of the proteins upregulated by KLF1 were protein-binding factors, some of which were associated with the cell membrane or extracellular vesicles We demonstrated that filtration of macrophage-conditioned media resulted in a reduction in the supportive effects on erythroid cell viability and maturation implying a role for extracellular vesicles but this was not KLF1 dependent. Pathway analyses of the proteomic data revealed that proteins upregulated by KLF1 were associated with the citric acid cycle, pyruvate metabolism and ATP synthesis indicating that KLF1-activated macrophages had a metabolic profile comparable to a pro-reparative phenotype. This study has generated a proteomic dataset that could provide new insights into the role of macrophages within the EBI niche and has indicated a potential role for extracellular vesicles in the differentiation and maturation of RBCs in vitro. Further research will aid in the production of RBCs in vitro for use in disease modelling and cell therapy.

Human cellular model systems of ß-thalassemia enable in-depth analysis of disease phenotype.

ß-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we create human disease cellular model systems for ß-thalassemia by gene editing the erythroid line BEL-A, which accurately recapitulate the phenotype of patient erythroid cells. We also develop a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilize the assay to demonstrate that the lines respond appropriately to verified reagents. We next use the lines to perform extensive analysis of the altered molecular mechanisms in ß-thalassemia erythroid cells, revealing upregulation of a wide range of biological pathways and processes along with potential novel targets for therapeutic investigation. Overall, the lines provide a sustainable supply of disease cells as research tools for identifying therapeutic targets and as screening platforms for new drugs and reagents.

A novel human cellular model of CDA IV enables comprehensive analysis revealing the molecular basis of the disease phenotype.

Red blood cell disorders can result in severe anemia. One such disease congenital dyserythropoietic anemia IV (CDA IV) is caused by the heterozygous mutation E325K in the transcription factor KLF1. However, studying the molecular basis of CDA IV is severely impeded by the paucity of suitable and adequate quantities of material from patients with anemia and the rarity of the disease. We, therefore, took a novel approach, creating a human cellular disease model system for CDA IV that accurately recapitulates the disease phenotype. Next, using comparative proteomics, we reveal extensive distortion of the proteome and a wide range of disordered biological processes in CDA IV erythroid cells. These include downregulated pathways the governing cell cycle, chromatin separation, DNA repair, cytokinesis, membrane trafficking, and global transcription, and upregulated networks governing mitochondrial biogenesis. The diversity of such pathways elucidates the spectrum of phenotypic abnormalities that occur with CDA IV and impairment to erythroid cell development and survival, collectively explaining the CDA IV disease phenotype. The data also reveal far more extensive involvement of KLF1 in previously assigned biological processes, along with novel roles in the regulation of intracellular processes not previously attributed to this transcription factor. Overall, the data demonstrate the power of such a model cellular system to unravel the molecular basis of disease and how studying the effects of a rare mutation can reveal fundamental biology.

Reproducible immortalization of erythroblasts from multiple stem cell sources provides approach for sustainable RBC therapeutics.

Developing robust methodology for the sustainable production of red blood cells in vitro is essential for providing an alternative source of clinical-quality blood, particularly for individuals with rare blood group phenotypes. Immortalized erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34+ cells that had improved differentiation and enucleation potential compared to other lines reported. In this study we show that our immortalization approach is reproducible for erythroid cells differentiated from bone marrow and also from far more accessible peripheral and cord blood CD34+ cells, consistently generating lines with similar improved erythroid performance. Extensive characterization of the lines shows them to accurately recapitulate their primary cell equivalents and provides a molecular signature for immortalization. In addition, we show that only cells at a specific stage of erythropoiesis, predominantly proerythroblasts, are amenable to immortalization. Our methodology provides a step forward in the drive for a sustainable supply of red cells for clinical use and for the generation of model cellular systems for the study of erythropoiesis in health and disease, with the added benefit of an indefinite expansion window for manipulation of molecular targets.

Identification of the transcription factor MAZ as a regulator of erythropoiesis.

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.

Characterization and evolutionary origin of novel C2H2 zinc finger protein (ZNF648) required for both erythroid and megakaryocyte differentiation in humans.

Human ZNF648 is a novel poly C-terminal C2H2 zinc finger protein identified amongst the most dysregulated proteins in erythroid cells differentiated from iPSC. Its nuclear localisation and structure indicate it is likely a DNA-binding protein. Using a combination of ZNF648 overexpression in an iPSC line and primary adult erythroid cells, ZNF648 knockdown in primary adult erythroid cells and megakaryocytes, comparative proteomics and transcriptomics we show that ZNF648 is required for both erythroid and megakaryocyte differentiation. Orthologues of ZNF648 were detected across Mammals, Reptilia, Actinopterygii, in some Aves, Amphibia and Coelacanthiformes suggesting the gene originated in the common ancestor of Osteichthyes (Euteleostomi or bony fish). Conservation of the C-terminal zinc finger domain is higher, with some variation in zinc finger number but a core of at least six zinc fingers conserved across all groups, with the N-terminus recognisably similar within but not between major lineages. This suggests the N-terminus of ZNF648 evolves faster than the C-terminus, however this is not due to exon-shuffling as the entire coding region of ZNF648 is within a single exon. As for other such transcription factors, the N-terminus likely carries out regulatory functions, but showed no sequence similarity to any known domains. The greater functional constraint on the zinc finger domain suggests ZNF648 binds at least some similar regions of DNA in the different organisms. However, divergence of the N-terminal region may enable differential expression, allowing adaptation of function in the different organisms.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.

In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

ZEB1-repressed microRNAs inhibit autocrine signaling that promotes vascular mimicry of breast cancer cells.

During normal tumor growth and in response to some therapies, tumor cells experience acute or chronic deprivation of nutrients and oxygen and induce tumor vascularization. While this occurs predominately through sprouting angiogenesis, tumor cells have also been shown to directly contribute to vessel formation through vascular mimicry (VM) and/or endothelial transdifferentiation. The extrinsic and intrinsic mechanisms underlying tumor cell adoption of endothelial phenotypes, however, are not well understood. Here we show that serum withdrawal induces mesenchymal breast cancer cells to undergo VM and that knockdown of the epithelial-to-mesenchymal transition (EMT) regulator, Zinc finger E-box binding homeobox 1 (ZEB1), or overexpression of the ZEB1-repressed microRNAs (miRNAs), miR-200c, miR-183, miR-96 and miR-182 inhibits this process. We find that secreted proteins Fibronectin 1 (FN1) and serine protease inhibitor (serpin) family E member 2 (SERPINE2) are essential for VM in this system. These secreted factors are upregulated in mesenchymal cells in response to serum withdrawal, and overexpression of VM-inhibiting miRNAs abrogates this upregulation. Intriguingly, the receptors for these secreted proteins, low-density lipoprotein receptor-related protein 1 (LRP1) and Integrin beta 1 (ITGB1), are also targets of the VM-inhibiting miRNAs, suggesting that autocrine signaling stimulating VM is regulated by ZEB1-repressed miRNA clusters. Together, these data provide mechanistic insight into the regulation of VM and suggest that miRNAs repressed during EMT, in addition to suppressing migratory and stem-like properties of tumor cells, also inhibit endothelial phenotypes of breast cancer cells adopted in response to a nutrient-deficient microenvironment.

The hydroxypyridinone iron chelator CP94 increases methyl-aminolevulinate-based photodynamic cell killing by increasing the generation of reactive oxygen species.

Methyl-aminolevulinate-based photodynamic therapy (MAL-PDT) is utilised clinically for the treatment of non-melanoma skin cancers and pre-cancers and the hydroxypyridinone iron chelator, CP94, has successfully been demonstrated to increase MAL-PDT efficacy in an initial clinical pilot study. However, the biochemical and photochemical processes leading to CP94-enhanced photodynamic cell death, beyond the well-documented increases in accumulation of the photosensitiser protoporphyrin IX (PpIX), have not yet been fully elucidated. This investigation demonstrated that MAL-based photodynamic cell killing of cultured human squamous carcinoma cells (A431) occurred in a predominantly necrotic manner following the generation of singlet oxygen and ROS. Augmenting MAL-based photodynamic cell killing with CP94 co-treatment resulted in increased PpIX accumulation, MitoSOX-detectable ROS generation (probably of mitochondrial origin) and necrotic cell death, but did not affect singlet oxygen generation. We also report (to our knowledge, for the first time) the detection of intracellular PpIX-generated singlet oxygen in whole cells via electron paramagnetic resonance spectroscopy in conjunction with a spin trap.

Cement-in-cement femoral revisions using a specially designed polished short revision stem; 24 consecutive stems followed for five to seven years.

Femoral cement-in-cement revisions are attractive if the cement bone mantle is well fixed. However, most available cemented stems are too long to fit in the existing cement mantle. We evaluated the medium-term outcomes of the 125 mm short tapered polished stem (Exeter Short Revision Stem (SRS)) with a 44 mm offset specifically designed to facilitate cement-in-cement revisions of hip arthroplasties. The Exeter SRS was clinically and radiographically evaluated in 24 consecutive femoral cement-in-cement revisions (11 men, 13 women) between July 2005 and February 2008 after a mean follow-up of six years (5-7). The mean age at operation was 67 years (54-83). No hip was lost to follow-up, but two patients (two hips) died. None of the deaths were related to the surgery. Kaplan Meier survival analysis was performed. Four femoral components (17%) were removed for septic loosening after a mean of 2.4 years (0.8-4.9). Three of these hips were revised again in a two-stage revision, and one was converted to a permanent excision arthroplasty. The probability of survival with re-revision for any reason was 82% (95% CI: 58-93) and survivorship with aseptic loosening as the endpoint was 100% at six years. There were no additional radiological failures. The Exeter Short Revision Stem is a valuable option for simplifying cement-in-cement revisions. Despite the short stem length, at mid-term there were no signs of instability or aseptic loosening.

Computer screen saver hand hygiene information curbs a negative trend in hand hygiene behavior.

BACKGROUND: Appropriate hand hygiene among health care workers is the most important infection prevention measure; however, compliance is generally low. Gain-framed messages (ie, messages that emphasize the benefits of hand hygiene rather than the risks of noncompliance) may be most effective, but have not been tested. METHODS: The study was conducted in a 27-bed neonatal intensive care unit. We performed an interrupted time series analysis of objectively measured hand disinfection events. We used electronic devices in hand alcohol dispensers, which continuously documented the frequency of hand disinfection events. In addition, hand hygiene compliance before and after the intervention period were directly observed. RESULTS: The negative trend in hand hygiene events per patient-day before the intervention (decrease by 2.3 [standard error, 0.5] per week) changed to a significant positive trend (increase of 1.5 [0.5] per week) after the intervention (P < .001). The direct observations confirmed these results, showing a significant improvement in hand hygiene compliance from 193 of 303 (63.6%) observed hand hygiene events at pretest to 201 of 281 (71.5%) at posttest. CONCLUSIONS: We conclude that gain-framed messages concerning hand hygiene presented on screen savers may improve hand hygiene compliance.

Total hip arthroplasties in young patients under 50 years: limited evidence for current trends. A descriptive literature review.

We examined all reported outcomes of uncemented and cemented total hip arthroplasty in patients younger than 50 years of age listed in Medline (1966- 1 January 2009) and PubMed, and scrutinised reference lists of relevant papers. In addition, we evaluated relevant data in the Swedish hip arthroplasty register. 109 relevant articles were identified, 37 of which had a mean follow-up longer than 10 years. Although uncemented implants are widely used in patients under 50 years of age, there are only 2 reports that fulfil the criteria published by the National Institute for Clinical Excellence (NICE) in the United Kingdom (follow-up of >10 yrs and survival of =90%). Current trends relating to implant selection remain unsupported by survival data, and additional information about the long-term results of newer implants is essential. As matters stand, the most reliable results relate to cemented implants.

Revisions of extensive acetabular defects with impaction grafting and a cement cup.

BACKGROUND: Loosening of acetabular components often leads to bony defects. Management of extensive acetabular bone loss in hip revision arthroplasty can be a tremendous challenge. QUESTIONS/PURPOSES: We asked whether a reconstruction with impacted bone grafts will provide a durable and pain-free function in extensive acetabular defects. We specifically determined the (1) survival rates with the end point of revision for any reason, aseptic revision, and radiographic loosening; (2) visual analog scale (VAS) pain score, Harris hip score (HHS), and the Oxford Hip Questionnaire score (OHQS); (3) number of repeat revisions; (4) complications; and (5) radiographic loosening, wear, and radiolucencies. PATIENTS AND METHODS: We retrospectively followed 25 patients (27 hips) with extensive acetabular defects. No patient was lost to followup. Two patients died during followup. Minimum followup was 3 years (mean, 8.8 years; range, 3-14.1 years). RESULTS: Three patients (three hips) underwent repeat revision surgery and another two patients (two hips) had radiographically loose hips. The 10-year survival rate was 88% (95% confidence interval, 74.2%-100%) with the end point acetabular revision for any reason and 95% (95% confidence interval, 86.0%-100%) with the end point acetabular revision for aseptic loosening. The mean HHSs were 55 points before surgery and 72 points postoperatively. CONCLUSIONS: Acetabular reconstruction with impaction bone grafting and a cemented cup is a reliable technique with a 10-year survival rate of 88% in patients with extensive acetabular deficiencies. LEVEL OF EVIDENCE: Level IV, case series. See the Guidelines for Authors for a complete description of levels of evidence.

[Total hip arthroplasty in patients under 40 years of age and, if indicated, reconstruction of acetabular defects with bone impaction grafting]. / Totaleheupprothesen bij patiënten jonger dan 40 jaar en reconstructie van botdefecten met botsnippers.

OBJECTIVE: To determine the long-term results of primary cemented total hip arthroplasty in patients under the age of 40. In cases of acetabular defects, initial reconstruction with bone impaction grafting was performed. DESIGN: Cohort analysis. METHODS: Details of patients under the age of 40 who underwent primary cemented total hip arthroplasty between 1 January 1988 and 30 June 2004 were analysed. The primary goal of this study was to determine the time until revision surgery. Kaplan Meier analysis was used to calculate prosthesis survival. RESULTS: A total of 175 total hip arthroplasties in 130 patients were included in the study. Acetabular reconstruction using bone impaction grafting was performed on 84 hips (48%). The average age at surgery was 31 years. Six patients (8 hips) died during follow-up; none of these had undergone revision surgery. Average follow-up was 8.1 years (range: 2.0-18.5). In total, 24 hips (14%) were revised. Reasons for revision were: septic loosening (n = 8), recurrent dislocations (n = 4), traumatic loosening (n = 1) and aseptic loosening (n = 11). The 10-year prosthesis survival was 83% (95% CI: 76-90) with the endpoint 'revision for any reason' and 92% (95% CI: 86-98) with the endpoint 'revision for aseptic loosening'. Aseptic survival of the cups with and without bone impaction grafting was 95% (95% CI: 89-100) and 90% (95% CI: 81-99) (p = 0.73), respectively. CONCLUSION: Hip replacement with cemented total hip arthroplasty in patients under the age of 40 produced good long-term results. Acetabular deficiencies reconstructed with bone impaction grafting also produced good results in this group of patients.

Good results with cemented total hip arthroplasty in patients between 40 and 50 years of age.

BACKGROUND AND PURPOSE: Total hip arthroplasties in young patients have lower long-term survival rates than in older patients. We evaluated the use of a unique treatment protocol in patients aged between 40 and 50 years. In all cases we used a cemented THA, and for acetabular deficiencies we also used impacted bone grafts together with a cemented cup. METHODS: In 140 consecutive patients who were between 40 and 50 years of age at index surgery, 168 cemented total hip prostheses were evaluated after a mean follow-up time of 10 (2-19) years. Acetabular deficiencies were reconstructed with wire meshes and impacted bone grafts with a cemented cup (70 hips). During follow-up, 18 patients died (27 hips); in this group 3 hips (3 patients) had been revised. None of the patients were lost to follow-up. In all surviving patients, clinical assessment was performed with hip-score questions and all radiographs were evaluated. RESULTS: All clinical questionnaires showed an improved clinical hip score. 29 hips (17%) were revised after a mean of 8 (0.3-18) years. Kaplan-Meier survival analysis showed a survival of 88% (95% CI: 82-94) after 10 years with revision of either component for any reason. Survival with endpoint revision for aseptic loosening of either component was 94% (95% CI: 90-99) after 10 years. INTERPRETATION: Cemented implants in young patients have satisfying long-term results. Reconstruction of acetabular deficiencies with impacted bone grafts show promising results.

Cemented polyethylene cups in patients younger than 40 years.

UNLABELLED: Although uncemented cup implants frequently are used in young patients, we believe long-term survival rates of cups in these patients are somewhat disappointing, and therefore we have continued to use cemented cups in primary THA, even in young patients. However, in cases of acetabular bone stock defects, we also use bone impaction grafting. We prospectively followed 130 patients with 175 cemented cups; no patients were lost to followup. The mean age of the patients at surgery was 31 years (range, 16-39 years). An acetabular reconstruction with bone impaction grafting was performed in 84 hips (48%). The minimum followup was 2 years (average, 8.1 years; range, 2.0-18.5 years). Twenty-one of the 175 cups (12%) were revised at an average of 8.1 years (range, 2.0-18.5 years). Reasons for revision were infection (one early, seven late), recurrent dislocations (two), traumatic loosening (one), and aseptic loosening (10). The 10-year survival rate of all cemented cups with end point of revision for any cause was 85%. Survival with end point of aseptic loosening of all cups was 92%. Survival with end point of revision for aseptic loosening was 90% for the cups without impaction grafting and 95% for the cups with impaction grafting. We believe cemented acetabular cups in young patients have acceptable midterm survival; however, in the case of acetabular bone defects, we recommend reconstruction with impaction grafting. LEVEL OF EVIDENCE: Level III, therapeutic study.

Bilateral forefoot ischemia as a premonitory symptom of mixed cryoglobulinemia.

Cryoglobulins are composed of cold-sensitive immunoglobulins that precipitate upon cooling. As the cutaneous vasculature of the extremities is commonly exposed to colder temperatures than the body core, this precipitation often occurs in cutaneous, or even digital vessels. Hyperviscosity from the precipitated proteins can incite local thrombosis in otherwise normal vessels, which is manifested clinically as ischemic ulceration. In previously injured vessels, as seen with atherosclerotic occlusive disease, cryoglobulin precipitation can lead to thrombosis of larger vessels, with the consequence being more severe ischemic necrosis. A case of bilateral forefoot ischemia is presented where the precipitating cause of the gangrenous changes appears to be the development of a mixed cryoglobulinemia and a B-cell lymphoma. Tibial angioplasty, plasmaphoresis, and chemotherapy directed at the B-cell lymphoma allowed limb salvage with bilateral transmetatarsal amputations.