Imaging Glioblastoma Metabolism by Using Hyperpolarized [1-13C]Pyruvate Demonstrates Heterogeneity in Lactate Labeling: A Proof of Principle Study.

Purpose To evaluate glioblastoma (GBM) metabolism by using hyperpolarized carbon 13 (13C) MRI to monitor the exchange of the hyperpolarized 13C label between injected [1-13C]pyruvate and tumor lactate and bicarbonate. Materials and Methods In this prospective study, seven treatment-naive patients (age [mean ± SD], 60 years ± 11; five men) with GBM were imaged at 3 T by using a dual-tuned 13C-hydrogen 1 head coil. Hyperpolarized [1-13C]pyruvate was injected, and signal was acquired by using a dynamic MRI spiral sequence. Metabolism was assessed within the tumor, in the normal-appearing brain parenchyma (NABP), and in healthy volunteers by using paired or unpaired t tests and a Wilcoxon signed rank test. The Spearman ρ correlation coefficient was used to correlate metabolite labeling with lactate dehydrogenase A (LDH-A) expression and some immunohistochemical markers. The Benjamini-Hochberg procedure was used to correct for multiple comparisons. Results The bicarbonate-to-pyruvate (BP) ratio was lower in the tumor than in the contralateral NABP (P < .01). The tumor lactate-to-pyruvate (LP) ratio was not different from that in the NABP (P = .38). The LP and BP ratios in the NABP were higher than those observed previously in healthy volunteers (P < .05). Tumor lactate and bicarbonate signal intensities were strongly correlated with the pyruvate signal intensity (ρ = 0.92, P < .001, and ρ = 0.66, P < .001, respectively), and the LP ratio was weakly correlated with LDH-A expression in biopsy samples (ρ = 0.43, P = .04). Conclusion Hyperpolarized 13C MRI demonstrated variation in lactate labeling in GBM, both within and between tumors. In contrast, bicarbonate labeling was consistently lower in tumors than in the surrounding NABP. Keywords: Hyperpolarized 13C MRI, Glioblastoma, Metabolism, Cancer, MRI, Neuro-oncology Supplemental material is available for this article. Published under a CC BY 4.0 license.

Deuterium metabolic imaging and hyperpolarized 13C-MRI of the normal human brain at clinical field strength reveals differential cerebral metabolism.

Deuterium metabolic imaging (DMI) and hyperpolarized 13C-pyruvate MRI (13C-HPMRI) are two emerging methods for non-invasive and non-ionizing imaging of tissue metabolism. Imaging cerebral metabolism has potential applications in cancer, neurodegeneration, multiple sclerosis, traumatic brain injury, stroke, and inborn errors of metabolism. Here we directly compare these two non-invasive methods at 3 T for the first time in humans and show how they simultaneously probe both oxidative and non-oxidative metabolism. DMI was undertaken 1-2 h after oral administration of [6,6'-2H2]glucose, and 13C-MRI was performed immediately following intravenous injection of hyperpolarized [1-13C]pyruvate in ten and nine normal volunteers within each arm respectively. DMI was used to generate maps of deuterium-labelled water, glucose, lactate, and glutamate/glutamine (Glx) and the spectral separation demonstrated that DMI is feasible at 3 T. 13C-HPMRI generated maps of hyperpolarized carbon-13 labelled pyruvate, lactate, and bicarbonate. The ratio of 13C-lactate/13C-bicarbonate (mean 3.7 ± 1.2) acquired with 13C-HPMRI was higher than the equivalent 2H-lactate/2H-Glx ratio (mean 0.18 ± 0.09) acquired using DMI. These differences can be explained by the route of administering each probe, the timing of imaging after ingestion or injection, as well as the biological differences in cerebral uptake and cellular physiology between the two molecules. The results demonstrate these two metabolic imaging methods provide different yet complementary readouts of oxidative and reductive metabolism within a clinically feasible timescale. Furthermore, as DMI was undertaken at a clinical field strength within a ten-minute scan time, it demonstrates its potential as a routine clinical tool in the future.

Hyperpolarized 13C-Pyruvate Metabolism as a Surrogate for Tumor Grade and Poor Outcome in Renal Cell Carcinoma-A Proof of Principle Study.

Differentiating aggressive clear cell renal cell carcinoma (ccRCC) from indolent lesions is challenging using conventional imaging. This work prospectively compared the metabolic imaging phenotype of renal tumors using carbon-13 MRI following injection of hyperpolarized [1-13C]pyruvate (HP-13C-MRI) and validated these findings with histopathology. Nine patients with treatment-naïve renal tumors (6 ccRCCs, 1 liposarcoma, 1 pheochromocytoma, 1 oncocytoma) underwent pre-operative HP-13C-MRI and conventional proton (1H) MRI. Multi-regional tissue samples were collected using patient-specific 3D-printed tumor molds for spatial registration between imaging and molecular analysis. The apparent exchange rate constant (kPL) between 13C-pyruvate and 13C-lactate was calculated. Immunohistochemistry for the pyruvate transporter (MCT1) from 44 multi-regional samples, as well as associations between MCT1 expression and outcome in the TCGA-KIRC dataset, were investigated. Increasing kPL in ccRCC was correlated with increasing overall tumor grade (ρ = 0.92, p = 0.009) and MCT1 expression (r = 0.89, p = 0.016), with similar results acquired from the multi-regional analysis. Conventional 1H-MRI parameters did not discriminate tumor grades. The correlation between MCT1 and ccRCC grade was confirmed within a TCGA dataset (p < 0.001), where MCT1 expression was a predictor of overall and disease-free survival. In conclusion, metabolic imaging using HP-13C-MRI differentiates tumor aggressiveness in ccRCC and correlates with the expression of MCT1, a predictor of survival. HP-13C-MRI may non-invasively characterize metabolic phenotypes within renal cancer.

Hyperpolarised 13C-MRI identifies the emergence of a glycolytic cell population within intermediate-risk human prostate cancer.

Hyperpolarised magnetic resonance imaging (HP 13C-MRI) is an emerging clinical technique to detect [1-13C]lactate production in prostate cancer (PCa) following intravenous injection of hyperpolarised [1-13C]pyruvate. Here we differentiate clinically significant PCa from indolent disease in a low/intermediate-risk population by correlating [1-13C]lactate labelling on MRI with the percentage of Gleason pattern 4 (%GP4) disease. Using immunohistochemistry and spatial transcriptomics, we show that HP 13C-MRI predominantly measures metabolism in the epithelial compartment of the tumour, rather than the stroma. MRI-derived tumour [1-13C]lactate labelling correlated with epithelial mRNA expression of the enzyme lactate dehydrogenase (LDHA and LDHB combined), and the ratio of lactate transporter expression between the epithelial and stromal compartments (epithelium-to-stroma MCT4). We observe similar changes in MCT4, LDHA, and LDHB between tumours with primary Gleason patterns 3 and 4 in an independent TCGA cohort. Therefore, HP 13C-MRI can metabolically phenotype clinically significant disease based on underlying metabolic differences in the epithelial and stromal tumour compartments.

Hyperpolarized Carbon-13 MRI for Early Response Assessment of Neoadjuvant Chemotherapy in Breast Cancer Patients.

Hyperpolarized 13C-MRI is an emerging tool for probing tissue metabolism by measuring 13C-label exchange between intravenously injected hyperpolarized [1-13C]pyruvate and endogenous tissue lactate. Here, we demonstrate that hyperpolarized 13C-MRI can be used to detect early response to neoadjuvant therapy in breast cancer. Seven patients underwent multiparametric 1H-MRI and hyperpolarized 13C-MRI before and 7-11 days after commencing treatment. An increase in the lactate-to-pyruvate ratio of approximately 20% identified three patients who, following 5-6 cycles of treatment, showed pathological complete response. This ratio correlated with gene expression of the pyruvate transporter MCT1 and lactate dehydrogenase A (LDHA), the enzyme catalyzing label exchange between pyruvate and lactate. Analysis of approximately 2,000 breast tumors showed that overexpression of LDHA and the hypoxia marker CAIX was associated with reduced relapse-free and overall survival. Hyperpolarized 13C-MRI represents a promising method for monitoring very early treatment response in breast cancer and has demonstrated prognostic potential. SIGNIFICANCE: Hyperpolarized carbon-13 MRI allows response assessment in patients with breast cancer after 7-11 days of neoadjuvant chemotherapy and outperformed state-of-the-art and research quantitative proton MRI techniques.

An international collaborative study to establish the WHO 3rd International Standard for Thrombin: Communication from the ISTH SSC subcommittee on factor XIII and fibrinogen.

The calibration of thrombin products relies on the World Health Organization (WHO) 2nd International Standard (IS) for Thrombin (01/580) which defines the international unit (IU) for thrombin potency. With stocks of the 2nd IS (01/580) running low, an international collaborative study was organized to calibrate a replacement. Twenty laboratories from 13 countries took part in the study and measured the potency of two candidate replacement standards (coded 01/578 and 19/188) relative to the 2nd IS. In total, 111 valid assays were returned, which were a combination of plasma/fibrinogen clotting assays and chromogenic assays. Variation between and within laboratories was low, with inter- and intra-laboratory geometric coefficient of variation (GCV) generally <5% for all assay methods and substrates. For 01/578, potency estimates by clotting assays (101.1 IU/ampoule) were significantly lower than estimates by chromogenic assays (111.5 IU/ampoule). Mean potency estimates for 19/188 were 90.4 IU/ampoule by clotting assay and 88.1 IU/ampoule by chromogenic assay, which was not a statistically significant difference. The close ratio between clotting and chromogenic assay potency estimates for 19/188 suggests it has a higher α-thrombin content than 01/578 and is equivalent to the current IS (01/580). Accelerated degradation studies predicted excellent long-term stability profiles for preparations 01/580, 01/578, and 19/188. Based on the results of this study, the WHO Expert Committee on Biological Standardization established 19/188 as the 3rd IS for Thrombin with a potency of 90 IU/ampoule in August 2020.

Extracellular Histones Inhibit Fibrinolysis through Noncovalent and Covalent Interactions with Fibrin.

Histones released into circulation as neutrophil extracellular traps are causally implicated in the pathogenesis of arterial, venous, and microvascular thrombosis by promoting coagulation and enhancing clot stability. Histones induce structural changes in fibrin rendering it stronger and resistant to fibrinolysis. The current study extends these observations by defining the antifibrinolytic mechanisms of histones in purified, plasma, and whole blood systems. Although histones stimulated plasminogen activation in solution, they inhibited plasmin as competitive substrates. Protection of fibrin from plasmin digestion is enhanced by covalent incorporation of histones into fibrin, catalyzed by activated transglutaminase, coagulation factor FXIII (FXIIIa). All histone subtypes (H1, H2A, H2B, H3, and H4) were crosslinked to fibrin. A distinct, noncovalent mechanism explains histone-accelerated lateral aggregation of fibrin protofibrils, resulting in thicker fibers with higher mass-to-length ratios and in turn hampered fibrinolysis. However, histones were less effective at delaying fibrinolysis in the absence of FXIIIa activity. Therapeutic doses of low-molecular-weight heparin (LMWH) prevented covalent but not noncovalent histone-fibrin interactions and neutralized the effects of histones on fibrinolysis. This suggests an additional antithrombotic mechanism for LMWH beyond anticoagulation. In conclusion, for the first time we report that histones are crosslinked to fibrin by FXIIIa and promote fibrinolytic resistance which can be overcome by FXIIIa inhibitors and histone-binding heparinoids. These findings provide a rationale for targeting the FXIII-histone-fibrin axis to destabilize fibrin and prevent potentially thrombotic fibrin networks.

Hyperpolarized 13C MRI of Tumor Metabolism Demonstrates Early Metabolic Response to Neoadjuvant Chemotherapy in Breast Cancer.

Purpose: To compare hyperpolarized carbon 13 (13C) MRI with dynamic contrast material-enhanced (DCE) MRI in the detection of early treatment response in breast cancer. Materials and Methods: In this institutional review board-approved prospective study, a woman with triple-negative breast cancer (age, 49 years) underwent 13C MRI after injection of hyperpolarized [1-carbon 13 {13C}]-pyruvate and DCE MRI at 3 T at baseline and after one cycle of neoadjuvant therapy. The 13C-labeled lactate-to-pyruvate ratio derived from hyperpolarized 13C MRI and the pharmacokinetic parameters transfer constant (K trans) and washout parameter (k ep) derived from DCE MRI were compared before and after treatment. Results: Exchange of the 13C label between injected hyperpolarized [1-13C]-pyruvate and the endogenous lactate pool was observed, catalyzed by the enzyme lactate dehydrogenase. After one cycle of neoadjuvant chemotherapy, a 34% reduction in the 13C-labeled lactate-to-pyruvate ratio resulted in correct identification of the patient as a responder to therapy, which was subsequently confirmed via a complete pathologic response. However, DCE MRI showed an increase in mean K trans (132%) and mean k ep (31%), which could be incorrectly interpreted as a poor response to treatment. Conclusion: Hyperpolarized 13C MRI enabled successful identification of breast cancer response after one cycle of neoadjuvant chemotherapy and may improve response prediction when used in conjunction with multiparametric proton MRI.Published under a CC BY 4.0 license.

Fibrinogen protects neutrophils from the cytotoxic effects of histones and delays neutrophil extracellular trap formation induced by ionomycin.

Neutrophils are pivotal players in immune defence which includes a process of release of histones and DNA as neutrophil extracellular traps (NETs). Histones, while toxic to invading pathogens, also kill host cells, including neutrophils. Bacteria have evolved mechanisms to escape neutrophils, including the secretion of leucocidins (e.g. ionomycin). Live cell video microscopy showed how fibrinogen and fibrin influence NETosis and neutrophil responses to extracellular histones. Histones were rapidly lethal to neutrophils after binding to cells, but formation of fibrinogen/fibrin-histone aggregates prevented cell death. Histone cytotoxicity was also reduced by citrullination by peptidyl arginine deiminase 4, or digestion by serine proteases. Ionomycin and phorbol 12-myristate 13 acetate (PMA) are used to trigger NETosis. Fibrinogen was responsible for a second distinct mechanism of neutrophil protection after treatment with ionomycin. Fibrinogen clustered on the surface of ionomycin-stimulated neutrophils to delay NETosis; and blocking the ß integrin receptor, αMß2, abolished fibrinogen protection. Fibrinogen did not bind to or protect neutrophils stimulated with PMA. Fibrinogen is an acute phase protein that will protect exposed cells from damaging circulating histones or leucocidins; but fibrinogen depletion/consumption, as in trauma or sepsis will reduce protection. It is necessary to consider the role of fibrinogen in NETosis.

An international collaborative study to establish the WHO 4th International Standard for Streptokinase: Communication from the SSC of the ISTH.

Streptokinase is used worldwide as a cost-effective treatment for acute myocardial infarction. Manufacturers use the World Health Organization (WHO) International Standard (IS) for Streptokinase to potency label their products, ensuring consistent, safe, and effective dosing. Stocks of the third IS for streptokinase (coded 00/464) are running low, and an international collaborative study was organized to calibrate a replacement. A total of 15 laboratories from nine countries took part, using chromogenic and/or fibrin clot lysis methods to determine the potency of two candidate preparations, coded 16/356 (sample B) and 16/358 (sample C), relative to the third IS (00/464). A third sample (88/824, sample A), which was used in the collaborative studies to establish the second and third IS, was also included. There was good agreement in potency estimates from different assay methods and low variability both within and between laboratories. Long-term stability modeling indicated the candidates are very stable. Comparison of potency estimates for 88/824 (sample A) with potencies calculated in previous studies revealed a variability of only 1.9% over the course of three collaborative studies spanning 30 years and more than 50 years of streptokinase standardization. This indicates excellent continuity of the International Unit (IU) and assay methods. Following agreement by study participants and Scientific and Standardization Committee experts of the International Society on Thrombosis and Haemostasis, the WHO Expert Committee on Biological Standardization established 16/358 (sample C) as the fourth IS for Streptokinase with a potency of 1013 IU per ampoule in October 2019.

Imaging breast cancer using hyperpolarized carbon-13 MRI.

Our purpose is to investigate the feasibility of imaging tumor metabolism in breast cancer patients using 13C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized 13C label exchange between injected [1-13C]pyruvate and the endogenous tumor lactate pool. Treatment-naïve breast cancer patients were recruited: four triple-negative grade 3 cancers; two invasive ductal carcinomas that were estrogen and progesterone receptor-positive (ER/PR+) and HER2/neu-negative (HER2-), one grade 2 and one grade 3; and one grade 2 ER/PR+ HER2- invasive lobular carcinoma (ILC). Dynamic 13C MRSI was performed following injection of hyperpolarized [1-13C]pyruvate. Expression of lactate dehydrogenase A (LDHA), which catalyzes 13C label exchange between pyruvate and lactate, hypoxia-inducible factor-1 (HIF1α), and the monocarboxylate transporters MCT1 and MCT4 were quantified using immunohistochemistry and RNA sequencing. We have demonstrated the feasibility and safety of hyperpolarized 13C MRI in early breast cancer. Both intertumoral and intratumoral heterogeneity of the hyperpolarized pyruvate and lactate signals were observed. The lactate-to-pyruvate signal ratio (LAC/PYR) ranged from 0.021 to 0.473 across the tumor subtypes (mean ± SD: 0.145 ± 0.164), and a lactate signal was observed in all of the grade 3 tumors. The LAC/PYR was significantly correlated with tumor volume (R = 0.903, P = 0.005) and MCT 1 (R = 0.85, P = 0.032) and HIF1α expression (R = 0.83, P = 0.043). Imaging of hyperpolarized [1-13C]pyruvate metabolism in breast cancer is feasible and demonstrated significant intertumoral and intratumoral metabolic heterogeneity, where lactate labeling correlated with MCT1 expression and hypoxia.

Maintaining peri-implant health: an evaluation of understanding among dental hygienists and therapists in Wales.

Objectives To describe the approach taken by dental hygienists and therapists (DH/Ts) in Wales regarding dental implant maintenance. To gather their opinions about the current level of implant education.Materials and methods Online questionnaires were distributed to 257 DH/Ts within Wales.Results The response rate was 35%. Dental implant care was within the remit of service for 92% of respondents. All respondents that provided implant care stated that they performed oral hygiene instruction, while 98% performed supragingival debridement, 85% subgingival debridement, and 64% clinical assessment of peri-implant health. A high proportion of DH/Ts in Wales did not feel entirely confident in carrying out procedures relating to peri-implant maintenance and only 27% felt confident in clinically assessing dental implants. The majority (83%) felt that postgraduate training in peri-implant maintenance should be obligatory. 'No available courses' was the main reason for not attending further postgraduate training in implantology.Conclusions A high proportion of responding DH/Ts practising in Wales do not feel entirely confident in carrying out procedures relating to peri-implant maintenance. Postgraduate training may be useful in addressing this issue and undergraduate training programmes may need to consider increasing trainees' exposure to dental implant maintenance.

Teaching of implant dentistry in dental hygiene and therapy schools in the UK and Ireland.

Objective The aim of this study was to assess the current status of implant teaching within dental hygiene and therapy schools in the UK and Ireland.Methods An online questionnaire relating to current and future possible trends in dental implantology education was developed and distributed to programme directors in each of the 23 dental hygiene and therapy schools in the UK and Ireland.Results All responding schools (response rate of 60%) provided implant training for their students. The teaching is mainly delivered in lecture-based or phantom head room settings. The majority of schools provided direct clinical experience in procedures relating to peri-implant maintenance, although in some schools it was stated that not every student was guaranteed to receive such experience. In 86% of schools, students gained experience in oral hygiene and scaling, while 71% and 64% provided experience of non-surgical management of patients with peri-implant mucositis and peri-implantitis, respectively. The main barrier to developing the implant programme was an insufficient number of suitable cases.Conclusions Although all responding schools provide implant training, the overall findings show that further development and improvement of implant teaching in dental hygiene and therapy schools within the UK and Ireland is required, particularly with respect to direct clinical experience. This will ensure that newly qualified dental hygienists and therapists are sufficiently prepared for managing implant patients in their clinical practice.

The Efficacy of Plasma Rich in Growth Factors for the Treatment of Alveolar Osteitis: A Randomized Controlled Trial.

PURPOSE: To investigate the efficacy of plasma rich in growth factors (PRGF; BTI Biotechnology Institute, San Antonio, Spain) for the treatment of alveolar osteitis compared with a positive control (Alvogyl; Septodont, Maidstone, Kent, UK). MATERIALS AND METHODS: This single-center, single-blinded, randomized, 2-treatment, parallel study was conducted in a UK dental hospital. All healthy adults who presented with alveolar osteitis after tooth extraction over a 3-month period were invited to participate. Each socket was randomized and treated with 1 of 2 treatment modalities, a test treatment (PRGF) or a positive control (Alvogyl). After treatment, patients were reviewed at 3 and 7 days by a second clinician blinded to the treatment given. Outcome measures included pain, exposed bone, inflammation, halitosis, dysgeusia, and quality-of-life assessment. RESULTS: Thirty-eight patients with data from 44 sockets (22 in the PRGF group and 22 in the Alvogyl group) were analyzed. The PRGF group showed significantly faster bone coverage and significantly decreased inflammation and halitosis (P < .05) compared with the control group receiving Alvogyl. There was no significant difference for pain, quality-of-life measures, or dysgeusia between groups. CONCLUSION: PRGF predictably treated alveolar osteitis after tooth extraction compared with the conventional standard treatment of Alvogyl, which has been used for many years. PRGF could be considered an alternative treatment for alveolar osteitis and indeed appears to have considerable advantages over Alvogyl.

Inhibition of the Polyamine Synthesis Pathway Is Synthetically Lethal with Loss of Argininosuccinate Synthase 1.

Argininosuccinate synthase 1 (ASS1) is the rate-limiting enzyme for arginine biosynthesis. ASS1 expression is lost in a range of tumor types, including 50% of malignant pleural mesotheliomas. Starving ASS1-deficient cells of arginine with arginine blockers such as ADI-PEG20 can induce selective lethality and has shown great promise in the clinical setting. We have generated a model of ADI-PEG20 resistance in mesothelioma cells. This resistance is mediated through re-expression of ASS1 via demethylation of the ASS1 promoter. Through coordinated transcriptomic and metabolomic profiling, we have shown that ASS1-deficient cells have decreased levels of acetylated polyamine metabolites, together with a compensatory increase in the expression of polyamine biosynthetic enzymes. Upon arginine deprivation, polyamine metabolites are decreased in the ASS1-deficient cells and in plasma isolated from ASS1-deficient mesothelioma patients. We identify a synthetic lethal dependence between ASS1 deficiency and polyamine metabolism, which could potentially be exploited for the treatment of ASS1-negative cancers.

Oral mucosal progenitor cell clones resist in vitro myogenic differentiation.

Progenitor cells derived from the oral mucosa lamina propria (OMLP-PCs) demonstrate an ability to differentiate into tissue lineages removed from their anatomical origin. This clonally derived population of neural-crest cells have demonstrated potential to differentiate along mesenchymal and neuronal cell lineages. OBJECTIVE: Significant efforts are being made to generate functioning muscle constructs for use in research and clinical tissue engineering. In this study we aimed to determine the myogenic properties of clonal populations of expanded OMLP-PCs. DESIGN: PCs were subject to several in vitro culture conditions in an attempt to drive myogenic conversion. Methodologies include use of demethylation gene-modifying reagents, mechanical conditioning of tissue culture substrates, tuneable polyacrylamide gels and a 3-dimensional construct as well as published myogenic media compositions. PCR and immunostaining for the muscle cell markers Desmin and MyoD1 were used to assess muscle differentiation. RESULTS: The clones tested did not intrinsically express myogenic lineage markers. Despite use of two and 3-dimensional pre-published in vitro culture protocols OMLP clones could not be differentiated down a myogenic lineage. CONCLUSIONS: Within the confines of these experimental parameters it was not possible to generate identifiable muscle using the clonal populations. When reviewing the previously successful reports of myogenic conversion, cells utilised have either been derived from tissues that are already 'primed' with the requisite myogenic genetic potential or have undergone specific genetic reprogramming to enhance the myogenic conversion rate. This, along with as yet unidentified stromal interplay, may therefore be required for positive myogenic differentiation to be realised.

Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?

We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze-thawed and freshly harvested cells. We found that freeze-thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze-thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti-inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze-thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement-mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation.

Lys11- and Lys48-linked ubiquitin chains interact with p97 during endoplasmic-reticulum-associated degradation.

The ATPase associated with various cellular activities p97 has a critical function in the cytoplasmic degradation of proteins misfolded in the ER (endoplasmic reticulum) through a mechanism known as ERAD (ER-associated degradation). During this process, p97 binds polyubiquitinated ERAD substrates and couples ATP hydrolysis to their dislocation from the ER as a prerequisite to destruction by the proteasome. The ubiquitin signals important for this process are not fully understood. In the present paper we report that p97 interacts with Lys11- and Lys48-linked ubiquitin polymers, but not those containing Lys63 linkages. Disruption of p97 through siRNA-mediated depletion, dominant-negative overexpression or chemical inhibition results in the accumulation of Lys11 and Lys48 ubiquitin chains predominantly at the ER membrane, and is associated with ER stress induction. We show that a catalytically inactive deubiquitinating enzyme and p97 cofactor YOD1 enhances the accumulation of Lys11- and Lys48-linked polyubiquitin in the cytoplasm, at the ER membrane and bound to p97. In addition to general effects on p97-associated ubiquitin polymers, the ERAD substrate CD3δ is modified with both Lys11 and Lys48 ubiquitin chains prior to p97-dependent dislocation. Collectively, the results of the present study are consistent with a major role for p97 in the recognition of Lys11 and Lys48 polyubiquitinated proteins before their degradation by the proteasome.