Summary Background A population-based cohort of children aged 1–18 years with acute lymphoblastic leukaemia (ALL) was treated with a dexamethasone-based protocol (Dutch Childhood Oncology Group DCOG ...ALL-9). We aimed to confirm the results of the most effective DCOG ALL protocol for non-high-risk (NHR) patients to date (ALL-6), compare results with ALL-7 and ALL-8, and study prognostic factors in a non-randomised setting. Methods From Jan 1, 1997, until Nov 1, 2004, patients with ALL were treated according to the ALL-9 protocol in eight Dutch academic centres with their affiliated peripheral hospitals. Patients were stratified into NHR and high risk (HR) groups. HR criteria were white-blood-cell count of 50 000 cells per μL or more, T-cell phenotype, mediastinal mass, CNS or testicular involvement, and Philadelphia chromosome or MLL rearrangement; patients who did not fulfil these criteria were deemed to be NHR. The NHR group was treated with a three-drug induction (dexamethasone, vincristine, and asparaginase) for 6 weeks, medium-dose methotrexate for 3 weeks, then maintenance therapy. HR patients received a four-drug induction (as for the NHR patients plus daunorubicin) for 6 weeks, high-dose methotrexate for 8 weeks, and two intensification courses before receiving maintenance therapy. Triple intrathecal medication was given 13 times in NHR patients, 15 times in HR patients (17 times for patients with initial CNS involvement). No patient received cranial irradiation. Maintenance therapy was given until 109 weeks for all patients and consisted of mercaptopurine and methotrexate for 5 weeks, alternated with dexamethasone and vincristine for 2 weeks. Kaplan–Meier analysis was done on an intention-to-treat basis with event-free survival as the primary endpoint. This trial is registered at trialregister.nl , number NTR460/SNWLK-ALL-9. Findings 859 patients were recruited to the study. Complete remission was achieved in 592 (98·5%) of the 601 patients in the NHR group and 250 (96·9%) of the 258 in the HR group. Five patients in the NHR group and four in the HR group died during induction. Median follow-up for patients alive was 72·2 (range 4·8–132·7) months as of August, 2008. 5-year event-free survival was 81% (SE 1%) in all patients: 84% (2%) in NHR patients, and 72% (3%) in HR patients. Isolated CNS relapses occurred in 22 (2·6%) of 842 patients. In a multivariate analysis, DNA index was the strongest predictor of outcome (<1·16 vs ≥1·16; relative risk 0·42, 95% CI 0·22–0·78), followed by age (1–9 vs ≥10 years; 2·23, 1·60–3·11) and white-blood-cell count (<50 000 vs ≥50 000 cells per μL; 1·60, 1·13–2·26). Interpretation The overall results of the dexamethasone-based DCOG ALL-9 protocol are better than those of our previous Berlin–Frankfurt–Münster-based protocols ALL-7 and ALL-8. The results for NHR patients were achieved with high cumulative doses of dexamethasone and vincristine, but without the use of anthracyclines, etoposide, cyclophosphamide, or cranial irradiation, therefore minimising the risk of side-effects. Funding Dutch Health Insurers.
The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being ...investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re‐endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re‐endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell –derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re‐endothelialized scaffold could, in contrast to the non‐re‐endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney.
Human decellularized kidney scaffolds can be functionally re‐endothelialized using expanded human pluripotent stem cell–derived endothelial cells.
Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss ...of chromosome 3. Inactivating mutations in BAP1 encoding the BRCA1-associated protein 1 and located on chromosome 3p21.1, have been described in uveal melanoma and other types of cancer. In this study, we determined the prevalence of somatic BAP1 mutations and examined whether these mutations correlate with the functional expression of BAP1 in uveal melanoma tissue and with other clinical, histopathological and chromosomal parameters. We screened a cohort of 74 uveal melanomas for BAP1 mutations, using different deep sequencing methods. The frequency of BAP1 mutations in our study group was 47%. The expression of BAP1 protein was studied using immunohistochemistry. BAP1 staining was absent in 43% of the cases. BAP1 mutation status was strongly associated with BAP1 protein expression (P<0.001), loss of chromosome 3 (P<0.001), and other aggressive prognostic factors. Patients with a BAP1 mutation and absent BAP1 expression had an almost eightfold higher chance of developing metastases compared with those without these changes (P=0.002). We found a strong correlation between the immunohistochemical and sequencing data and therefore propose that, immunohistochemical screening for BAP1 should become routine in the histopathological work-up of uveal melanoma. Furthermore, our analysis indicates that loss of BAP1 may be particularly involved in the progression of uveal melanoma to an aggressive, metastatic phenotype.
Ex situ dual hypothermic oxygenated machine perfusion (DHOPE) and normothermic machine perfusion (NMP) of donor livers may have a complementary effect when applied sequentially. While DHOPE ...resuscitates the mitochondria and increases hepatic adenosine triphosphate (ATP) content, NMP enables hepatobiliary viability assessment prior to transplantation. In contrast to DHOPE, NMP requires a perfusion solution with an oxygen carrier, for which red blood cells (RBC) have been used in most series. RBC, however, have limitations and cannot be used cold. We, therefore, established a protocol of sequential DHOPE, controlled oxygenated rewarming (COR), and NMP using a new hemoglobin‐based oxygen carrier (HBOC)‐based perfusion fluid (DHOPE‐COR‐NMP trial, NTR5972). Seven livers from donation after circulatory death (DCD) donors, which were initially declined for transplantation nationwide, underwent DHOPE‐COR‐NMP. Livers were considered transplantable if perfusate pH and lactate normalized, bile production was ≥10 mL and biliary pH > 7.45 within 150 minutes of NMP. Based on these criteria five livers were transplanted. The primary endpoint, 3‐month graft survival, was a 100%. In conclusion, sequential DHOPE‐COR‐NMP using an HBOC‐based perfusion fluid offers a novel method of liver machine perfusion for combined resuscitation and viability testing of suboptimal livers prior to transplantation.
This clinical cohort study indicates that a combination of hypo‐ and normothermic machine perfusion, using a preservation fluid containing an hemoglobin‐based oxygen carrier, is feasible and provides a tool to resuscitate and select initially declined high‐risk donor livers that can be transplanted successfully.
Summary Objective Interleukin-1 (IL-1) is an alleged important cytokine in osteoarthritis (OA), although the exact contribution of IL-1 to joint destruction remains unclear. Here we investigated the ...involvement of IL-1α and IL-1β in joint pathology during collagenase-induced OA (CiOA). Methods CiOA was induced in WT and IL-1αβ-/- mice. Additionally, IL-1 signaling was inhibited in WT mice with CiOA using osmotic pumps containing IL-1RA. Joint pathology was assessed using histology. Activity of cartilage-degrading enzymes was determined using antibodies against aggrecan neo-epitopes VDIPEN and NITEGE. Synovial gene expression was analyzed using qRT-PCR. Serum protein levels were measured with Luminex or ELISA. Results Synovial IL-1β expression was strongly elevated 7 days after induction of CiOA in WT mice but decreased afterwards, whereas S100A8/A9, previously described to aggravate OA, remained elevated for 21 days. Remarkably, synovial inflammation was comparable between WT and IL-1αβ-/- mice on day 7 of CiOA. In line, synovial mRNA expression of genes involved in IL-1 signaling and inflammatory mediators was comparable between WT and IL-1αβ-/- mice, and serum levels for KC/IL-6/S100A8/S100A9/IL-10 were equal. Synovial MMP/aggrecanase expression and activity in cartilage was not different in WT and IL-1αβ-/- mice on day 7 of CiOA. Cartilage destruction on day 42 was not different between WT and IL-1αβ-/- mice, which was supported by our finding that IL-1RA treatment in WT mice with CiOA did not alter joint destruction. Conclusions IL-1α and IL-1β are not involved in synovial inflammation and cartilage destruction during CiOA, implicating that other mediators are responsible for the joint damage.
Background
Short‐chain fatty acids (SCFAs) are fermented dietary components that regulate immune responses, promote colonic health, and suppress mast cell–mediated diseases. However, the effects of ...SCFAs on human mast cell function, including the underlying mechanisms, remain unclear. Here, we investigated the effects of the SCFAs (acetate, propionate, and butyrate) on mast cell–mediated pathology and human mast cell activation, including the molecular mechanisms involved.
Method
Precision‐cut lung slices (PCLS) of allergen‐exposed guinea pigs were used to assess the effects of butyrate on allergic airway contraction. Human and mouse mast cells were co‐cultured with SCFAs and assessed for degranulation after IgE‐ or non–IgE‐mediated stimulation. The underlying mechanisms involved were investigated using knockout mice, small molecule inhibitors/agonists, and genomics assays.
Results
Butyrate treatment inhibited allergen‐induced histamine release and airway contraction in guinea pig PCLS. Propionate and butyrate, but not acetate, inhibited IgE‐ and non–IgE‐mediated human or mouse mast cell degranulation in a concentration‐dependent manner. Notably, these effects were independent of the stimulation of SCFA receptors GPR41, GPR43, or PPAR, but instead were associated with inhibition of histone deacetylases. Transcriptome analyses revealed butyrate‐induced downregulation of the tyrosine kinases BTK, SYK, and LAT, critical transducers of FcεRI‐mediated signals that are essential for mast cell activation. Epigenome analyses indicated that butyrate redistributed global histone acetylation in human mast cells, including significantly decreased acetylation at the BTK, SYK, and LAT promoter regions.
Conclusion
Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation.
Short‐chain fatty acids suppress primary human mast cell activation via HDAC inhibition and not via PPAR or GPR signaling. Butyrate induces transcriptional changes of genes important for signaling and activation of human mast cells. Butyrate triggers a redistribution of global H3K27 acetylation levels, leading to decreased acetylation at the transcription start site of genes encoding key FcεRI‐mediated signaling molecules.
The clinical outcomes of noncompaction cardiomyopathy (NCCM) range from asymptomatic to heart failure, arrhythmias, and sudden cardiac death. Genetics play an important role in NCCM.
This study ...investigated the correlations among genetics, clinical features, and outcomes in adults and children diagnosed with NCCM.
A retrospective multicenter study from 4 cardiogenetic centers in the Netherlands classified 327 unrelated NCCM patients into 3 categories: 1) genetic, with a mutation in 32% (81 adults; 23 children) of patients; 2) probably genetic, familial cardiomyopathy without a mutation in 16% (45 adults; 8 children) of patients; or 3) sporadic, no family history, without mutation in 52% (149 adults; 21 children) of patients. Clinical features and major adverse cardiac events (MACE) during follow-up were compared across the children and adults.
MYH7, MYBPC3, and TTN mutations were the most common mutations (71%) found in genetic NCCM. The risk of having reduced left ventricular (LV) systolic dysfunction was higher for genetic patients compared with the probably genetic and sporadic cases (p = 0.024), with the highest risk in patients with multiple mutations and TTN mutations. Mutations were more frequent in children (p = 0.04) and were associated with MACE (p = 0.025). Adults were more likely to have sporadic NCCM. High risk for cardiac events in children and adults was related to LV systolic dysfunction in mutation carriers, but not in sporadic cases. Patients with MYH7 mutations had low risk for MACE (p = 0.03).
NCCM is a heterogeneous condition, and genetic stratification has a role in clinical care. Distinguishing genetic from nongenetic NCCM complements prediction of outcome and may lead to management and follow-up tailored to genetic status.
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Summary Objective To review the literature on the role and regulation of chondrocyte terminal differentiation (hypertrophy-like changes) in osteoarthritis (OA) and to integrate this in a conceptual ...model of primary OA development. Methods Papers investigating chondrocyte terminal differentiation in human OA cartilage and experimental models of OA were recapitulated and discussed. Focus has been on the occurrence of hypertrophy-like changes in chondrocytes and the factors described to play a role in regulation of chondrocyte hypertrophy-like changes in OA. Results Chondrocyte hypertrophy-like changes are reported in both human OA and experimental OA models by most investigators. These changes play a crucial part in the OA disease process by protease-mediated cartilage degradation. We propose that altered chondrocyte behavior and concomitant cartilage degradation result in a disease-amplifying loop, leading to a mixture of disease stages and cellular responses within an OA joint. Conclusion Chondrocyte hypertrophy-like changes play a role in early and late stage OA. Since not all cells in an OA joint are synchronized, inhibition of hypertrophy-like changes might be a therapeutic target to slow down further OA progression.
The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, ...the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm
2
(± 0.9 Ω.cm
2
) and 28.2 Ω.cm
2
(± 1.3 Ω.cm
2
) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm
2
. Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm
2
. This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.
Alarmins S100A8 and S100A9 are major products of activated macrophages regulating cartilage damage and synovial activation during murine and human osteoarthritis (OA). In the current study, we ...investigated whether S100A8 and S100A9 are involved in osteophyte formation during experimental OA and whether S100A8/A9 predicts osteophyte progression in early human OA.
OA was elicited in S100A9-/- mice in two experimental models that differ in degree of synovial activation. Osteophyte size, S100A8, S100A9 and VDIPEN neoepitope was measured histologically. Chondrogenesis was induced in murine mesenchymal stem cells in the presence of S100A8. Levels of S100A8/A9 were determined in plasma of early symptomatic OA participants of the Cohort Hip and Cohort Knee (CHECK) cohort study and osteophytes measured after 2 and 5 years.
Osteophyte size was drastically reduced in S100A9-/- mice in ligaments and at medial femur and tibia on days 21 and 42 of collagenase-induced OA, in which synovial activation is high. In contrast, osteophyte size was not reduced in S100A9-/- mice during destabilised medial meniscus OA, in which synovial activation is scant. S100A8 increased expression and activation of matrix metalloproteinases during micromass chondrogenesis, thereby possibly increasing cartilage matrix remodelling allowing for larger osteophytes. Interestingly, early symptomatic OA participants of the CHECK study with osteophyte progression after 2 and 5 years had elevated S100A8/A9 plasma levels at baseline, while C-reactive protein, erythrocyte sedimentation rate and cartilage oligomeric matrix protein were not elevated at baseline.
S100A8/A9 aggravate osteophyte formation in experimental OA with high synovial activation and may be used to predict osteophyte progression in early symptomatic human OA.
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