Prospects and Potential for Chimerism Analysis after Allogeneic Hematopoietic Stem Cell Transplantation.

Chimerism analysis after allogeneic hematopoietic stem cell transplantation serves to confirm engraftment, indicate relapse of hematologic malignancy, and attribute graft failure to either immune rejection or poor graft function. Short tandem repeat PCR (STR-PCR) is the prevailing method, followed by quantitative real-time PCR (qPCR), with detection limits of 1-5% and 0.1%, respectively. Chimerism assays using digital PCR or next-generation sequencing, both of which are more sensitive than STR-PCR, are increasingly used. Stable mixed chimerism is usually not associated with poor outcomes in non-malignant diseases, but recipient chimerism may foretell relapse of hematologic malignancies, so higher detection sensitivity may be beneficial in such cases. Thus, the need for and the type of intervention, e.g., immunosuppression regimen, donor lymphocyte infusion, and/or salvage second transplantation, should be guided by donor chimerism in the context of the feature and/or residual malignant cells of the disease to be treated.

Cellular carcinogenesis in preleukemic conditions:drivers and defenses.

Acute myeloid leukemia (AML) arises from preleukemic conditions. We have investigated the pathogenesis of typical preleukemia, myeloproliferative neoplasms, and clonal hematopoiesis. Hematopoietic stem cells in both preleukemic conditions harbor recurrent driver mutations; additional mutation provokes further malignant transformation, leading to AML onset. Although genetic alterations are defined as the main cause of malignant transformation, non-genetic factors are also involved in disease progression. In this review, we focus on a non-histone chromatin protein, high mobility group AT-hook2 (HMGA2), and a physiological p53 inhibitor, murine double minute X (MDMX). HMGA2 is mainly overexpressed by dysregulation of microRNAs or mutations in polycomb components, and provokes expansion of preleukemic clones through stem cell signature disruption. MDMX is overexpressed by altered splicing balance in myeloid malignancies. MDMX induces leukemic transformation from preleukemia via suppression of p53 and p53-independent activation of WNT/ß-catenin signaling. We also discuss how these non-genetic factors can be targeted for leukemia prevention therapy.

Prenatal vitamin D deficiency alters immune cell proportions of young adult offspring through alteration of long-term stem cell fates.

Vitamin D deficiency is a common deficiency worldwide, particularly among women of reproductive age. During pregnancy, it increases the risk of immune-related diseases in offspring later in life. However, exactly how the body remembers exposure to an adverse environment during development is poorly understood. Herein, we explore the effects of prenatal vitamin D deficiency on immune cell proportions in offspring using vitamin D deficient mice established by dietary manipulation. We show that prenatal vitamin D deficiency alters immune cell proportions in offspring by changing the transcriptional properties of genes downstream of vitamin D receptor signaling in hematopoietic stem and progenitor cells of both the fetus and adults. Further investigations of the associations between maternal vitamin D levels and cord blood immune cell profiles from 75 healthy pregnant women and their term babies also confirm that maternal vitamin D levels significantly affect immune cell proportions in the babies. Thus, lack of prenatal vitamin D, particularly at the time of hematopoietic stem cell migration from the liver to the bone marrow, has long-lasting effects on immune cell proportions. This highlights the importance of providing vitamin D supplementation at specific stages of pregnancy.

Impaired Repopulating Ability of Uhrf2-/- Hematopoietic Progenitor Cells in Mice.

UHRF proteins catalyze the ubiquitination of target proteins and are involved in regulating gene expression. Some studies reported a reduced expression of UHRF2 in acute leukemia cells, but the role of UHRF2 in hematopoiesis remains unknown. Here, we generated Uhrf2-/- mice to clarify the role of UHRF2 deletion in hematopoiesis. Compared to Uhrf2+/+ mice, Uhrf2-/- mice showed no differences in complete blood counts, as well as bone marrow (BM) findings and spleen weights. Proportions of cells in progenitor fractions in BM were comparable between Uhrf2+/+ mice and Uhrf2-/- mice. However, in competitive repopulation assays with BM transplants (BMT), the proportions of Uhrf2-/- cells were decreased relative to Uhrf2+/+ cells in all lineages. After the second BMT, Uhrf2-/- neutrophils were few, while 20-30% of Uhrf2-/- T cells and B cells were still detected. RNA sequencing showed downregulation of some genes associated with stem-cell function in Uhrf2-/- hematopoietic stem/progenitor cells (HSPCs). Interestingly, trimethylated histone H3 lysine 9 was increased in Uhrf2-/- HSPCs in a cleavage under targets and tagmentation assay. While UHRF2 deletion did not cause hematologic malignancy or confer a growth advantage of HSPCs, our results suggest that UHRF2 may play a role in the regulation of hematopoiesis.

Extreme Medialized Repair for Challenging Large and Massive Rotator Cuff Tears Reveals Healing and Significant Functional Improvement.

PURPOSE: To evaluate range of motion, muscle strength, clinical outcomes, and radiographic results of the extreme medialized procedure on rotator cuff tears that were initially irreparable. METHODS: From arthroscopic rotator cuff repair cases performed at our institution (June 2017 and August 2020), we retrospectively reviewed cases in which the rotator cuff was (1) unable to be withdrawn to the greater tuberosity, (2) repaired using the extreme medialized procedure, and (3) followed up for a minimum of 2 years. Patients with a history of previous surgery were excluded. Preoperative and postoperative scores were used for clinical evaluation. Imaging evaluation used 2-year postoperative magnetic resonance (MR) images. RESULTS: Sixty-four patients met the criteria; mean age 68.2 ± 7.9 (range 51-82) years; mean follow-up period 26 ± 2 (24-37) months. Tear size: 45 ± 7.1 (30-70) mm in medial to lateral diameters, 40 ± 9.3 (30-60) mm in anteroposterior diameter; suture anchor number: 5.5 ± 1.2 (4-8). The visual analog scale score (50.7 to 11.8), the University of California, Los Angeles, score (12 to 31), constant score (45 to 31), and the American Shoulder and Elbow Surgeons score (53 to 31) at the final follow-up improved compared with preoperative values (all P < .0001). Preoperative and postoperative changes in range of motion also showed improvement in anterior elevation (107° to 151°, P < .0001), abduction (100° to 154°, P < .0001), external rotation (41° to 47°, P = .0238), and internal rotation (L1 to Th10, P < .0001). Muscle strength was also improved in abduction (from 1.9 kg to 5.0 kg, P < .0001) and external rotation (from 3.5 kg to 7.7 kg, P < .0001). MR imaging evaluation revealed 2 cases (3.1%) of retears that fell into type 4 Sugaya classification. CONCLUSIONS: Extremely medialized repair of large and massive tears not able to be repaired using conventional techniques led to improved clinical outcomes compared to preoperative conditions. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Murine double minute X plays a central role in leukemic transformation and may be a promising target for leukemia prevention strategies.

Acute myeloid leukemia (AML) is a fatal disease resulting from preleukemic hematopoietic conditions, including asymptomatic clonal hematopoiesis. The accumulation of genetic changes is one of the causes of leukemic transformation. However, nongenetic factors, including the overexpression of specific genes also contribute to preleukemic to leukemic transition. Among them, the p53 inhibitor murine double minute X (MDMX) plays crucial roles, especially in leukemia initiation. MDMX is broadly overexpressed in the vast majority of AML cases, including in hematopoietic stem/progenitor cell (HSPC) level. Recently, high expression of MDMX in HSPC has been shown to be associated with leukemic transformation in patients with myelodysplastic syndromes, and preclinical studies have demonstrated that MDMX overexpression accelerates the transformation of preleukemic murine models, including models of clonal hematopoiesis. MDMX inhibition, through activation of cell-intrinsic p53 activity, shows antileukemic effects. However, the molecular mechanisms of MDMX in provoking leukemic transformation are complicated. Both p53-dependent and -independent mechanisms are involved in the progression of the disease. This review discusses the canonical and noncanonical functions of MDMX and how these functions are involved in the maintenance, expansion, and progression to malignancy of preleukemic stem cells. Moreover, strategies on how leukemic transformation could be prevented by targeting MDMX in preleukemic stem cells are discussed.

Evaluation of a quantitative PCR-based method for chimerism analysis of Japanese donor/recipient pairs.

Chimerism analysis is a surrogate indicator of graft rejection or relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Although short tandem repeat PCR (STR-PCR) is the usual method, limited sensitivity and technical variability are matters of concern. Quantitative PCR-based methods to detect single nucleotide polymorphisms (SNP-qPCR) are more sensitive, but their informativity and quantitative accuracy are highly variable. For accurate and sensitive chimerism analysis, a set of KMR kits (GenDx, Utrecht, Netherlands), based on detection of insertions/deletions (indels) by qPCR, have been developed. Here, we investigated informativity and validated the accuracy of KMR kits in Japanese donor/recipient pairs and virtual samples of DNA mixtures representative of Japanese genetic diversity. We found that at least one recipient-specific marker among 39 KMR-kit markers was informative in all of 65 Japanese donor/recipient pairs. Moreover, the percentage of recipient chimerism estimated by KMRtrack correlated well with ratios of mixed DNA in virtual samples and with the percentage of chimerism in HSCT recipients estimated by STR-PCR/in-house SNP-qPCR. Moreover, KMRtrack showed better sensitivity with high specificity when compared to STR-PCR to detect recipient chimerism. Chimerism analysis with KMR kits can be a standardized, sensitive, and highly informative method to evaluate the graft status of HSCT recipients.

Comparison of cryoprotectants in hematopoietic cell infusion-related adverse events.

BACKGROUND: The standard cryoprotectant for human cellular products is dimethyl sulfoxide (DMSO), which is associated with hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantation including peripheral blood stem cell (PBSC) transplantation (PBSCT). DMSO is often used with hydroxyethyl starch (HES), which reduces DMSO concentration while maintaining the postthaw cell recovery. The cryoprotectant medium CP-1 (Kyokuto Pharmaceutical Industrial) is widely used in Japan. After mixture of a product with CP-1, DMSO and HES concentrations are 5% and 6%, respectively. However, the safety profile of CP-1 in association with HCI-AEs has not been investigated. STUDY DESIGN AND METHODS: To compare CP-1 with other cryoprotectants, we conducted a subgroup analysis of PBSCT recipients in a prospective surveillance study for HCI-AEs. Moreover, we validated the toxicity of CP-1 in 90 rats following various dose administration. RESULTS: The PBSC products cryopreserved with CP-1 (CP-1 group) and those with other cryoprotectants, mainly 10% DMSO (non-CP-1 group), were infused into 418 and 58 recipients, respectively. The rate of ≥grade 2 HCI-AEs was higher in the CP-1 group, but that of overall or ≥grade 3 HCI-AEs was not significantly different, compared to the non-CP-1 group. Similarly, after propensity score matching, ≥grade 2 HCI-AEs were more frequent in the CP-1 group, but the ≥grade 3 HCI-AE rate did not differ significantly between the groups. No significant toxicity was detected regardless of the CP-1 dose in the 90 rats. CONCLUSIONS: Infusion of a CP-1-containing PBSC product is feasible with the respect of HCI-AEs.

Clonal hematopoiesis with JAK2V617F promotes pulmonary hypertension with ALK1 upregulation in lung neutrophils.

Pulmonary hypertension (PH) is a progressive cardiopulmonary disease characterized by pulmonary arterial remodeling. Clonal somatic mutations including JAK2V617F, the most frequent driver mutation among myeloproliferative neoplasms, have recently been identified in healthy individuals without hematological disorders. Here, we reveal that clonal hematopoiesis with JAK2V617F exacerbates PH and pulmonary arterial remodeling in mice. JAK2V617F-expressing neutrophils specifically accumulate in pulmonary arterial regions, accompanied by increases in neutrophil-derived elastase activity and chemokines in chronic hypoxia-exposed JAK2V617F transgenic (JAK2V617F) mice, as well as recipient mice transplanted with JAK2V617F bone marrow cells. JAK2V617F progressively upregulates Acvrl1 (encoding ALK1) during the differentiation from bone marrow stem/progenitor cells peripherally into mature neutrophils of pulmonary arterial regions. JAK2V617F-mediated STAT3 phosphorylation upregulates ALK1-Smad1/5/8 signaling. ALK1/2 inhibition completely prevents the development of PH in JAK2V617F mice. Finally, our prospective clinical study identified JAK2V617F-positive clonal hematopoiesis is more common in PH patients than in healthy subjects. These findings indicate that clonal hematopoiesis with JAK2V617F causally leads to PH development associated with ALK1 upregulation.

Myeloproliferative neoplasm-driving Calr frameshift promotes the development of pulmonary hypertension in mice.

Frameshifts in the Calreticulin (CALR) exon 9 provide a recurrent driver mutation of essential thrombocythemia (ET) and primary myelofibrosis among myeloproliferative neoplasms (MPNs). Here, we generated knock-in mice with murine Calr exon 9 mimicking the human CALR mutations, using the CRISPR-Cas9 method. Knock-in mice with del10 [Calrdel10/WT (wild-type) mice] exhibited an ET phenotype with increases of peripheral blood (PB) platelets and leukocytes, and accumulation of megakaryocytes in bone marrow (BM), while those with ins2 (Calrins2/WT mice) showed a slight splenic enlargement. Phosphorylated STAT3 (pSTAT3) was upregulated in BM cells of both knock-in mice. In BM transplantation (BMT) recipients from Calrdel10/WT mice, although PB cell counts were not different from those in BMT recipients from CalrWT/WT mice, Calrdel10/WT BM-derived macrophages exhibited elevations of pSTAT3 and Endothelin-1 levels. Strikingly, BMT recipients from Calrdel10/WT mice developed more severe pulmonary hypertension (PH)-which often arises as a comorbidity in patients with MPNs-than BMT recipients from CalrWT/WT mice, with pulmonary arterial remodeling accompanied by an accumulation of donor-derived macrophages in response to chronic hypoxia. In conclusion, our murine model with the frameshifted murine Calr presented an ET phenotype analogous to human MPNs in molecular mechanisms and cardiovascular complications such as PH.

MDMX acts as a pervasive preleukemic-to-acute myeloid leukemia transition mechanism.

MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/ß-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of ß-Catenin in a p53-independent manner. Wnt/ß-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/ß-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/ß-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

Stump classification was correlated with retear in the suture-bridge and double-row repair techniques for arthroscopic rotator cuff repair.

PURPOSE: The Stump classification is significantly correlated with a retear after arthroscopic rotator cuff repair. However, no study has evaluated whether or not the stump classification is correlated with retear in the suture-bridge or double-row repair techniques. The aim of this study was to evaluate the relationship between a retear and the stump classification in the suture-bridge and double-row repair techniques. METHODS: Among 389 patients who underwent arthroscopic repairs of full-thickness rotator cuff tears using suture-bridge or double-row repair techniques, 326 patients (median age 67.0 years; range 25-85) were included. There were 51 small, 172 medium, 83 large, and 20 massive tears. Two hundred forty patients were treated with the suture-bridge technique, and 86 patients were treated with the double-row technique. The following variables were analyzed: age, sex, the Cofield classification, anteroposterior and mediolateral tear size on preoperative MRI, global fatty degeneration index, and the stump classification. Cuff integrity was evaluated on magnetic resonance imaging at 6 months after surgery. The patients were divided into the intact and retear groups and the relationship between the variables and retear was evaluated by multivariate logistic regression analysis. RESULTS: The overall retear rate was 10.1%. In the multivariate logistic regression analysis, the independent predictors of a retear were the stump classification type 3 (Odds ratio: 4.71, p = 0.0246), global fatty degeneration index (Odds ratio: 3.87, p = 0.0030), and anteroposterior tear size (Odds ratio: 1.07, p = 0.0077) in the suture bridge technique. In the double-row technique, the independent predictors of retear were stump classification type 3 (Odds ratio: 7.82, p = 0.0348), and age (Odds ratio: 1.22, p = 0.0163). CONCLUSION: The stump classification was significantly correlated with retear in the suture-bridge and double-row repair technique. Stump classification type 3 was indicated to be an important risk factor for predicting retear. LEVEL OF EVIDENCE: III.

[Examination of Application to Radiation Protection Education by Four-dimensional Visualization of Scatter Distribution in Radiological Examination Using Virtual Reality].

PURPOSE: When working on fluoroscopy and patient assistance in a healthcare facility, workers need to understand how to properly protect scattered radiation. In this study, we examined a four-dimensional visualization method to make it easy to understand the spread of scattered radiation visually, and proposed its application to radiation protection education. METHODS: We constructed the X-ray room, X-ray CT room, and angiography room using Particle Heavy Ion Transport code System (PHITS), and calculated the scattered radiation distribution when the patient was irradiated with X-rays. The three-dimensional distribution of each moment was continuously displayed to create a four-dimensional distribution. Using the created data, we conducted radiation protection education including exercises to make the students confirm the scatter distribution from any direction. The effectiveness of the scattered radiation visualization data was evaluated by a questionnaire. RESULTS: The position of assistance for standing chest radiograph was less scattered radiation at the side and below the patient. As a result of the questionnaire, this education has confirmed the effect of attracting attention about radiation protection. The fourdimensional visualization allowed students to understand the behavior of radiation and the source of scattered radiation. CONCLUSION: Visualization of three- and four-dimensional scattered radiation distribution in the radiological examination room can intuitively enhance the understanding of the invisible radiation spread and appropriate aids.

Neuroprotective effects of different frequency preconditioning exercise on neuronal apoptosis after focal brain ischemia in rats.

OBJECTIVE: Preconditioning exercise can exert neuroprotective effects after stroke; however, the effects of exercise intensity, frequency, duration are unknown. We investigated the neuroprotective effect of different frequency preconditioning exercise on neuronal apoptosis after cerebral ischemia in rats. METHODS: Rats were divided into the following five groups: 5 times a week of exercise (5/w-Ex) group, 3 times a week of exercise (3/w-Ex) group, once a week of exercise (1/w-Ex) group, no exercise (No-Ex) group, and intact control (control) group. Rats were made to run on a treadmill for 30 min per day at a speed of 25 m/min for 3 weeks. After the running program, the rats were subjected to 60-min left middle cerebral artery occlusion. Two days after ischemia, the cerebral infarct volume, neurological and motor function, Bcl-2-associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2) ratio, expression of caspase-3, and TUNEL positive cells were examined in the cerebral cortex surrounding the ischemic zone. RESULTS: The 3/w-Ex and 5/w-Ex groups showed significantly reduced infarct volumes compared with the No-Ex group, but the 1/w-Ex group did not. In addition, the 3/w-Ex and 5/w-Ex groups had improved neurological scores and sensorimotor function compared with the No-Ex group. The Bax/Bcl-2 ratio, expression of caspase-3, and TUNEL-positive cells significantly decreased in the penumbra area in the 3/w-Ex or 5/w-Ex groups compared with the No-Ex group. DISCUSSION: Our findings suggested that three times or more per week of high-intensity preconditioning exercise exert neuroprotective effects through the downregulation of the Bax/Bcl-2 ratio and caspase-3 activation after stroke. ABBREVIATIONS: TUNEL: terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick and labeling; MCAO:middle cerebral artery occlusion; BAX:Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; TTC: 2,3,5-triphenyltetrazorlium chloride.

Lenalidomide as a Beneficial Treatment Option for Renal Impairment Caused by Light Chain Deposition Disease.

Light chain deposition disease (LCDD) is a rare systemic disorder caused by the deposition of light chain immunoglobulins, which often results in renal impairment associated with either nephrotic syndrome or asymptomatic proteinuria. B-cell neoplasms, such as multiple myeloma and lymphoproliferative disorders, are well-known underlying diseases in LCDD. Some chemotherapy regimens have been reported, but both evidence-based treatment and management for LCDD have yet to be established. We herein report three cases of LCDD treated with lenalidomide-based therapy, resulting in hematologic responses accompanied by a significant reduction in proteinuria and improvement in the renal function. We recommend lenalidomide-based therapy for renal impairment caused by LCDD.

Application of a Novel Anti-Adhesive Membrane, E8002, in a Rat Laminectomy Model.

Neuropathic pain after spinal surgery, so-called failed back surgery syndrome, is a frequently observed common complication. One cause of the pain is scar tissue formation, observed as post-surgical epidural adhesions. These adhesions may compress surrounding spinal nerves, resulting in pain, even after successful spinal surgery. E8002 is an anti-adhesive membrane. In Japan, a clinical trial of E8002 is currently ongoing in patients undergoing abdominal surgery. However, animal experiments have not been performed for E8002 in spinal surgery. We assessed the anti-adhesive effect of E8002 in a rat laminectomy model. The dura matter was covered with an E8002 membrane or left uncovered as a control. Neurological evaluations and histopathological findings were compared at six weeks postoperatively. Histopathological analyses were performed by hematoxylin⁻eosin and aldehyde fuchsin-Masson Goldner staining. Three assessment areas were selected at the middle and margins of the laminectomy sites, and the numbers of fibroblasts and inflammatory cells were counted. Blinded histopathological evaluation revealed that adhesions and scar formation were reduced in the E8002 group compared with the control group. The E8002 group had significantly lower numbers of fibroblasts and inflammatory cells than the control group. The present results indicate that E8002 can prevent epidural scar adhesions after laminectomy.

The Amelioration of Myelofibrosis with Thrombocytopenia by a JAK1/2 Inhibitor, Ruxolitinib, in a Post-polycythemia Vera Myelofibrosis Patient with a JAK2 Exon 12 Mutation.

Less than 5% of patients with polycythemia vera (PV) show JAK2 exon 12 mutations. Although PV patients with JAK2 exon 12 mutations are known to develop post-PV myelofibrosis (MF) as well as PV with JAK2V617F, the role of JAK inhibitors in post-PV MF patients with JAK2 exon 12 mutations remains unknown. We describe how treatment with a JAK1/2 inhibitor, ruxolitinib, led to the rapid amelioration of marrow fibrosis, erythrocytosis and thrombocytopenia in a 77-year-old man with post-PV MF who carried a JAK2 exon 12 mutation (JAK2H538QK539L). This case suggests that ruxolitinib is a treatment option for post-PV MF in patients with thrombocytopenia or JAK2 exon 12 mutations.