Busulfan Treatment Research Articles

Automation in Busulfan Therapeutic Drug Monitoring: Evaluation of an Immunoassay on two Routine Chemistry Analyzers.

Therapeutic drug monitoring (TDM) of busulfan is recommended for hematopoietic stem cell transplant recipients. Timely reporting of these TDM results is essential given the short administration period and the planned dose adjustments on day 2. The authors evaluated the performance of a new nanoparticle-based competitive immunoassay on two routine clinical chemistry analyzers and compared its performance to that of an in-house high-resolution mass spectrometry (HRMS) method. The MyCare Oncology Busulfan Assay Kit (Saladax Biomedical) was applied on two routine clinical chemistry analyzers (Abbott Architect c8000 and Roche Cobas c502) with a linearity range of 187-2000 ng/mL. The study evaluation measured imprecision and accuracy, sample probe carry-over, and dilution integrity. Method comparison with liquid chromatography (LC)-HRMS was performed using samples from patients undergoing busulfan treatment. Within- and between-run coefficient of variations for both analyzers were ≤5.23% and ≤8.45%, respectively, across the busulfan concentration range. The obtained biases were ≤10.3%. Both analyzers met the acceptance criteria for sample probe carry-over and dilution integrity. Agreement between the immunoassay and LC-HRMS was high: 92% and 89% of the samples measured on Architect and Cobas, respectively, were within the ±15% limit compared to the corresponding LC-HRMS results. Overall, good analytical performance and high agreement with LC-HRMS results were obtained for the immunoassay installed on both routine clinical chemistry analyzers. Therefore, this assay could be implemented as a valid alternative for LC methods in clinical laboratories on different open-channel clinical chemistry analyzers, resulting in shorter turn-around times for reporting busulfan TDM results with subsequent faster dosage adjustments.

Successful in vivo Transplantation of Cultured and Enriched Testicular Germ Cells of Pre-Pubertal Bucks to Busulfan-Treated Homologous Recipients

The objective of the present study was to establish a workable approach for the production of germ cell (GC)-depleted recipient goat model using intra-testicular busulfan treatment and transplantation of cultured and enriched caprine-male GC (cmGCs) into the homologous recipients under ultrasonography (USG) guidance. The evaluation of post-transplantation colonization of donor cmGCs and restoration of the normal architecture of seminiferous tubules (ST) was performed. For this, the cmGCs of pre-pubertal male goats were isolated and enriched by differential platting for culture until the third passage. Thereafter, cells were harvested and further enriched by magnetic-activated cell sorting using rabbit-anti-CD90 antibody. After confirmation of metabolic viability (MTT-assay) and cluster-forming ability (crystal violet staining) of CD90⁺ cmGCs, the cells were labeled with a lipophilic red-fluorescent dye (PKH26) before transplanted into the recipient male goats by injection directly into the mediastinum testes under USG guidance. The colonization and repopulation of transplanted CD90⁺ cmGCs into the recipient ST was observed up to 8 weeks post-transplantation. The PKH26-labeled donor cell-derived colonies were identified in enzymatically digested ST and cryosections of recipient testes. Moreover, histochemical analyses revealed the restoration of the normal architecture of ST of recipient testis after GC transplantation. Therefore, the results suggest that the reproductive competence of infertile animals can be restored through mGC therapy and thus the methodology presented herein could be useful to obtain donor mGCs-derived functional male gametes in the recipient animal testis.

Effect of GSTA1 Variants on Busulfan-Based Conditioning Regimen Prior to Allogenic Hematopoietic Stem-Cell Transplantation in Pediatric Asians.

Busulfan is widely used as a chemotherapy treatment before hematopoietic stem-cell transplantation (HSCT). However, the response of busulfan is highly variable and unpredictable, whereby the pharmacogenetic interference of glutathione S-transferase (GST) has strong evidence in Caucasians and some adult Asians but not in pediatric Asian patients. This study was aimed at investigating the associations of GST genetic polymorphisms with variations in the pharmacokinetic (PK) properties of busulfan in pediatric Asian patients. This retrospective cohort study recruited 92 pediatric patients. The polymorphism of GSTA1 was genotyped by Sanger sequencing, and GSTM1 and GSTP1 were genotyped by real-time PCR. Drug concentration and PK estimation were identified using an LC-MS/MS method and a noncompartmental model. Statistical analysis was performed by R software. Out of 92 patients, 48 (53%) were males, the mean age was 8.4 ± 5.12 years old, and the average weight was 26.52 ± 14.75 kg. The allele frequencies of GSTA1*B and of GSTM1 and GSTP1* deletions were 16.9%, 68.5%, and 21.2%, respectively. Patients with GSTA1*B had a statistically significant impact on the PK of busulfan, whereas those with GSTM1 and GSTP1 did not (p > 0.05). The carriers of GSTA1*B showed a significant difference compared to noncarriers in terms of t1/2 (for first dose: 161.9 vs. 134.3 min, p = 0.0016; for second dose: 156.1 vs. 129.8, p = 0.012), CL (88.74 vs. 124.23 mL/min, p = 0.0089), Cmax (4232.6 vs. 3675.5 ng/mL, p = 0.0021), and AUC (5310.6 vs. 4177.1 µM/min, p = 0.00033). The augmentation of AUC was around 27.1% in patients carrying the GSTA1*B variant. The GSTA1 polymorphism was significantly associated with variations of the pharmacokinetic properties of busulfan treatment in pediatric Asian patients.

Busulfan impairs blood–testis barrier and spermatogenesis by increasing noncollagenous 1 domain peptide via matrix metalloproteinase 9

Sterility induced by anti-cancer treatments has caused significant concern, yet the mechanism and treatment exploration are little for male infertility after cancer therapy. Busulfan, the antineoplastic that was widely applied before bone marrow transplantation, was known to induce male reproductive disorder. To investigate the effect of busulfan on blood-testis barrier function in adult rats and determine whether noncollagenous 1 domain peptide, the biologically active fragment proteolyzed from the collagen α3 chain (IV) by matrix metalloproteinase 9, was involved during this process. Adult male rats were treated with one-dose or double-dose of busulfan (10mg/kg) before euthanized at day 35. Blood-testis barrier integrity assay, HE staining, immunofluorescence, and Western blot were used to validate the effect of busulfan on blood-testis barrier permeability and spermatogenesis. JNJ0966 was applied to specifically inhibit the matrix metalloproteinase 9 activity. The polymerization activity of F-actin/G-actin and microtubule/tubulin in the testis were assessed by using commercial kits. A noteworthy blood-testis barrier injury and significant up-regulation of matrix metalloproteinase 9 activity and noncollagenous 1 level after a single-dose busulfan (10mg/kg) treatment in adult rat testis were revealed. The application of JNJ0966 was found to decrease noncollagenous 1 level and rescue the busulfan-induced blood-testis barrier injury including the mis-localization of junction proteins across the seminiferous epithelium, by recovering the organization and polymerization of both F-actin and microtubule. The busulfan-induced spermatogenesis impairment was also improved by JNJ0966. These findings thus demonstrate that the elevation in matrix metalloproteinase 9 and noncollagenous 1 might participate in busulfan-induced blood-testis barrier disruption in adult male rats. As such, busulfan-induced male infertility could possibly be managed through interventions on noncollagenous 1 production.

Microbiota-derived lactate promotes hematopoiesis and erythropoiesis by inducing stem cell factor production from leptin receptor+ niche cells

Although functional interplay between intestinal microbiota and distant sites beyond the gut has been identified, the influence of microbiota-derived metabolites on hematopoietic stem cells (HSCs) remains unclear. This study investigated the role of microbiota-derived lactate in hematopoiesis using mice deficient in G-protein-coupled receptor (Gpr) 81 (Gpr81−/−), an established lactate receptor. We detected significant depletion of total HSCs in the bone marrow (BM) of Gpr81−/− mice compared with heterogenic (Gpr81+/−) mice in a steady state. Notably, the expression levels of stem cell factor (SCF), which is required for the proliferation of HSCs, decreased significantly in leptin receptor-expressing (LepR+) mesenchymal stromal cells (MSCs) around the sinusoidal vessels of the BM from Gpr81−/− mice compared with Gpr81+/− mice. Hematopoietic recovery and activation of BM niche cells after irradiation or busulfan treatment also required Gpr81 signals. Oral administration of lactic acid-producing bacteria (LAB) activated SCF secretion from LepR+ BM MSCs and subsequently accelerated hematopoiesis and erythropoiesis. Most importantly, LAB feeding accelerated the self-renewal of HSCs in germ-free mice. These results suggest that microbiota-derived lactate stimulates SCF secretion by LepR+ BM MSCs and subsequently activates hematopoiesis and erythropoiesis in a Gpr81-dependent manner.

Reversal of Aplastic Anemia By Mesenchymal Stem Cell-Derived Extracellular Vesicles

Aplastic anemia is a disease of bone marrow failure. Aplastic anemia remains a challenging disease as far as etiology and therapeutic approaches. Extracellular vesicles (EVs) have been studied for many years. These were initially felt to be cell junk largely from platelets and erythrocytes. But, more recently, intense interest has been focused on their potential for altering cell fate and reversing tissue injury. Vesicles derived from mesenchymal stem cells (MSC) have been shown to affect the phenotype and induce healing of many different cell types. Our recent published work has shown that human or murine MSC-EV treatment dramatically reversed radiation damage to bone marrow both in vitro and in vivo by inhibition of DNA damage and apoptosis. In this study, we evaluated whether human MSC-EVs could reverse marrow failure in a Busulfan induced aplastic anemia mouse model. We have carried out studies on Busulfan (BU) induced marrow aplasia by administering Busulfan in doses of 9.25 mg/kg intraperitoneally (9 times over 20 days) to induce bone marrow aplasia in female Balb/cByJ mice. Mice were then infused with 4x108 human MSC-EVs at 1, 2 and 3 days after the final BU dose. We observed that the human MSC EV treatment significantly prolonged survival in these mice. 50% of the BU treated mice died by 30 days after BU injection, whereas only 30% of EV-injected mice died by this time. The mice were sacrificed to harvest WBM cells and peripheral blood for cell count at 10 days after the final BU dose. There were significant decreases in marrow cellularity from tibiae and femurs and complete blood count (CBC) including RBC, WBC, HBG and PLT in BU treated mice compared to vehicle control mice. The vesicle treatment led to a statistically significant (p<0.05) increase in bone marrow cell count with the average of 3.30 x106 ±0.46 x106 cells /tibia and femur for the vesicle treated mice compared to 2.05 x106 ± 0.46 x 106 cells for the non-vesicle treated mice. The restoration growth of bone marrow stem cells was further confirmed by WBM proliferation assessment in cell culture. We further investigated the effect of MSC-EVs on the microRNA expression in bone marrow cells from Busulfan induced aplastic anemia mice by microRNA realtime PCR array. 151 microRNAs were altered by Busulfan treatment. 88 showed partial or complete reversal of these alterations after EV treatment. In summary, our data suggest that MSC-EV treatment could partially reverse Busulfan induced marrow failure at early time points. The reversal of the Busulfan induced chronic bone marrow failure by MSC-EVs needs further investigation. DisclosuresNo relevant conflicts of interest to declare.

Risk Assessment for BK Virus-Associated Hemorrhagic Cystitis After Pediatric Hematopoietic Stem Cell Transplant: A Single-Center Retrospective Cross-Sectional Study.

BK virus-associated hemorrhagic cystitis is a common complication of allogeneic hematopoietic stem cell transplant. It is known to be associated with cyclophosphamide therapy and the intensity of the conditioning regimen as well as infection with the BK virus. Data are limited for BK virus-associated hemorrhagic cystitis in pediatric recipients of allogeneic hematopoietic stem cell transplant. Therefore, we aimed to identify the risk factors and etiology of BK virus-associated hemorrhagic cystitis and determine the factors that may improve the treatment efficacy. Data from recipients of allogeneic hematopoietic stem cell transplant were retrospectively analyzed. These data included information about age, sex, underlying disease, the details of ablative conditioning, graft-versus-host disease prophylaxis, donor type, stem cell source, history of acute graft-versus-host disease, and cytomegalovirus reactivation. A total of 50 patients developed BK virusassociated hemorrhagic cystitis among 334 patients. Symptoms associated with BK virus-associated hemorrhagic cystitis manifested an average of 45.3 days after transplant. Most of the patients had grade 2 and grade 3 hemorrhagic cystitis. Risk factor analysis revealed that haploidentical donor type, treatment with busulfan and cyclophosphamide as part of conditioning regimen, and history of total body irradiation increased the risk of BK virus-associated hemorrhagic cystitis in the pediatric recipient population. We found that, despite current conditioning regimens, BK virus-associated infection still leads to a considerable incidence rate of hemorrhagic cystitis in pediatric recipients of allogeneic hematopoietic stem cell transplant. Patients with a haploidentical donor and a history of busulfan and cyclophosphamide treatment or total body irradiation had a higher risk of BK virus-associated hemorrhagic cystitis. Thus, we suggest that patients with these factors should be followed closely after allogeneic hematopoietic stem cell transplant.

Leukemia after gene therapy for sickle cell disease: insertional mutagenesis, busulfan, both, or neither

Recently, encouraging data provided long-awaited hope for gene therapy as a cure for sickle cell disease (SCD). Nevertheless, the transient suspension of the bluebird bio gene therapy trial (clinicaltrials.gov: NCT02140554) after participants developed acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) raised concerns. Potential possibilities for these cases include busulfan, insertional mutagenesis, both, or neither. Busulfan was considered the cause in the first reported case because the transgene was not present in the AML/MDS. However, busulfan is unlikely to have contributed to the most recent case. The transgene was present in the patient's malignant cells, indicating they were infused after busulfan treatment. Several lines of evidence suggest an alternative explanation for events in the bluebird bio trial, including that SCD population studies show an increased relative, but a low absolute, risk of AML/MDS. We propose a new hypothesis: after gene therapy for SCD, the stress of switching from homeostatic to regenerative hematopoiesis by transplanted cells drives clonal expansion and leukemogenic transformation of preexisting premalignant clones, eventually resulting in AML/MDS. Evidence validating our hypothesis will support prescreening individuals with SCD for preleukemic progenitors before gene therapy. While presumed viable, safe strategy has been implemented to resume gene therapy in adults with severe SCD, reasonable alternative curative therapy should be considered for children and adults with severe SCD. Currently, open multicenter clinical trials are incorporating nonmyeloablative conditioning, related haploidentical donors, and posttransplantation cyclophosphamide. Preliminary results from these trials appear promising, and National Institutes of Health–sponsored trials are ongoing in individuals with SCD using this platform.

Human placental mesenchymal stem cells ameliorate chemotherapy-induced damage in the testis by reducing apoptosis/oxidative stress and promoting autophagy

BackgroundThe side effects of busulfan on male reproduction are serious, so fertility preservation in children undergoing busulfan treatment is a major worldwide concern. Human placental mesenchymal stem cells (hPMSCs) have advantages such as stable proliferation and lower immunogenicity that make them an ideal material for stimulating tissue repair, especially restoring spermatogenesis. The protective effects of hPMSCs in busulfan-induced Sertoli cells and in busulfan-treated mouse testes have not been determined. Our study aimed to elaborate the protective effect and potential mechanisms of hPMSCs in busulfan-treated testes and Sertoli cells.MethodsFirst, we developed a mouse model of busulfan-induced testicular toxicity in vivo and a mouse Sertoli cell line treated with busulfan in vitro to assess the protective effect and mechanisms of hPMSC treatment on spermatogenesis. Then, the length, width, and weight of the testes were monitored using Vernier calipers. Furthermore, at 1 week and 4 weeks after the transplantation of hPMSCs, histological sections of testes were stained with hematoxylin-eosin, and the seminiferous tubules with fluid-filled cavities were counted. Through ELISA analysis, testosterone levels and MDA, SOD, LDH, and CAT activities, which are associated with ROS, were detected. Markers of ROS, proliferation (Ki67), and apoptosis (Annexin V) were evaluated by FACS. Next, the fluorescence intensity of proliferation markers (BrdU and SCP3), an antioxidant marker (SIRT1), a spermatogenesis marker (PLZF), and autophagy-related genes (P62 and LC3AB) were detected by fluorescence microscopy. The mRNA expression of γ-H2AX, BRCA1, PARP1, PCNA, Ki67, P62, and LC3 was determined by qRT-PCR.ResultshPMSCs restored disrupted spermatogenesis, promoted improved semen parameters, and increased testosterone levels, testis size, and autophagy in the testis toxicity mouse model induced by busulfan. hPMSCs suppressed the apoptosis of Sertoli cells and enhanced their rate of proliferation in vitro. Additionally, hPMSCs protected against oxidative stress and decreased oxidative damage in the testis toxicity mouse model induced by busulfan. Furthermore, hPMSCs increased the expression of proliferation genes (PCNA and KI67) and decreased the mRNA levels of apoptotic genes such as γ-H2AX, BRCA1, and PARP1.ConclusionsThis research showed that hPMSC injection ameliorated busulfan-induced damage in the testis by reducing apoptosis/oxidative stress and promoting autophagy. The present study offers an idea for a new method for clinical treatment of chemotherapy-induced spermatogenesis.

Intratesticular injection of busulfan for producing recipient male pigs for spermatogonial stem cell transplantation

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and fertility in male animals. In agricultural perspectives, SSC transplantation holds potential in expanding the availability of gametes from elite livestock rapidly, thus offering a highly efficient reproductive technique for livestock production. A limiting factor in application of this technique is acquisition of non-endogenous-SSC recipients, especially from large domestic animals. To explore a simple method to ablate pig endogenous SSCs and prepare transplant recipients, we directly injected different doses of busulfan into the testis tissue of Duroc pigs. Following injection, no significant health problems and body weight loss were observed in pigs treated with 3 mg/kg busulfan. Morphological analysis and gene expression assays showed that busulfan treatment efficiently removed endogenous SSCs, but sustained an intact Sertoli cells structure in pig testis. Compared with 5 mg/kg busulfan treatment group, 3 mg/kg busulfan treatment resulted in a complete SSCs ablation with less toxicity, providing a suitable environment for transplantation of exogenous SSCs. This study showed that intratesticular injection of busulfan in pigs to prepare recipients was an efficient and safe method, which lays the groundwork for the application of pig SSC transplantation.

Testicular quantitative ultrasound: A noninvasive monitoring method for evaluating spermatogenic function in busulfan-induced testicular injury mouse models.

Busulfan-induced testicular injury mouse models are commonly used for experiments on spermatogonial stem cell transplantation, treatments for azoospermia due to spermatogenic failure and preserving male fertility after chemotherapy. Here, we investigated the value of testicular quantitative ultrasound for evaluating spermatogenic function in this model. In this study, testicular ultrasound was performed on mice from day 0 to 126 after busulfan treatment (n=48), and quantitative data, including the testicular volume, mean pixel intensity and pixel uniformity, were analysed. The results revealed that from day 0 to 36, the testicular volume was positively associated with the testicle-to-body weight ratio (r=.92). On day 63, the pixel uniformity, which remained stable from day 0 to 36, declined significantly compared with that on day 36 (p<.01). On day 126, when the whole progression of spermatogenesis could be observed in most tubules, the mean pixel intensity also returned to normal (p>.05). In conclusion, testicular quantitative ultrasound could be used as a noninvasive and accurate monitoring method for evaluating spermatogenic function in busulfan-induced testicular injury mouse models.

Effect of Gosha-Jinki-Gan on Levels of Specific mRNA Transcripts in Mouse Testes after Busulfan Treatment.

With the increase in survival rates of cancer patients in recent years, infertility caused by anticancer treatments has become a significant concern for cancer survivors. Some studies have suggested that Sertoli cells play a key role in mediating testicular immunology in busulfan-induced aspermatogenesis. We recently demonstrated that Gosha-jinki-gan (TJ107), a traditional Japanese medicine, can completely recover injured spermatogenesis in mice 60 days after busulfan injection. In the present study, we sought to examine the levels of mRNA transcripts encoding markers of 25 Sertoli cell-specific products and 10 markers of germ cell differentiation. Our results demonstrated that only supplementation of TJ107 at day 60 after busulfan injection could significantly recover the increase in five mRNA species (Amh, Clu, Shbg, Testin, and Il1a) and the decrease in four mRNA species (Aqp8, CST9, Wnt5a, and Tjp1) in response to Busulfan (BSF) at day 120, with the increase of all examined spermatogenic markers.

Dnmt3a-null hematopoietic stem and progenitor cells expand after busulfan treatment

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) comprise the majority of mutations found in clonal hematopoiesis (CH), an age-related condition that was recently found to affect outcomes in patients undergoing hematopoietic stem cell transplant (HSCT). Recent studies have indicated that patients with CH have worse prognoses after HSCT, suggesting stress imposed by HSCT preconditioning agents may impact hematopoietic stem cell (HSC) dynamics in transplant recipients. In this study, we used a competitive transplantation mouse model to investigate how treatment with the common preconditioning agents 5-fluorouracil (5-FU) and busulfan (BU) affect the prevalence of Dnmt3a-/- HSCs and progenitor cells in competition with wild-type cells. We found that, though sufficient to deplete peripheral blood counts, 5-FU preconditioning did not significantly alter the frequency of Dnmt3a-null hematopoietic stem and progenitor cells (HSPCs) in mosaic mice. In contrast, mice treated with BU had a sevenfold decline in total bone marrow cells and an increase in Dnmt3a-null HSPCs that was detectable in peripheral blood. Indeed, even though all mosaic mice had a starting engraftment of ∼10%-40%, 85%-100% of HSPCs were Dnmt3a-null in four of seven mice after BU treatment, indicating these cells expand dramatically during recovery. Overall, these results suggest that individual preconditioning regimens have different effects on the expansion of Dnmt3a-mutant cells in patients with pre-existing CH. Thus, the presence of CH-associated mutants should be evaluated prior to selecting preconditioning regimens for HSCT.

Two resveratrol analogs, pinosylvin and 4,4′-dihydroxystilbene, improve oligoasthenospermia in a mouse model by attenuating oxidative stress via the Nrf2-ARE pathway

Two synthesized resveratrol analogs from our laboratory, namely pinosylvin (3,5-dihydroxy-trans-stilbene, PIN) and 4,4′-dihydroxystilbene (DHS), have been carefully evaluated for treatment of oligoasthenospermia. Recent studies have demonstrated that PIN and DHS improved sperm quality in the mouse. However, the mechanism of action of PIN and DHS on oligoasthenospermia remains unknown. Herein, we investigated the mechanistic basis for improvements in sperm parameters by PIN and DHS in a mouse model of oligoasthenospermia induced by treatment with busulfan (BUS) at 6 mg/kg b.w.. Two weeks following busulfan treatment, mice were administered different concentrations of PIN or DHS daily for 2 consecutive weeks. Thereafter, epididymal sperm concentration and motility were determined, and histopathology of the testes was performed. Serum hormone levels including testosterone (T), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were measured using corresponding specific enzyme-linked immunosorbent assay (ELISA) kits. Testicular mRNA expression profiles were determined by RNA sequencing analysis. These findings were validated by quantitative real-time PCR, western blotting and ELISA. Both PIN and DHS improved the epididymal sperm concentration and motility, enhanced testosterone levels, and promoted testicular morphological recovery following BUS treatment. PIN treatment was found to significantly reduce oxidative stress via the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE)-dependent antioxidant, glutathione peroxidase 3. DHS treatment significantly reduced oxidative stress via the Nrf2-ARE-dependent antioxidants glutathione S-transferase theta 2 and glutathione S-transferase omega 2. In summary, PIN and DHS ameliorated oligoasthenospermia in this mouse model by attenuating oxidative stress via the Nrf2-ARE pathway.

MPN-214: Chronic Myeloproliferative Neoplasms in Elderlies: Challenging Issues of Diagnosis and Treatment

Context: Chronic myeloproliferative neoplasms (CMN) in elderlies are the frequently registered clonal stem cell malignancies, being characterized in the advanced phases by recurrent evolution and negative socio-economic sequels. OBJECTIVE: To assert the diagnosis and treatment options in elderlies with CMN. DESIGN: This clinico-analytic study included 78 elderly patients with chronic myeloid leukemia (CML), primary myelofibrosis (PMF) and polycythemia vera (PV), treated at the Institute of Oncology from Moldova between 1995–2020. The diagnosis was proved by histopathological, cytological, molecular examinations and FISH. SETTING: The follow-up and study were completed at the comprehensive cancer center. The study is related to the ambulatory and hospitalized care. PATIENTS: Prefibrotic PMF stage was confirmed in 8 (42.1%) cases, fibrotic stage – in 11 (57.9%). CML was proved in chronic phase in 25 (89.3%) patients, in the accelerated phase in 3 (10.7%). PV was diagnosed in stage IIA in 27 (87.1%) patients, in stage IIB in 4 (12.9%). INTERVENTIONS: TKIs were administered in the newly diagnosed CML patients, in cases of resistance to non-TKIs chemotherapy and interferon-α. PV and PMF patients were treated with busulfan, hydroxycarbamide and interferon-α, combined with phlebotomies in PV. RESULTS: There was no difference of the hematological response (HR) and molecular response rates under TKIs therapy between elderlies and CML totality. In patients ≥60 years 5-year overall survival (OS) was 69% after TKIs therapy, being slightly lower than in CML totality (74%). HR was achieved in all PV patients under chemotherapy and phlebotomies. In relapses, interferon-α provided HR for 61 and 79 months. 10-year OS of elderly PV patients constituted 76.4%, being lower than in PV totality (85.9%). There was no difference of the OS in elderlies under chemotherapy with busulfan and hydroxycarbamide. HR rate and OS were lower under busulfan and hydroxycarbamide treatment in PMF patients, than in PV. CONCLUSIONS: No difference of the OS was revealed in PV elderlies under busulfan and hydroxycarbamide therapy, surpassing the OS in PMF patients. TKIs therapy is a curable option for CML patients regardless the age. The OS in elderlies decays due to more frequent vascular-thrombotic events owing to thrombocytosis.

Microbiota-Derived Lactate Controls Hematopoiesis and Erythropoiesis Through Regulating Stem Cell Factor from Leptin Receptor+ Niche Cells in the Bone Marrow

Although functional interplay between intestinal microbiota and the distant sites beyond the gut has been identified, it is unclear how microbiota-derived metabolites influence the fate of hematopoietic stem cells (HSCs). In this study, we investigated the role of microbiota-derived lactate in hematopoiesis using G-protein–coupled receptor (Gpr) 81, a known lactate receptor, deficient (Gpr81-/-) mice. We found that the total number of HSCs was significantly reduced in the bone marrow (BM) of Gpr81-/- mice as compared with heterogenic (Gpr81+/-) mice in a steady state. Notably, the expression levels of stem cell factor (SCF), which is required for proliferation of HSCs, were significantly decreased in leptin-receptor expressing (LepR+) mesenchymal stromal cells (MSCs) around the sinusoidal vessels of BM obtained from Gpr81-/- mice as compared with Gpr81+/- mice. Hematopoietic recovery and activation of BM niche cells after irradiation or busulfan treatment required Gpr81 signals. Oral administration of lactic acid-producing bacteria (LAB) activated the secretion of SCF from LepR+ BM MSCs and subsequently accelerated hematopoiesis and erythropoiesis. Most importantly, LAB feeding accelerated the self-renewal of HSCs in germ-free mice. These results suggested that microbiota-derived lactate stimulates SCF secretion by LepR+ BM MSCs and subsequently activates hematopoiesis and erythropoiesis in a Gpr81-dependent manner.

Busulfan Suppresses Autophagy in Mouse Spermatogonial Progenitor Cells via mTOR of AKT and p53 Signaling Pathways.

In testis, a rare undifferentiated germ cell population with the capacity to regenerate robustly and support spermatogenesis, is defined as spermatogonial progenitor cells (SPCs) population. As a widely used drug for tumor therapy or bone marrow transplantation, busulfan has a severe side effect on SPCs population and causes a consequent infertility. Recently, accumulating evidence revealed the protective role of autophagy in stem cell maintenance under exogenous stress. To better understand the role of autophagy in SPCs fates, we investigated the potential function of autophagy in SPCs under busulfan stress, and found that treatment of busulfan induced the formation of autophagic vesicles and autophagosomes in mouse SPCs. Subsequently, a connection of autophagy and SPCs maintenance and survival was demonstrated in a dose-dependent manner. Moreover, mTOR was identified as an essential factor for autophagy in SPCs with a complicated mechanism: (1) mTOR is phosphorylated by AKT to activate its target genes, p70s6 kinase, resulting in the inhibition of autophagy during short-term busulfan treatment. (2) mTOR mediates autophagy with p53 together, to regulate the fate of SPCs. Collectively, observations from this study indicate that moderate autophagy effectively protects SPCs from the stress of chemotherapy, which may provide an important hint for fertility protection in clinic.

Chestnut polysaccharides benefit spermatogenesis through improvement in the expression of important genes.

Recently there has been a continuing worldwide decrease in the quality of human spermatozoa, especially in spermatozoa motility and concentration. Many factors are involved in this decline, and great efforts have been made to rescue spermatogenesis; however, there has been little progress in the improvement of sperm quality. Chestnuts are used in traditional Chinese medicine; their major active components are chestnut polysaccharides (CPs). CPs have many biological activities but their effects on spermatogenesis are unknown. The current investigation was designed to explore the impact of CPs on spermatogenesis and the underlying mechanisms. We demonstrated that CPs significantly increased sperm motility and concentration (4-fold and 12-fold, respectively), and improved seminiferous tubule development by increasing the number of germ cells after busulfan treatment. CPs dramatically rescued the expression of important genes and proteins (STRA8, DAZL, SYCP1, SYCP3, TNP1 etc.) in spermatogenesis. Furthermore, CPs increased the levels of hormone synthesis proteins such as CYP17A1 and HSD17β1. All the data suggested that CPs improved the testicular microenvironment to rescue spermatogenesis. With CPs being natural products, they may be an attractive alternative for treating infertile patients in the future. At the same time, the deep underlying mechanisms of their action need to be explored.

Inhibition of ferroptosis attenuates busulfan-induced oligospermia in mice

Busulfan is commonly used for cancer chemotherapy, nevertheless it cause male infertility via damaging the germ cells. Therefore, the underlying mechanism should be explored. In the present study, we demonstrated for the first time that ferroptosis was involved in busulfan-induced oligospermia in mice. Mice were given testicular injection of busulfan on both sides at the dose of 4 mg/kg body weight to establish the model of oligospermia. Four weeks later, the results showed that busulfan-treated mice exhibited decreased sperm concentration and motility, along with features of typical ferroptosis in testis, such as increased malondialdehyde (MDA) content and prostaglandin-endoperoxide synthase (PTGS2) mRNA expression, and decreased NADPH content. Inhibition of ferroptosis by ferrostatin-1 (Fer-1) or deferoxamine (DFO) partially alleviated busulfan-induced oligospermia in mice. Additionally, we also revealed that busulfan treatment induced spermatogenic cells ferroptosis by down-regulating nuclear factor-E2-related factor 2 (Nrf2) and glutathione peroxidase 4 (GPX4) expressions, and decreasing iron efflux through reduction of ferroportin 1 (FPN1) expression. Fer-1 or DFO obviously reversed busulfan-induced ferroptosis by increasing Nrf2, GPX4 and FPN1 expressions. Furthermore, after activation of Nrf2 by sulforaphane, sperm concentration and motility in busulfan-treated mice increased, accompanied by enhanced expressions of GPX4 and FPN1. These findings imply that busulfan-induced ferroptosis might be mediated via inhibition of Nrf2-GPX4 (FPN1) signaling pathway, and highlight that targeting ferroptosis serves as a potential strategy for prevention of busulfan-induced damage and male infertility.

Inhibiting Necroptosis of Spermatogonial Stem Cell as a Novel Strategy for Male Fertility Preservation.

Fertility preservation is a common concern for male cancer survivors of reproductive age. However, except for testicular tissue cryopreservation, which is not very effective, there is no feasible and precise therapy capable of protecting spermatogenesis for prepubertal boys before or during gonadotoxic treatment. This study aims to investigate the effects of inhibiting necroptosis of spermatogonial stem cell (SSC) in fertility preservation. Male mice 12 weeks of age were used to establish gonadotoxicity with two intraperitoneal injections of busulfan at a total dose of 40 mg kg-1. The mouse model and the primary cultured mouse SSCs were used to characterize the relationship between necroptosis of SSC and gonadotoxicity. Meanwhile, the effects of an inhibitor of necroptosis pathway, RIPA-56, were observed on day 36 in the mouse model of busulfan-induced gonadotoxicity. We found that the number of SSCs was decreased, but the level of necroptosis was upregulated on day 18 after busulfan treatment in testes from gonadotoxic mice. Further experiments in primary cultured cells showed that the necroptosis caused cell death in busulfan-treated SSCs and could be inhibited by RIPA-56. After suppressing the necroptosis of SSCs, the busulfan-induced mice had a decreased loss of spermatogenic cells as shown by histology and an increased Johnsen's score. Moreover, the quantities of SSCs and epididymal spermatozoa were restored after intervention with RIPA-56, indicating a series of beneficial effects by targeting the necroptosis of SSCs in mice undergoing busulfan treatment. In conclusion, our findings reveal that the necroptosis of SSCs plays a critical role in busulfan-induced gonadotoxicity and may be a potential target for male fertility preservation.