Inhibition of CXCR4 in Spinal Cord and DRG with AMD3100 Attenuates Colon-Bladder Cross-Organ Sensitization.

BACKGROUND: Cross-sensitization of pelvic organs is one theory for why symptoms of gut sickness and interstitial cystitis/bladder pain syndrome overlap. Experimental colitis has been shown to trigger bladder hyperactivity and hyperalgesia in rats. The chemokine receptor CXCR4 plays a key role in bladder function and central sensitization. We aim to study the role of CXCR4 and its inhibitor AMD3100 in colon-bladder cross-organ sensitization. METHODS: The colitis model was established by rectal infusion of trinitrobenzene sulfonic acid. Western blot and immunofluorescence were used to assess the expression and distribution of CXCR4. Intrathecal injection of AMD3100 (a CXCR4 inhibitor) and PD98059 (an ERK inhibitor) were used to inhibit CXCR4 and downstream extracellular signal-regulated kinase (ERK) in the spinal cord and dorsal root ganglion (DRG). Intravesical perfusion of resiniferatoxin was performed to measure the pain behavior counts of rats, and continuous cystometry was performed to evaluate bladder voiding function. RESULTS: Compared to the control group, CXCR4 was expressed more in bladder mucosa and colon mucosa, L6-S1 dorsal root ganglion (DRG), and the corresponding segment of the spinal dorsal horn (SDH) in rats with colitis. Moreover, intrathecal injection of the AMD3100 suppressed bladder overactivity, bladder hyperalgesia, and mastocytosis symptoms caused by colitis. Furthermore, AMD3100 effectively inhibited ERK activation in the spinal cord induced by experimental colitis. Finally, treatment with PD98059 alleviated bladder overactivity and hyperalgesia caused by colitis. CONCLUSION: Increased CXCR4 in the DRG and SDH contributes to colon inflammation-induced bladder overactivity and hyperalgesia partly via the phosphorylation of spinal ERK. Treatment targeting the CXCR4/ERK pathway might provide a potential new approach for the comorbidity between the digestive system and the urinary system.

Subacute AMD3100 Treatment Is Not Efficient in Neonatal Hypoxic-Ischemic Rats.

BACKGROUND AND PURPOSE: Despite the advances in treating neonatal hypoxic-ischemic encephalopathy (HIE) with induced hypothermia, the rates of severe disability are still high among survivors. Preclinical studies have indicated that cell therapies with hematopoietic stem/progenitor cells could improve neurological outcomes in HIE. In this study, we investigated whether the administration of AMD3100, a CXCR4 antagonist that mobilizes hematopoietic stem/progenitor cells into the circulation, has therapeutic effects in HIE. METHODS: P10 Wistar rats of both sexes were subjected to right common carotid artery occlusion or sham procedure, and then were exposed to hypoxia for 120 minutes. Two subcutaneous injections of AMD3100 or vehicle were given on the third and fourth day after HIE. We first assessed the interindividual variability in brain atrophy after experimental HIE and vehicle treatment in a small cohort of rats. Based on this exploratory analysis, we designed and conducted an experiment to test the efficacy of AMD3100. Brain atrophy on day 21 after HIE was defined as the primary end point. Secondary efficacy end points were cognitive (T-water maze) and motor function (rotarod) on days 17 and 18 after HIE, respectively. RESULTS: AMD3100 did not decrease the brain atrophy in animals of either sex. Cognitive impairments were not observed in the T-water maze, but male hypoxic-ischemic animals exhibited motor coordination deficits on the rotarod, which were not improved by AMD3100. A separate analysis combining data from animals of both sexes also revealed no evidence of the effectiveness of AMD3100 treatment. CONCLUSIONS: These results indicate that the subacute treatment with AMD3100 does not improve structural and functional outcomes in a rat HIE model.

Chemokine receptor antagonists enhance morphine's antinociceptive effect but not respiratory depression.

AIMS: We have shown that chemokines injected into the periaqueductal gray region of the brain blocks opioid-induced analgesia in the rat cold-water tail flick test (CWTF). The present experiments tested whether chemokine receptor antagonists (CRAs), in combination with sub-analgesic doses of morphine, would provide maximal analgesia in the CWTF test and the mouse formalin pain assay. The effect of CRAs on respiratory depression was also evaluated. MAIN METHODS: One, two or four CRAs (AMD3100/CXCR4, maraviroc/CCR5, RS504393/CCR2 orAZD8797/CX3CR1) were used in combination with sub-analgesic doses of morphine, all given systemically. Pain was assessed using the rat CWTF test or formalin injection into the paw of mice scored by licking. Respiration and oxygen saturation were measured in rats using a MouseOX® Plus - pulse oximeter. KEY FINDINGS: In the CWTF test, a sub-maximal dose of morphine in combination with maraviroc alone, maraviroc plus AMD3100, or with the four chemokine receptor antagonists, produced synergistic increases in antinociception. In the formalin test, the combination of four CRAs plus a sub-maximal dose of morphine resulted in increased antinociception in both male and female mice. AMD3100 had an additive effect with morphine in both sexes. Coadministration of CRAs with morphine did not potentiate the opioid respiratory depressive effect. SIGNIFICANCE: These results support the conclusion that combinations of CRAs can increase the potency of sub-analgesic doses of morphine analgesia without increasing respiratory depression. The results support an "opioid sparing" strategy for alleviation of pain using reduced doses of opioids in combination with CRAs to achieve maximal analgesia.

An innovation in stem cell harvesting: Heparin use.

BACKGROUND AND OBJECTIVES: Stem cell transplantation is a growing treatment strategy for most malignant and non- malignant hematological diseases. Plerixafor and granulocyte colony stimulating factor (G-CSF) are usually used in mobilization regimens to increase the CD34+ cell count in the harvest. Heparin is a sulphated glycosaminoglycated polymer with 12-15 kDa mass. Heparin inhibits the CXCR4/SDF1 axis, as does plerixafor. In this study, our aim was to investigate the effect of using heparin on stem cell mobilization and harvesting. MATERIALS AND METHODS: We administered 5000 units of unfractioned heparin intravenously in 150 mL (mL) of isotonic sodium chloride solution, 15 min before the stem cell harvesting procedure to 141 patients who underwent bone marrow transplantation between the years of 2018 and 2019 at our Stem Cell Transplantation Unit. Thirty patients were included as a control group, and they were not given heparin. The study population included patients with multiple myeloma and lymphoma equally in each group. RESULTS: In all patients hematopoeitic stem cells were successfully harvested in a single cycle of apheresis. In multiple myeloma patients who received heparin, the mean collected CD34+ cell number was 8 × 106/kg, and the mean CD34+ cell number yield was 12,555/µl. In the control group, the mean collected CD34+ cell number was 4,2 × 106/kg, and mean CD34+ cell number in yield was 492/µl. In lymphoma patients who received heparin, the mean collected CD34+ cell number was 6,8 × 106/kg, and the mean CD34+ cell number was 1421/µl. In the control group the mean collected CD34+ cell number was 4,3 × 106/kg, and the mean CD34+ cell number was 358/µl. The effect of heparin on the collected stem cell number in both myeloma and lymphoma patients was statistically significant (p < 0.01). CONCLUSIONS: Our results have shown that heparin increases harvested stem cell numbers significantly. Heparin may be a promising agent for stem cell harvesting.

CXCL12/CXCR4 axis as a key mediator in atrial fibrillation via bioinformatics analysis and functional identification.

Atrial fibrillation (AF) is an increasingly prevalent arrhythmia with significant health and socioeconomic impact. The underlying mechanism of AF is still not well understood. In this study, we sought to identify hub genes involved in AF, and explored their functions and underlying mechanisms based on bioinformatics analysis. Five microarray datasets in GEO were used to identify the differentially expressed genes (DEGs) by Robust Rank Aggregation (RRA), and hub genes were screened out using protein-protein interaction (PPI) network. AF model was established using a mixture of acetylcholine and calcium chloride (Ach-CaCl2) by tail vein injection. We totally got 35 robust DEGs that mainly involve in extracellular matrix formation, leukocyte transendothelial migration, and chemokine signaling pathway. Among these DEGs, we identified three hub genes involved in AF, of which CXCL12/CXCR4 axis significantly upregulated in AF patients stands out as one of the most potent targets for AF prevention, and its effect on AF pathogenesis and underlying mechanisms were investigated in vivo subsequently with the specific CXCR4 antagonist AMD3100 (6 mg/kg). Our results demonstrated an elevated transcription and translation of CXCL12/CXCR4 axis in AF patients and mice, accompanied with the anabatic atrial inflammation and fibrosis, thereby providing the substrate for AF maintenance. Blocking its signaling via AMD3100 administration in AF model mice reduced AF inducibility and duration, partly ascribed to decreased atrial inflammation and structural remodeling. Mechanistically, these effects were achieved by reducing the recruitment of CD3+ T lymphocytes and F4/80+ macrophages, and suppressing the hyperactivation of ERK1/2 and AKT/mTOR signaling in atria of AF model mice. In conclusion, this study provides new evidence that antagonizing CXCR4 prevents the development of AF, and suggests that CXCL12/CXCR4 axis may be a potential therapeutic target for AF.

Evidence for continued dose escalation of plerixafor for hematopoietic progenitor cell collections in sickle cell disease.

We present data from our study of plerixafor mobilization (NCT02193191) relevant to the question of whether further dose escalation of plerixafor can address inconsistent adequacy of CD34+ mobilization for gene therapy of sickle cell disease (SCD). We found that, in the same patient, higher plerixafor dosing was associated with higher fold increases in PB CD34+ count, but not necessarily higher absolute CD34+ counts. Variation in pre-apheresis absolute CD34+ counts was related to intra-individual variation in baseline PB CD34+ counts and inter-individual variation in responsiveness to plerixafor. Overall, our results support further studies of continued dose escalation of plerixafor for autologous HPC collection in SCD.

Process and procedural adjustments to improve CD34+ collection efficiency of hematopoietic progenitor cell collections in sickle cell disease.

BACKGROUND: Adequate CD34+ collection efficiency (CE) is critical to achieve target CD34+ cell doses in hematopoietic progenitor cell (HPC) collections. Autologous HPC collection in sickle cell disease (SCD) is associated with unstable collection interfaces and low CD34+ CEs. We hypothesized that variables specific to SCD, activation of blood cells and elevated viscosity, might contribute to these issues and made adjustments to the collection process and procedure to address our hypothesis. STUDY DESIGN AND METHODS: In two patients with SCD undergoing autologous HPC collection on our clinical trial (NCT02193191), we therefore implemented adjustments to the process and procedure in the following areas: proximity of RBC exchange to HPC collection, the type of anticoagulation, and the packing factor setting. RESULTS: There was no collection interface instability. Our CD34+ CE1s were high at 70% and 51%, and granulocyte CE, platelet CE, and product granulocyte % were remarkably low. Product hematocrits were not as high as previously reported to be required to obtain adequate CEs. Interestingly, one HPC product showed a hemoglobin S (HbS) of 91% at the same time that the peripheral blood (PB) showed a HbS of 22%. DISCUSSION: Adjustments to the HPC collection process and procedure were associated with adequate CD34+ CEs and low granulocyte and platelet contamination in HPC products from SCD patients. Given the discrepancy in the percentage of sickle RBCs in the product versus the PB, we hypothesize that CD34+ cells and RBCs may aggregate. Our interventions and hypothesis should be further investigated in larger studies.

CXCR4 blockade sensitizes osteosarcoma to doxorubicin by inducing autophagic cell death via PI3K­Akt­mTOR pathway inhibition.

Doxorubicin is one of the most frequently used chemotherapy drugs in the treatment of osteosarcoma (OS), but the emergence of chemoresistance often leads to treatment failure. C­X­C motif chemokine receptor 4 (CXCR4) has been demonstrated to regulate OS progression and metastasis. However, whether CXCR4 is also involved in OS chemoresistance and its molecular mechanisms has yet to be fully elucidated. In the present study, CXCR4­mediated autophagy for OS chemotherapy was investigated by western blot analysis, transmission electron microscopy and confocal microscopy. CXCR4 silencing enhanced doxorubicin­induced apoptosis by reducing P­glycoprotein in CXCR4+ LM8 cells, while CXCR4 overexpression promoted OS doxorubicin resistance in CXCR4­ Dunn cells. Furthermore, CXCR4 silencing with or without doxorubicin increased the expression of beclin 1 and light chain 3B, and the number of autophagosomes and autolysosomes, as well as induced autophagic flux activation by suppressing the PI3K/AKT/mTOR signaling pathway. In addition, pretreatment with the autophagy inhibitor bafilomycin A1 attenuated CXCR4 abrogation­induced cell death. Finally, the CXCR4 antagonist AMD3100 synergistically reinforced the antitumor effect of doxorubicin in an orthotopic OS mouse model. Taken together, the present study revealed that CXCR4 inhibition sensitizes OS to doxorubicin by inducing autophagic cell death. Therefore, targeting the CXCR4/autophagy axis may be a promising therapeutic strategy to overcome OS chemotherapy resistance.

Phase 1 study of plerixafor in combination with total body irradiation-based myeloablative conditioning for allogeneic hematopoietic stem cell transplantation.

Plerixafor, a CXCR4 inhibitor, has the potential to mobilize leukemic cells, which may contribute to their chemosensitization. This phase 1 study evaluated the safety of myeloablative conditioning combined with plerixafor for allogeneic hematopoietic stem cell transplantation (HSCT). Patients with high-risk leukemia undergoing allogeneic HSCT after total body irradiation (TBI, 12 Gy)-based myeloablative conditioning were eligible; 9 patients were enrolled. The study was performed using a 3 + 3 design with an escalating total dose of plerixafor. Plerixafor was given subcutaneously 8 h before TBI and chemotherapeutic agents. Plerixafor was successfully escalated to the maximum dose (0.72 mg/kg) without dose-limiting toxicities. Underlying diseases were acute myelogenous and lymphoblastic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome. As adverse events, plerixafor administration was associated with transient Grades 2-3 diarrhea (n = 7) and abdominal pain (n = 4). In 6 patients, leukemic cell mobilization into the peripheral blood by plerixafor was confirmed by a morphological or molecular method. All patients achieved neutrophil engraftment and 5 were alive in remission at a follow-up after 30-40 months. Plerixafor-combined myeloablative conditioning for allogeneic HSCT was well tolerated. Leukemic-cell mobilization into peripheral blood was observed in half of the patients. Further study is required to evaluate the efficacy and safety of this concept.

Plerixafor added to G-CSF allows mobilization of a sufficient number of hematopoietic progenitors without impacting the efficacy of TCR-alpha/beta depletion in pediatric haploidentical and genoidentical donors failing to mobilize with G-CSF alone.

BACKGROUND: Collection of a large number of early hematopoietic progenitors is essential for allogeneic apheresis products intended for TCR-alpha/beta depletion. MATERIALS AND METHODS: We added plerixafor 0.24 mg/kg body weight (bw) on day 4 of high-dose filgrastim mobilization 10 hours prior to apheresis in 16 (30.5%) pediatric allogeneic donors who failed to recover a sufficient number of CD34+ cells. RESULTS: On day 4 of G-CSF, the median CD34+ cell count in peripheral blood was 6 per µL (range 4-9 per µL) in 6 poor mobilizers and 16 per µL (range 12-19 per µL) in insufficient mobilizers. In all donors, the threshold of 50 CD34+ cells/µL was achieved, and the median increase was 14.8-fold in poor mobilizers and 6.5-fold in insufficient mobilizers, whereas it was 3.45-fold increase in those mobilized with G-CSF alone. DISCUSSION: In all donors, a predefined number of >10 × 106 CD34+ cells/kg of recipient bw before depletion was reached in the apheresis product. The use of plerixafor did not affect the purity of further TCR-alpha/beta depletion. Side effects were mild to moderate and consisted of nausea and vomiting. CONCLUSION: Thus, the safety and high efficacy of plerixafor was proven in healthy pediatric allogeneic hematopoietic cell donors.

Threshold for optimal administration of plerixafor in autologous peripheral blood stem cell collections through CD34+ cell monitoring based on the experience from two Japanese university hospitals.

Plerixafor was introduced to Japan in 2017 as a stem cell mobilization enhancement reagent, but the threshold for its use remains unclear. In this study, we assessed 57 patients treated with plerixafor (33 patients with multiple myeloma (MM) and 24 with malignant lymphoma (ML) and 152 patients without plerixafor administration. When CD34+ cell pre-counts were between 5.5 and 20 cells/µL in MM or 6 and 21 cells/µL in ML, the CD34+ cell count increased significantly, attaining the highest yield in response to plerixafor (achievement rate by one leukapheresis is 93.3% and 91.7% in MM and ML, at P < .001 and P = .012, respectively). In case the CD34+ cell pre-count was less than 5.5 cells/µL, an increase of at least 7 cells/µL from baseline by plerixafor was the necessary condition to achieve successful collection through a two-time leukapheresis. Monitoring CD34+ cell numbers might improve the collection efficiency and reduce the cost.

AMD3100 redosing fails to repeatedly mobilize hematopoietic stem cells in the nonhuman primate and humanized mouse.

AMD3100 (plerixafor) is a vital component of many clinical and preclinical transplant protocols, facilitating harvest of hematopoietic stem and progenitor cells through mobilization into the peripheral blood circulation. Repeat mobilization with AMD3100 is also necessary for many patients with suboptimal first stem cell collection or those requiring repeat transplantation. In this study we investigated the mobilization efficacy of repeated AMD3100 dosages in the nonhuman primate and humanized mouse models. In nonhuman primates, we observed effective mobilization after the first AMD3100 administration but a significantly poorer response in CD34+ and hematopoietic stem cell-enriched CD90+ cells with subsequent doses of the drug. A similar loss of efficacy with repeated administration was noted in immunodeficient mice engrafted with human CD34+ cells, in whom the total human white cell population, and particularly human hematopoietic stem and progenitor cells, mobilized significantly less effectively following a second AMD3100 administration when compared with the first dose. Together, our results are expected to inform future mobilization protocols for the purposes of peripheral blood hematopoietic stem cell extraction or for applications in which hematopoietic stem cells must be made accessible for in vivo-delivered gene targeting agents.

Subcutaneous Administration of AMD3100 into Mice Models of Alzheimer's Disease Ameliorated Cognitive Impairment, Reduced Neuroinflammation, and Improved Pathophysiological Markers.

BACKGROUND: Alzheimer's disease (AD), the prevalent dementia in the elderly, involves many related and interdependent pathologies that manifest simultaneously, leading to cognitive impairment and death. Amyloid-ß (Aß) accumulation in the brain triggers the onset of AD, accompanied by neuroinflammatory response and pathological changes. The CXCR4/CXCL12 (SDF1) axis is one of the major signal transduction cascades involved in the inflammation process and regulation of homing of hematopoietic stem cells (HSCs) within the bone marrow niche. Inhibition of the axis with AMD3100, a reversible antagonist of CXCR4 mobilizes endogenous HSCs from the bone marrow into the periphery, facilitating the recruitment of bone marrow-derived microglia-like cells into the brain, attenuates the neuroinflammation process that involves release of excitotoxic markers such as TNFα, intracellular Ca2 +, and glutamate and upregulates monocarboxylate transporter 1, the major L-lactate transporter in the brain. OBJECTIVE: Herein, we investigate if administration of a combination of AMD3100 and L-lactate may have beneficial effects in the treatment of AD. METHODS: We tested the feasibility of the combined treatment for short- and long-term efficacy for inducing endogenous stem cells' mobilization and attenuation of neuroinflammation in two distinct amyloid-ß-induced AD mouse models. RESULTS: The combined treatment did not demonstrate any adverse effects on the mice, and resulted in a significant improvement in cognitive/memory functions, attenuated neuroinflammation, and alleviated AD pathologies compared to each treatment alone. CONCLUSION: This study showed AMD3100's beneficial effect in ameliorating AD pathogenesis, suggesting an alternative to the multistep procedures of transplantation of stem cells in the treatment of AD.

AMD3100 and SDF­1 regulate cellular functions of endothelial progenitor cells and accelerate endothelial regeneration in a rat carotid artery injury model.

The present study was conducted to assess the effects of AMD3100 and stromal cell-derived factor 1 (SDF-1) on cellular functions and endothelial regeneration of endothelial progenitor cells (EPCs). The cell proliferation and adhesion capacity of EPCs were evaluated in vitro following treatment with AMD3100 and SDF­1 using a Cell Counting Kit­8 assay. Furthermore, the expression levels of C­X­C motif chemokine receptor 4 (CXCR4) and C­X­C motif chemokine receptor 7 (CXCR7) were detected before and after treatment with AMD3100 and SDF­1 to elucidate their possible role in regulating the cellular function of EPCs. A rat carotid artery injury model was established to assess the influences of AMD3100 and SDF­1 on endothelial regeneration. AMD3100 reduced the proliferation and adhesion capacity of EPCs to fibronectin (FN), whereas it increased the adhesion capacity of EPCs to human umbilical vein endothelial cells (HUVECs). However, SDF­1 stimulated the proliferation and cell adhesion capacity of EPCs to HUVECs and FN. Additionally, the expression levels of CXCR7 but not CXCR4 were upregulated following AMD3100 treatment, whereas the expression levels of both CXCR4 and CXCR7 were upregulated after SDF­1 treatment. In vivo results demonstrated that AMD3100 increased the number of EPCs in the peripheral blood and facilitated endothelial repair at 7 days after treatment. However, local administration of SDF­1 alone did not enhance reendothelialization 7 and 14 days after treatment. Importantly, the combination of AMD3100 with SDF­1 exhibited superior therapeutic effects compared with AMD3100 treatment alone, accelerated reendothelialization 7 days after treatment, and attenuated neointimal hyperplasia at day 7 and 14 by recruiting more EPCs to the injury site. In conclusion, AMD3100 could positively regulate the adhesion capacity of EPCs to HUVECs via elevation of the expression levels of CXCR7 but not CXCR4, whereas SDF­1 could stimulate the proliferation and adhesion capacity of EPCs to FN and HUVECs by elevating the expression levels of CXCR4 and CXCR7. AMD3100 combined with SDF­1 outperformed AMD3100 alone, promoted early reendothelialization and inhibited neointimal hyperplasia, indicating that early reendothelialization attenuated neointimal hypoplasia following endothelial injury.

Third time's a charm? Mobilization of autologous peripheral blood stem cells in patients with two previous failed mobilizations with plerixafor.

BACKGROUND: Patients who respond inadequately to plerixafor salvage during autologous peripheral blood stem cell (PBSC) collection are frequently remobilized with plerixafor to collect additional stem cells. However, in patients who fail remobilization, it is unclear whether additional mobilization efforts with plerixafor are useful. STUDY DESIGN AND METHODS: We retrospectively examined the PBSC collections of 15 consecutive patients with lymphoma and multiple myeloma who underwent three mobilizations with plerixafor. RESULTS: Of the 821 patients who underwent autologous stem cell collections, 15 patients were mobilized three times with plerixafor (1.8%), which enabled 11 (73.3%) patients to reach 2.0 × 106 CD34+ cells/kg or greater. Among patients who eventually collected successfully the median yields from the three collection attempts were 0.46, 0.76, and 1.54 × 106 CD34+ cells/kg, respectively. Among those who collected less than 2.0 × 106 CD34+ cells/kg cumulatively, the median yields were 0.14, 0.33, and 0.22 × 106 CD34+ cells/kg from the three collection attempts. The combined collection yields from the first two mobilization attempts were significantly lower (p = 0.003; range, 0.09-0.73 vs. 0.63-1.84; median, 0.51 vs. 1.36) in those who failed collection. CONCLUSIONS: The majority (73.3%) of patients who underwent three mobilization attempts were eventually able to collect enough cells to permit autologous transplantation. Extremely low peripheral blood CD34+ count after the first dose of plerixafor and collection yields during the first two attempts were associated with a poor collection yield on the third attempt. The risks and benefits of a third mobilization should be weighed to facilitate judicious use of resources.

Chemokine receptor CXCR4 activates the RhoA/ROCK2 pathway in spinal neurons that induces bone cancer pain.

BACKGROUND: Chemokine receptor CXCR4 has been found to be associated with spinal neuron and glial cell activation during bone cancer pain. However, the underlying mechanism remains unknown. Furthermore, the RhoA/ROCK2 pathway serves as a downstream pathway activated by CXCR4 during bone cancer pain. We first validated the increase in the expressions of CXCR4, p-RhoA, and p-ROCK2 in the spinal dorsal horn of a well-characterized tumor cell implantation-induced cancer pain rat model and how these expressions contributed to the pain behavior in tumor cell implantation rats. We hypothesized that spinal blockade of the CXCR4-RhoA/ROCK2 pathway is a potential analgesic therapy for cancer pain management. METHODS: Adult female Sprague-Dawley rats (body weight of 180-220 g) and six- to seven-week old female Sprague-Dawley rats (body weight of 80-90 g) were taken. Ascitic cancer cells were extracted from the rats (body weight of 80-90 g) with intraperitoneally implanted Walker 256 mammary gland carcinoma cells. Walker 256 rat mammary gland carcinoma cells were then injected (tumor cell implantation) into the intramedullary space of the tibia to establish a rat model of bone cancer pain. RESULTS: We found increased expressions of CXCR4, p-RhoA, and p-ROCK2 in the neurons in the spinal cord. p-RhoA and p-ROCK2 were co-expressed in the neurons and promoted by overexpressed CXCR4. Intrathecal delivery of CXCR4 inhibitor Plerixafor (AMD3100) or ROCK2 inhibitor Fasudil abrogated tumor cell implantation-induced pain hypersensitivity and tumor cell implantation-induced increase in p-RhoA and p-ROCK2 expressions. Intrathecal injection of stromal-derived factor-1, the principal ligand for CXCR4, accelerated p-RhoA expression in naive rats, which was prevented by postadministration of CXCR4 inhibitor Plerixafor (AMD3100) or ROCK2 inhibitor Fasudil. CONCLUSIONS: Collectively, the spinal RhoA/ROCK2 pathway could be a critical downstream target for CXCR4-mediated neuronal sensitization and pain hypersensitivity in bone cancer pain, and it may serve as a potent therapeutic target for pain treatment.

CD34+ cell mobilization, blood graft composition, and posttransplant recovery in myeloma patients compared to non-Hodgkin's lymphoma patients: results of the prospective multicenter GOA study.

BACKGROUND: Autologous stem cell transplantation is an established treatment option for patients with multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL). STUDY DESIGN AND METHODS: In this prospective multicenter study, 147 patients with MM were compared with 136 patients with NHL regarding the mobilization and apheresis of blood CD34+ cells, cellular composition of infused blood grafts, posttransplant recovery, and outcome. RESULTS: Multiple myeloma patients mobilized CD34+ cells more effectively (6.3 × 106 /kg vs. 3.9 × 106 /kg, p = 0.001). The proportion of poor mobilizers (peak blood CD34+ cell count <20 × 106 /L) was higher in NHL patients (15% vs. 3%, p < 0.001). Plerixafor was added to rescue the mobilization failure in 17 MM patients (12%) and in 35 NHL patients (26%; p = 0.002). The infused grafts contained more natural killer (NK) and CD19+ cells in MM patients. Blood platelet and NK-cell counts were higher in MM patients posttransplant. Early treatment-related mortality was low in both groups, but NHL patients had a higher late (>100 days) nonrelapse mortality (NRM; 6% vs. 0%, p = 0.003). CONCLUSIONS: Non-Hodgkin's lymphoma and MM patients differ in terms of mobilization of CD34+ cells, graft cellular composition, and posttransplant recovery. Thus, the optimal graft characteristics may also be different.

Use of polymeric CXCR4 inhibitors as siRNA delivery vehicles for the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and is associated with poor long-term survival often owing to relapse. Current treatments for AML are associated with considerable toxicity and are frequently not effective after relapse. Thus, it is important to develop novel therapeutic strategies. Short interfering RNA (siRNA)-based therapeutics targeting key oncogenes have been proposed as treatments for AML. We recently developed novel siRNA delivery polycations (PCX) based on AMD3100 (plerixafor), an FDA-approved inhibitor of the CXC chemokine receptor 4 (CXCR4). Inhibitors of CXCR4 have been shown to sensitize leukemia cells to chemotherapy. Therefore, PCX has the potential to target leukemia cells via two mechanisms: inhibition of CXCR4 and delivery of siRNAs against critical genes. In this report, we show that PCX exerts a cytotoxic effect on leukemia cells more effectively than other CXCR4 inhibitors, including AMD3100. In addition, we show that PCX can deliver siRNAs against the transcription factor RUNX1 to mouse and human leukemia cells. Overall, our study provides the first evidence that dual-function PCX/siRNA nanoparticles can simultaneously inhibit CXCR4 and deliver siRNAs, targeting key oncogenes in leukemia cells and that PCX/siRNA has clinical potential for the treatment of AML.

CXCL12 promotes proliferation of radial glia like cells after traumatic brain injury in rats.

To investigate the effect of CXCL12 on regeneration of radial glia like cells after traumatic brain injury (TBI). We randomly divided 48 rats into 4 groups: (1) the sham group, rats were performed craniotomy only, (2) the control group, saline were injected into the ipsilateral cortex after TBI, (3) the CXCL12 group, CXCL12 were injected, and (4) the CXCL12 + AMD3100 group, a mixture of CXCL12 and AMD3100 were injected. Seven days after TBI, the brain tissues were subjected to immunofluorescence double-labeled staining of BrdU/Nestin, BLBP/Nestin, BLBP/Vimentin, BLBP/SOX2, BLBP/CXCR4, BLBP/DCX. Western Blot assay was used to measure the levels of Nestin, BLBP, and Vimentin. Compared with the control group, CXCL12 treatment significantly increased the number of cells stained with BrdU/Nestin, BLBP/Nestin, and BLBP/Vimentin around the injured cortex and corpus callosum areas. CXCL12 + AMD3100 treatment significantly decreased the number of these cells compared with the CXCL12 treatment and control group. The protein levels of Nestin, BLBP, and Vimentin had the same change trends as those of the immunofluorescence staining. The BLBP/Vimentin positive cells presented with the astrocyte pattern around the injured cortex area but with the RGCs pattern around the injured corpus callosum area. The BLBP positive cells also expressed CXCR4 and SOX2. Altogether, CXCL12 promotes the proliferation of neural precursor cells after TBI by combing to its receptor, CXCR4. The proliferating neural precursor cells presents radial glial cell like cells. The RGCs-like cells can differentiate into immature neurons and promote the migration of immature neurons.