Prognostication refinement in NPM1-mutated acute myeloid leukemia stratified by FLT3-ITD status with different induction doses of cytarabine.

OBJECTIVE: We aimed to retrospectively discern the heterogeneity of outcomes from clinicopathological characteristics and next-generation sequencing (NGS) data in adult patients with NPM1-mutated (NPM1mut ) acute myeloid leukemia (AML) induced with standard-dose (SD, 100-200 mg/m2 ) and intermediate-dose (ID, 1000-2000 mg/m2 ) cytarabine arabinose (Ara-C). METHODS: In the entire cohort and FLT3-ITD subgroups, multivariate Logistic and Cox regression analyses were used to analyze the comprehensive complete remission (cCR) rate after one or two induction cycles, event-free survival (EFS), and overall survival (OS). RESULTS: Among a total of 203 NPM1mut patients evaluable for clinical outcome, 144 (70.9%) received a first SD-Ara-C induction and 59 (29.1%) received ID-Ara-C induction. Early death was recorded in seven (3.4%) after one or two cycles of induction. Focusing analysis on the NPM1mut /FLT3-ITD(-) subgroup, independent factors showing inferior outcome were presence of TET2 mutation [cCR rate, OR = 12.82 (95%CI 1.93-85.28), p = 0.008; EFS, HR = 2.92 (95%CI 1.46-5.86), p = 0.003], increasing age [EFS, HR = 1.49 (95%CI 1.10-2.02), p = 0.012 by every 10-years elevation], white blood cell count ≥60 × 109 /L [EFS, HR = 3.30 (95%CI 1.63-6.70), p = 0.001], and ≥4 mutated genes at initial diagnosis [OS, HR = 5.54 (95%CI 1.77-17.33), p = 0.003]. In contrast, when focusing on the NPM1mut /FLT3-ITD(+) subgroup, factors showing superior outcome were ID-Ara-C induction [cCR rate, OR = 0.20 (95%CI 0.05-0.81), p = 0.025; EFS, HR = 0.27 (95%CI 0.13-0.60), p = 0.001] and allo-transplantation [OS, HR = 0.45 (95%CI 0.21-0.94), p = 0.033]. Factors showing inferior outcome included CD34(+) [cCR rate, OR = 6.22 (95%CI 1.86-20.77), p = 0.003; EFS, HR = 2.01 (95%CI 1.12-3.61), p = 0.020] and ≥5 mutated genes [OS, HR = 2.85 (95%CI 1.33-6.10), p = 0.007]. CONCLUSION: We conclude that TET2(+) , age, and white blood cell count convey an outcome risk modulation for AML with NPM1mut /FLT3-ITD(-) , as does CD34 and ID-Ara-C induction for NPM1mut /FLT3-ITD(+) . The findings permit re-stratification of NPM1mut AML into distinct prognostic subsets to guide risk-adapted individualized treatment.

[Efficacy Analysis of Bendamustine-Based Combination Regimen in Treatment of Patients with Relapsed/Refractory Non-Hodgkin Lymphoma].

OBJECTIVE: To investigate the efficacy and safety of bendamustine combined with gemcitabine, vinorelbine，glucocorticoids (BeGEV)±X regimen in treatment of patients with relapsed/refractory non-Hodgkin lymphoma. METHODS: A total of 18 relapsed/ refractory non-Hodgkin lymphoma patients at the age of 18 years or older hospitalized in the First People's Hospital of Changzhou from March 2020 to March 2021 were selected. They received two or more cycles of BeGEV±X regimen. X could be anti-CD20 monoclonal antibody, PD-1-blocking antibodies, lenalidomide, BTK inhibitor, Bcl-2 inhibitor and so on according to patients' disease feature. The clinical efficacy and adverse effects were observed. RESULTS: In total, 18 patients completed two or more cycles of BeGEV±X regimen, including 14 with diffuse large B-cell lymphoma, one with low-grade follicular lymphoma, one with follicular lymphoma grade 3b, one with angioimmunoblastic T-cell lymphoma and one with peripheral T-cell lymphoma, not otherwise specified. 11 patients were male. The median age of the patients was 64 years old. 17 patients had modified Ann Arbor stage Ⅲ/Ⅳ disease. 13 patients had high- intermediate risk or high risk IPI score, while 15 patients had high-intermediate high risk or high risk NCCN-IPI score. 14 cases had extranodal sites of disease. And 6 cases had bulky disease. 12 patients experienced refractory disease, while 8 patients had received 3 line or more prior treatment. After two or three cycles of chemotherapy, the complete response rate was 6/18, the partial response rate was 3/18, and the objective response rate was 9/18. From the beginning of salvage chemotherapy to the end of follow-up, the median progression-free survival time was 130 days, and the median overall survival was 152 days. The most common grade 3 to 4 adverse events were hematologic toxicities, infection and febrile neutropenia. CONCLUSION: BeGEV±X is an effective salvage regimen in treatment of patients with relapsed/refractory non-Hodgkin lymphoma, while adverse events such as hematologic toxicities and infection should be closely monitored.

Role of CD19 and specific KIT-D816 on risk stratification refinement in t(8;21) acute myeloid leukemia induced with different cytarabine intensities.

High-dose cytarabine (Ara-C) has been reported with increased treatment-related mortality, whereas few data are available concerning intermediate-dose Ara-C for induction of acute myeloid leukemia (AML) with t(8;21) translocation. We retrospectively analyzed factors impacting complete remission (CR), event-free survival (EFS), cumulative incidence of relapse (CIR), and overall survival (OS) in 197 adults with t(8;21) AML, of whom 107 cases were induced with intermediate-dose and 90 with standard-dose Ara-C (as part of 3 + 7 protocol). After a single induction course, the overall CR rate was 87.6% (170/194), with a significant difference between the standard-dose (83/105, 79.0%) and intermediate-dose (87/89, 97.8%) groups (p < 0.001). Rather than general KITmut, the specific KIT-D816 independently led to a lower probability of achieving CR (HR = 3.29 [1.18-9.24], p = 0.023), worse EFS (HR = 3.53 [1.82-6.84], p < 0.001), and OS (HR = 5.45 [1.77-16.84], p = 0.003) in the standard-dose group, but not in the intermediate-dose group. CD19(+) represented the only independent factor predicting lower CIR both in the standard-dose group (HR = 0.32 [0.10-1.00], p = 0.050) and in the intermediate-dose group (HR = 0.11 [0.03-0.40], p = 0.001). When combined, KIT(+) plus CD19(-) conferred the most increased relapse risk (3-year CIR 60%; SE 0.12). Specific KIT-D816, instead of general KITmut, may be incorporated in prognostication model for t(8;21) AML. Combination of CD19 with KIT provides a more definite risk stratification profile for t(8;21) AML.

Clinical heterogeneity under induction with different dosages of cytarabine in core binding factor acute myeloid leukaemia.

Repeated cycles of post-remission high-dose cytarabine (Ara-C) have been suggested to improve survival in core binding factor (CBF) acute myeloid leukaemia (AML). High-dose Ara-C used for induction regimens has also been reported to be associated with increased treatment-related mortality (TRM). Few data are available about intermediate-dose Ara-C serving as induction therapy. The aim of our study was to compare the tolerance and outcomes of standard- and intermediate-dose levels of Ara-C as induction in CBF AML and to analyse the clinical heterogeneity of the two AML entities under these induction settings. We retrospectively investigated the outcomes in adults with CBF AML induced with regimens based on standard-dose Ara-C at 100 to 200 mg/m2 or intermediate-dose Ara-C at 1,000 mg/m2. In total, 152 patients with t(8; 21) and 54 patients with inv(16) AML were administered an induction regimen containing anthracyclines plus either standard- or intermediate-dose Ara-C. After a single course of induction, the complete remission (CR) rate in the inv(16) cohort was 52/52 (100%), higher than the 127/147 (86.4%) in the t(8; 21) cohort (P = 0.005). Intermediate-dose Ara-C (HR = 9.931 [2.135-46.188], P = 0.003) and negative KITmut (HR = 0.304 [0.106-0.874], P = 0.027) independently produced an increased CR rate in the t(8; 21) cohort. Positive CD19 expression (HR = 0.133 [0.045-0.387], P = 0.000) and sex (male) (HR = 0.238 [0.085-0.667], P = 0.006) were associated with superior leukaemia-free survival (LFS) in the t(8; 21) cohort independently of KITmut status or the induction regimen. We conclude that intermediate-dose Ara-C is superior to standard-dose Ara-C for induction of remission in t(8; 21) AML, and CD19 status and sex independently confer prognostic significance for LFS. The KITmut status alone does not have an independent effect on survival in t(8; 21) AML. More intensive induction therapy is unnecessary in inv(16) AML.

An outline for the pharmacological effect of icariin in the nervous system.

Icariin is a major active component of the traditional herb Epimedium, also known as Horny Goat Weed. It has been extensively studied throughout the past several years and is known to exert anti-oxidative, anti-neuroinflammatory, and anti-apoptotic effects. It is now being considered as a potential therapeutic agent for a wide variety of disorders, ranging from neoplasm to cardiovascular disease. More recent studies have shown that icariin exhibits potential preventive and/or therapeutic effects in the nervous system. For example, icariin can prevent the production of amyloid ß (1-42) and inhibit the expression of amyloid precursor protein (APP) and ß-site APP cleaving enzyme 1 (BACE-1) in animal models of Alzheimer's disease (AD). Icariin has been shown to mitigate pro-inflammatory responses of microglia in culture and in animal models of cerebral ischemia, depression, Parkinson's disease (PD), and multiple sclerosis (MS). Icariin also prevents the neurotoxicity induced by hydrogen peroxide (H2O2), endoplasmic reticulum (ER) stress, ibotenic acid, and homocysteine. In addition, icariin is implicated in facilitating learning and memory in both normal aging animals and disease models. To date, we still have no consolidated source of knowledge about the pharmacological effects of icariin in the nervous system, though its roles in other tissues have been reviewed in recent years. Here, we summarize the pharmacological development of icariin as well as its possible mechanisms in prevention and/or therapy of disorders afflicting the nervous system in hope of expanding the knowledge about the preventive and/or therapeutic effect of icariin in brain disorders.

The clinical characteristics and prognostic significance of AID, miR-181b, and miR-155 expression in adult patients with de novo B-cell acute lymphoblastic leukemia.

This study aimed to investigate clinical characteristics and prognostic significance of activation-induced cytidine deaminase (AID) gene, miR-181b and miR-155 expression in de novo adult B-cell acute lymphoblastic leukemia (B-ALL) patients. Results showed that AID and miR-155 expression were higher in B-ALL patients than healthy controls, while miR-181b expression was lower in B-ALL patients. In addition, Ph+ B-ALLs had higher AID expression than Ph- B-ALLs, and its high expression was associated with BCR-ABL. Moreover, B-ALL patients with AIDhigh or miR-181blow expression had a shorter overall survival (OS). AIDhigh with miR-181blow, AIDhigh with miR-155low, miR-181blow, miR-155low, AIDhigh with miR-181blow and miR-155low expression were associated with shorter OS. Combination of the three molecules are more accurate predictors for unfavorable OS compared with univariate group. Therefore, AID, miR-181b and miR-155 provide clinical prognosis of adult de novo B-ALL patients and may refine their molecular risk classification.

Low-dose decitabine plus all-trans retinoic acid in patients with myeloid neoplasms ineligible for intensive chemotherapy.

In our previous in vitro trials, decitabine and all-trans retinoic acid (ATRA) demonstrated synergistic effects on growth inhibition, differentiation, and apoptosis in SHI-1 cells; in K562 cells, ATRA enhanced the effect of decitabine on p16 demethylation, and the combination of the two drugs was found to activate RAR-ß expression (p16 and RAR-ß are two tumor suppressor genes). On the rationale of our in vitro trials, we used low-dose decitabine and ATRA to treat 31 myeloid neoplasms deemed ineligible for intensive chemotherapy. The regimen consisted of decitabine at the dose of 15 mg/m(2) intravenously over 1 h daily for consecutive 5 days and ATRA at the dose of 20 mg/m(2) orally from day 1 to 28 except day 4 to 28 in the first cycle, and the regimen was repeated every 28 days. After 6 cycles, decitabine treatment was stopped, and ATRA treatment was continued for maintenance treatment. Treated with a median of 2 cycles (range 1-6), 7 patients (22.6 %) achieved complete remission (CR), 7 (22.6 %) marrow CR (mCR), and 4 (12.9 %) partial remission (PR). The overall remission (CR, mCR, and PR) rate was 58.1 %, and the best response (CR and mCR) rate was 45.2 %. The median overall survival (OS) was 11.0 months, the 1-year OS rate was 41.9 %, and the 2-year OS rate was 26.6 %. In univariate analyses, age, performance status, comorbidities, white blood cell counts and platelets at diagnosis, percentage of bone marrow blasts, karyotype, and treatment efficacy demonstrated no impacts on OS (P > 0.05, each). Main side effects were tolerable hematologic toxicities. In conclusion, low-dose decitabine plus ATRA is a promising treatment for patients with myeloid neoplasms judged ineligible for intensive chemotherapy.

Allogeneic hemopietic stem cell transplants for the treatment of B cell acute lymphocytic leukemia.

OBJECTIVE: Explore the feasibility of allo- hemopietic stem cell transplants in treating patients with B cell acute lymphocytic leukemia. METHODS: Between september 2006 and February 2011, fifteen patients with B cell acute lymphocytic leukemia (ALL) were treated by allo-hemopietic stem cell transplants (HSCT). Stem cell sources were peripheral blood. Six patients were conditioned by busulfan (BU) and cyclophosphamide (CY) and nine patients were conditioned with TBI and cyclophosphamide (CY). Graft versus host disease (GVHD) prophylaxis regimen consisted of cyclosporine A (CSA), methotrex ate (MTX) and mycophenolatemofetil (MMF). RESULTS: Patients received a median of 7.98×108·kg⁻¹ (5.36-12.30×108·kg⁻¹) mononuclear cells (MNC). The median time of ANC>0.5×109/L was day 12 (10-15), and PLT>20.0×109/L was day 13 (11-16). Extensive acute GVHD occurred in 6 (40.0%) patients, and extensive chronic GVHD was recorded in 6 (40.0%) patients. Nine patients were alive after 2.5-65 months follow-up. CONCLUSION: Allogeneic stem cell transplant could be effective in treating patients with B cell acute lymphocytic leukemia.

Suppressive effects of simvastatin on the human acute promyelocytic leukemia NB4 cell line through the regulation of the nuclear factor-κB signaling pathway.

The present study examined the effects of simvastatin on the proliferation, apoptosis and gene expression levels involved in the nuclear factor-κB (NF-κB) signaling pathway in the human acute promyelocytic leukemia NB4 cell line by methyl thiazolyl tetrazolium assay, flow cytometry and the Human NF-κB Signaling Pathway RT2 Profiler™ PCR Array profiles. The results showed that simvastatin significantly inhibited proliferation and induced apoptosis of the NB4 cells in a time- and dose-dependent manner. Changes were noted in the expression levels of 56 genes involved in the NF-κB signaling pathways in the NB4 cells treated with 15 µm simvastatin at 48 h post-incubation, among which, 47 genes were downregulated and 9 were upregulated. In conclusion, simvastatin potentially inhibits the proliferation and induces the apoptosis of NB4 cells through the regulation of the expression levels of genes involved in the NF-κB signaling pathway.

Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors.

Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA(3) and HXB(3)). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB(3) at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA(3), or HXB(3) evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA(3) produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA(3) correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA(3) triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA(3)-evoked allodynia. These data indicate that spinal HXA(3) is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.

In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.

We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.

[Effects of simvastatin plus all-trans retinoic acid on WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4].

OBJECTIVE: To investigate the effects of simvastatin (SV) plus all-trans retinoic acid (ATRA) on the proliferation, differentiation, apoptosis and WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4. METHODS: The NB4 cell was incubated with simvastatin and ATRA alone or in combination. And the NB4 cell without any treatment was adopted as a normal control. The cells of different groups were collected at 24, 48 and 72 h post-incubation. Their morphological changes were observed after Wright staining. The method of MTT was employed to assay the growth inhibition rate and flow cytometry was used to detect the early-stage ratios of apoptosis and cell necrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the WT1/hDMP1 gene expression levels. RESULTS: The cell inhibition rates increased gradually (F = 7.15, P = 0.000) at 15, 10 and 5 µmol/L SV respectively. And so did the expression levels of CD11b (F = 3.41, P = 0.014) and Annexin-V (F = 43.38, P = 0.000). However the expression levels of WT1 decreased gradually (F = 5.35, P = 0.001) reversely with the elevated levels of hDMP1 (F = 22.61, P = 0.000). Furthermore the NB4 cell exhibited the most significant changes at 15 µmol/L SV. After a 72-hour incubation, the expression levels of CD11b (89.46% ± 9.13%)and hDMP1 (626.9 ± 56.9) in NB4 cells at 15 µmol/L SV plus 0.5 µmol/L ATRA were significantly higher than those with ATRA(71.27% ± 7.27%, P = 0.000 and 421.8 ± 38.3, P = 0.003 in each) and SV alone(62.41% ± 6.37%, P = 0.003 and 241.4 ± 21.9, P = 0.003 in each). A combination of 15 µmol/L SV with 0.5 µmol/L ATRA displayed obvious interactions with the expressions of CD11b and hDMP1 (F = 4.09, P = 0.025 and F = 29.58, P = 0.000 in each). And there was no significant interaction for cell inhibition rates and Annexin-V expression. CONCLUSION: Simvastatin in vitro inhibits the proliferation of NB4 cell, induces its differentiation and promotes its apoptosis. And the lowered expression of WT1 has a dose-dependent correlation with the elevated expression of hDMP1. It indicates that simvastatin has the synergistic in vitro anti-promyelocytic potency.

Spinal phosphinositide 3-kinase-Akt-mammalian target of rapamycin signaling cascades in inflammation-induced hyperalgesia.

Phosphinositide 3-kinase (PI3K), Akt, and their downstream kinase, mammalian target of rapamycin (mTOR), are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work, we identified the following characteristics of this cascade. (1) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. (2) Increased pAkt and pmTOR were prominent in neurons in laminae I, III, and IV, whereas pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. (3) Intrathecal treatment with inhibitors to PI3K or Akt attenuated Formalin-induced second-phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. (4) Intrathecal rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, intrathecal wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). (5) pAkt and pmTOR are expressed in neurokinin 1 receptor-positive neurons in laminae I-III after peripheral inflammation. Intrathecal injection of Substance P activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by intrathecal wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K-Akt-mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.

Release of prostaglandin E(2) and nitric oxide from spinal microglia is dependent on activation of p38 mitogen-activated protein kinase.

BACKGROUND: The spinal release of prostaglandins (PGs), nitric oxide (NO), and cytokines has been implicated in spinal nociceptive processing. Microglia represent a possible cell of origin for these proexcitatory mediators. Spinal microglia possess Toll-like receptor 4 (TLR4) and neurokinin 1 (NK1) receptors, and both receptors play a significant role in peripheral nerve injury- and inflammation-induced spinal sensitization. Accordingly, we examined the properties of the cascades activated by the respective targets, which led to the release of PGE(2) and an increase in nitrite (NO(2)(-)) (a marker of NO) from cultured rat spinal microglia. METHODS: Spinal microglia isolated from Sprague-Dawley neonatal rats were cultured with lipopolysaccharide (LPS) or substance P (SP) alone, with LPS in combination with SP, and with LPS in the presence of each inhibitor of cyclooxygenase (COX), NO synthase 2 (NOS2) or p38 mitogen-activated protein kinase (p38), or minocycline for 24 hours and 48 hours. Concentrations of PGE(2) and NO(2)(-) in culture supernatants were measured using an enzyme immunoassay and a colorimetric assay, respectively. RESULTS: Application of LPS (a TLR4 ligand, 0.1 to 10 ng/mL) to cultured microglia produced a dose- and time-dependent increase in PGE(2) and NO(2)(-) production, whereas no effects were observed after incubation with SP (an NK1 agonist, up to 10(-5) M) alone or in combination with LPS. Antagonist studies with SC-560 (COX-1 inhibitor) and SC-236 (COX-2 inhibitor) showed that LPS-induced PGE(2) release was generated from both COX-1 and COX-2. LPS-induced NO release was suppressed by 1400W, an inhibitor of NOS2. Minocycline, an agent blocking microglial activation, and SB203580, an inhibitor of p38, both attenuated the LPS-induced PGE(2) and NO release. The 1400W, at the doses that suppressed NO release, also blocked increased PGE(2) release. CONCLUSIONS: Our findings suggest that (a) activation of spinal microglia via TLR4 but not NK1 receptors produces PGE(2) and NO release from these cells; (b) the evoked PGE(2) release is generated by both COX-1 and COX-2, and (c) the COX-PGE(2) pathway is regulated by p38 and NOS2. Taken together with our previous in vivo work, the current findings emphasize that p38 in spinal microglia is a key player in regulating production of pronociceptive molecules, such as PGE(2) and NO.

Inflammatory hyperalgesia induces essential bioactive lipid production in the spinal cord.

Lipid molecules play an important role in regulating the sensitivity of sensory neurons and enhancing pain perception, and growing evidence indicates that the effect occurs both at the site of injury and in the spinal cord. Using high-throughput mass spectrometry methodology, we sought to determine the contribution of spinal bioactive lipid species to inflammation-induced hyperalgesia in rats. Quantitative analysis of CSF and spinal cord tissue for eicosanoids, ethanolamides and fatty acids revealed the presence of 102 distinct lipid species. After induction of peripheral inflammation by intra-plantar injection of carrageenan to the ipsilateral hind paw, lipid changes in cyclooxygenase (COX) and 12-lipoxygenase (12-LOX) signaling pathways peaked at 4 h in the CSF. In contrast, changes occurred in a temporally disparate manner in the spinal cord with LOX-derived hepoxilins followed by COX-derived prostaglandin E(2), and subsequently the ethanolamine anandamide. Systemic treatment with the mu opioid agonist morphine, the COX inhibitor ketorolac, or the LOX inhibitor nordihydroguaiaretic acid significantly reduced tactile allodynia, while their effects on the lipid metabolites were different. Morphine did not alter the lipid profile in the presence or absence of carrageenan inflammation. Ketorolac caused a global reduction in eicosanoid metabolism in naïve animals that remained suppressed following injection of carrageenan. Nordihydroguaiaretic acid-treated animals also displayed reduced basal levels of COX and 12-LOX metabolites, but only 12-LOX metabolites remained decreased after carrageenan treatment. These findings suggest that both COX and 12-LOX play an important role in the induction of carrageenan-mediated hyperalgesia through these pathways.

Role of spinal p38alpha and beta MAPK in inflammatory hyperalgesia and spinal COX-2 expression.

Pharmacological studies indicate that spinal p38 mitogen-activated protein kinase plays a role in the development of hyperalgesia. We investigated whether either the spinal isoform p38alpha or p38beta is involved in peripheral inflammation evoked pain state and increased expression of spinal COX-2. Using intrathecal antisense oligonucleotides, we show that hyperalgesia is prevented by downregulation of p38beta but not p38alpha, whereas increases in spinal COX-2 protein expression at 8 hours are mediated by both p38alpha and beta isoforms. These data suggest that early activation of spinal p38beta isoform may affect acute facilitatory processing, and both p38beta and alpha isoforms mediate temporally delayed upregulation of spinal COX-2.

Acetaminophen prevents hyperalgesia in central pain cascade.

Acetaminophen is an analgesic and antipyretic drug believed to exert its effect through interruption of nociceptive processing. In order to determine whether this effect is due to peripheral or central activity, we studied the efficacy of systemic (oral) and intrathecal (IT) application of acetaminophen in preventing the development of hyperalgesia induced through the direct activation of pro-algogenic spinal receptors. Spinal administration of substance P (SP, 30 nmol, IT) in rats produced a decreased thermal threshold, indicating centrally mediated hyperalgesia. Pretreatment of rats with oral acetaminophen (300 mg/kg), but not vehicle, significantly attenuated IT SP-induced hyperalgesia. Acetaminophen given IT also produced a dose-dependent (10-200 microg) antinociceptive effect. In addition, oral acetaminophen suppressed spinal PGE(2) release evoked by IT SP in an in vivo IT dialysis model. The ability of IT as well as oral acetaminophen to reverse this spinally initiated hyperalgesia emphasizes the likely central action and bioavailability of the systemically delivered drug. Jointly, these data argue for an important central antihyperalgesic action of acetaminophen.

Acute thrombosis in superior mesenteric artery as first symptom in a AML patient.

It is well known that acute leukemia may accompany thromboembolic events; even severe thrombocytopenia does not prevent thrombosis. Coagulation dysfunction is the major pathophysiological background for thromboembolism in these patients. Most thromboembolism is localized in venous vessels in acute leukemic patients and it happens rarely in the artery. We report a case of acute thrombosis in the superior mesenteric artery as the first symptom in a patient suffering from acute myeloid leukemia (FAB M4).

Inhibition of spinal constitutive NOS-2 by 1400W attenuates tissue injury and inflammation-induced hyperalgesia and spinal p38 activation.

Nitric oxide (NO) and its synthesizing enzymes, including NO synthase-2 (NOS-2, also called inducible NOS, iNOS), have been implicated in spinal nociception. 1400W is a highly selective NOS-2 inhibitor, as compared with either NOS-1 (neuronal NOS, nNOS) or NOS-3 (endothelial NOS). Here we examined the anti-nociceptive effects of intrathecal (IT) administration of 1400W in two experimental models of hyperalgesia (formalin and carrageenan models), in addition to the effect of 1400W on stimulation-induced activation of spinal p38 mitogen-activated protein kinase (p38). IT treatment of rats with 1400W produced a dose-dependent inhibition of paw formalin-induced phase II flinches, and attenuated carrageenan-induced thermal hyperalgesia. In contrast, IT injection of a selective inhibitor of NOS-1, nNOS inhibitor-I, had no effect in either model. Furthermore, 1400W at a dose that suppressed formalin-induced flinching behavior also blocked formalin-evoked p38 phosphorylation (activation) in the spinal cord, while nNOS inhibitor-I displayed no activity. The prompt effects of IT 1400W suggest involvement of constitutively expressed NOS-2 in spinal nociception. The NOS-2 protein in rat spinal cords was undetectable by Western blotting. However, when the protein was immunoprecipitated prior to Western blotting, NOS-2-immunoreactive bands were detected in the tissues, including naïve spinal cords. The presence of constitutive spinal NOS-2 was further confirmed by reverse transcriptase-polymerase chain reaction. Taken together, the present studies suggest that constitutively expressed spinal NOS-2 mediates tissue injury and inflammation-induced hyperalgesia, and that activation of p38 is one of the downstream factors in NO-mediated signaling in the initial processing of spinal nociception.

Descending serotonergic facilitation of spinal ERK activation and pain behavior.

Serotonin (5-HT) derived from bulbo-spinal projection is released by nociceptive input into the spinal dorsal horn. Here we report that formalin injection in the paw produced pain behavior (flinching) and phosphorylation of spinal ERK1/2 (P-ERK1/2, indicating activation) in rats. Depletion of spinal 5-HT by intrathecal (IT) 5,7-DHT, a serotonergic neurotoxin, profoundly reduced formalin evoked flinching and the increase in P-ERK1/2. Ondansetron (a 5-HT3 receptor antagonist) at IT doses that inhibited flinching also attenuated spinal ERK activation. These findings reveal that primary afferent-evoked activation of spinal ERK requires the input from an excitatory 5-HT descending pathway.