Normal Blood Cell Count Research Articles

Transcriptome characterization of immune suppression from battlefield-like stress

Transcriptome alterations of leukocytes from soldiers who underwent 8 weeks of Army Ranger training (RASP, Ranger Assessment and Selection Program) were analyzed to evaluate impacts of battlefield-like stress on the immune response. About 1400 transcripts were differentially expressed between pre- and post-RASP leukocytes. Upon functional analysis, immune response was the most enriched biological process, and most of the transcripts associated with the immune response were downregulated. Microbial pattern recognition, chemotaxis, antigen presentation and T-cell activation were among the most downregulated immune processes. Transcription factors predicted to be stress-inhibited (IRF7, RELA, NFκB1, CREB1, IRF1 and HMGB) regulated genes involved in inflammation, maturation of dendritic cells and glucocorticoid receptor signaling. Many altered transcripts were predicted to be targets of stress-regulated microRNAs. Post-RASP leukocytes exposed ex vivo to Staphylococcal enterotoxin B showed a markedly impaired immune response to this superantigen compared with pre-RASP leukocytes, consistent with the suppression of the immune response revealed by transcriptome analyses. Our results suggest that suppression of antigen presentation and lymphocyte activation pathways, in the setting of normal blood cell counts, most likely contribute to the poor vaccine response, impaired wound healing and infection susceptibility associated with chronic intense stress.

Demodicidosis: an uncommon erythema after cranio-maxillofacial surgery

Demodex mites are commonly found in the healthy population, but the pathogenesis of demodicidosis has still not been clarified, though it is usually found in cases of immune deficiency. A 45-year-old man presented with an unusual outbreak of erythema and swelling 6 months after resection and chemoradiotherapy for a squamous cell carcinoma of the anterior floor of the mouth. The cheek was biopsied and histological examination showed demodicidosis. In cases of erythema with a normal blood cell count and no history of allergy, particularly in patients with reduced immunity, demodicidosis should be considered as a diagnosis and should be confirmed by examination of a biopsy specimen.

Index of Suspicion

Index of Suspicion

Pancytopenia, mucositis, and hepatotoxicity after intralesional methotrexate injection in a patient treated with peritoneal dialysis

A case of methotrexate toxicity from intralesional treatment of squamous cell carcinoma of the hands in a peritoneal dialysis patient is reported. A 68-year-old Caucasian man receiving peritoneal dialysis for end-stage renal disease complained to his primary care physician of shortness of breath, mouth and hand sores, nausea, and general malaise. Four days before his office visit, the patient received 25 mg of methotrexate intralesionally for refractory squamous cell carcinoma of his hands. The patient had several pustular lesions on both hands. On admission, the patient had a normal complete blood cell count and elevated liver enzyme levels. On hospital day 3, the patient's white blood cell count was 1,300 cells/μL, platelet count was 134,000 platelets/μL, hemoglobin value was 12.4 g/dL, and hematocrit was 37%. Folic acid 1 mg i.v. every six hours and leucovorin 10 mg/m(2) (20 mg) i.v. every six hours was initiated. His serum methotrexate concentration was 0.03 μmol/L on hospital day 6. The leucovorin dosage was increased to 200 mg i.v. every six hours. Platelets were administered, and the patient was switched to four-hour treatments of high-flux membrane hemodialysis for two consecutive days. The patient was discharged on hospital day 14. Mild mucositis was still present, but the patient had improved substantially and was discharged to a rehabilitation facility. A 68-year-old peritoneal dialysis patient who was treated with 25 mg of intralesional methotrexate for squamous cell carcinoma of the hands developed pancytopenia, mucositis, and hepatotoxicity as a result of systemic absorption and prolonged elimination.

Normal White Blood Cell Count Does Not Rule Out Bacteremia

Despite multiple studies showing that a normal white blood cell (WBC) count does not exclude serious disease, physicians in all specialties continue to behave as if it did. To assess whether a normal WBC count or absence of fever reliably excludes bacteremia in patients with suspected infection, investigators …

Autologous Hematopoietic Recovery with Aberrant Antigen Expression after Allogeneic Bone Marrow Transplantation

A 50-year-old woman was admitted in December 2006 with progressive petechiae. Bone marrow (BM) aspiration showed massive proliferation of leukemic myeloblasts with myeloperoxidase staining. Three-color flow cytometry (FCM) with a CD45 gate for the BM cells was performed in our laboratory ; the blasts were positive for CD7, CD11c, CD13, CD15, CD33, myeloperoxidase and HLA-DR and negative for CD34 and CD117. Karyotypes of the BM cells were normal. A diagnosis of acute myeloblastic leukemia (AML)M2 was made on the basis of French-American-British classification. The patient needed standard induction chemotherapy twice to achieve complete remission. Refractoriness to platelet transfusions due to anti-HLA antibody developed. She received an allogeneic BM transplant from an ABOmatched and HLA-DR-mismatched unrelated female donor in September 2007. The conditioning regimen consisted of total body irradiation (2 Gy twice daily for 3 days) followed by cyclophosphamide (60 mg/kg/day for 2 days). On day 0, 4.0 × 10 BM cells per recipient body weight were infused. Tacrolimus and short-course methotrexate were used as prophylaxis for graft-versus-host disease. After BM transplantation, severe pancytopenia persisted. BM aspiration on day 22 revealed marked hypocellularity, suggesting graft rejection. Peripheral blood neutrophils gradually increased from day 50. BM aspiration smears on day 75 showed recovery of the BM cells, especially myeloid cells, without significant morphologic abnormalities and proliferation of myeloblasts, indicating that complete remission was maintained. Chromosomal analysis of the BM cells showed various cytogenetic abnormalities (Table 1). A chimerism-based analysis of the BM cells using short tandem repeat-polymerase chain reaction showed that 100% of the cells originated from the recipient. A diagnosis of autologous (recipient) hematopoietic recovery after graft rejection was made. Multiparametric FCM based on a four-color method (ReproCELL, Yokohama, Japan), which had been approved by the Jichi Medical University Institutional Review Board (no. 06-70), did not show the abnormal expression of antigens in the blasts in the BM on days 75 and 183. However, it showed small populations of CD34CD7 cells and CD34CD15 cells in the blasts on day 253 (Fig. 1). The latter cells were characterized by a high intensity of CD34, which indicates abnormal expression of the antigen. BM aspiration was performed every three to six months ; various chromosomal abnormalities of BM cells were found in each sample (Table 1). After day 253, routine three-color FCM instead of the multiparametric FCM was conducted to analyze phenotypes of the blasts in the BM. This FCM can detect CD34CD7 cells but not CD34CD15 cells because of antibody combinations. The proportions of CD34CD7 cells among the blasts of the BM were as follows : on day 323, 37.2% ; on day 421, 30.9% ; on day 603, 35.5% ; on day 785, 21.5% ; on day 975, 49.2% ; on day 1,149, 36.0% ; and on day 1,232, 24.5%. Although chromosomal abnormalities were detected on and after day 75, obvious dysplastic features associated with myelodysplastic syndrome (MDS) were not found. The patient is clinically well with normal peripheral blood cell counts. There are several reports on autologous (recipient) hematopoietic recovery after allogeneic hematopoietic stem cell transplantation. All patients received total body irradiation

A Novel Observation That Heme Oxygenase-1 (HO-1) Deficient Mice Are Easy Mobilizers and That HO-1 Plays An Important Role in Maintaining Expression of SDF-1 in Bone Marrow (BM) Stroma and Promotes Retention of Hematopoietic Stem/Progenitor Cells (HSPCs) in the Bone Marrow Microenvironment

Abstract 315Background. Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. Two distinct HO isoforms have been identified: HO-2, which is constitutively expressed, and HO-1, which is stress-responsive and plays an important function in various physiological and pathophysiological states associated with cellular stress. HO-1 plays a role in ischemic/reperfusion injury, atherosclerosis, and cancer. It has also been reported that HO-1 regulates expression of a-chemokine stromal derived factor-1 (SDF-1) in myocardium (J Mol Cell Cardiol.2008;45:44–55). Aim of study. Since SDF-1 plays a crucial role in retention and survival of hematopoietic stem cell/progenitor cells (HSPCs) in BM, we become interested in whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach. To address this issue, we employed several complementary strategies to investigate HO-1−/−, HO+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) chemotactic responsiveness of HSPCs to an SDF-1 gradient, iv) adhesiveness of clonogenic progenitors, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100 assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. Results: Our data indicate that under normal, steady-state conditions, HO-1−/− and HO+/– mice have normal peripheral blood cell counts and numbers of circulating CFU-GM. Interestingly, lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. Next, BMMNCs from HO-1−/−have normal expression of the SDF-1-binding receptor, CXCR4, but a 5-times lower level of CXCR7, which is another SDF-1-binding receptor. Of note, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1−/−animals responded more robustly to an SDF-1 gradient, and HO-1−/−animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into peripheral blood in response to G-CSF and AMD3100. Conclusions: Our data demonstrate for the first time that heme oxygenase plays an important and underappreciated role in BM retention of HSPCs and may affect their trafficking. Since small non-toxic molecular inhibitors of HO-1 have been developed for clinical use (e.g., metaloporhirins), blockage of HO-1 could be a novel strategy for mobilizing HSPCs. Our recent in vivo mobilization studies lend support to this hypothesis. Disclosures:No relevant conflicts of interest to declare.

Loss of Stem Cell Quiescence and Impaired Stem Cell Function of CD34+/CD38low Cells One Year Following Autologous Stem Cell Transplantation

Abstract 1893Autologous stem cell transplantation (ASCT) allows the application of high-dose chemotherapy and is included in the standard treatment regimens for multiple myeloma and relapsing lymphoma. The application of ASCT has considerably improved treatment outcome but in 30–50% of the patients the underlying malignant disorder relapses. In that case treatment options are limited, in part due to a diminished capacity of the transplanted bone marrow to tolerate chemotherapy. The transplanted bone marrow seems to be more vulnerable to chemotoxic stress. This is supported by our recent observations that progenitor cells from post-ASCT bone marrow demonstrate altered phenotypic and functional properties (Woolthuis et al. BMT 2011:110-115). In the present study, a more detailed examination of the hematopoietic stem cell (HSC) compartment post-ASCT was performed. The examined bone marrow cells were obtained from patients with normal peripheral blood cell counts one year after ASCT. First it was studied whether stem cell quiescence had been changed due to the ASCT procedure. Therefore the percentage of cells in the G0 phase was measured by staining the cells with Hoechst and Pyronin Y followed by flowcytometric analysis. Both the stem cell (CD34+/CD38low) and progenitor (CD34+/CD38+) fractions were analyzed. Post-ASCT bone marrow cells (n=6) were compared with normal bone marrow cells (n=9) and mobilized peripheral blood stem cells (PBSC) (n=7). Interestingly, post-ASCT bone marrow contained a significantly lower percentage of quiescent cells in the CD34+/CD38low fraction compared to normal bone marrow (mean percentage 23.6% (95%CI: 6.5–40.8) vs. 48.6% (95%CI: 31.4–65.9), p=0.045). In contrast no differences were observed in the CD34+/CD38+ fraction. To examine whether the loss of stem cell quiescence is associated with loss of stem cell function, stem cell frequency was analyzed in vitro by the long-term colony-initiating cell assay. These analyses revealed a strongly decreased stem cell frequency in post-ASCT CD34+ cells compared to normal CD34+ bone marrow cells (mean frequency 0.0016 (95%CI: 0.0003–0.0028) vs. 0.0206 (95%CI: 0.0162–0.0250), p=0.002). Importantly, CD34+ PBSC cells demonstrated a stem cell frequency comparable with the frequency observed in normal bone marrow, suggesting that the decrease in stem cell frequency observed in post-ASCT bone marrow can not simply be explained by the previous chemotherapy and the mobilization procedure. To obtain more insight into the mechanisms explaining our observations, levels of reactive oxygen species (ROS) were measured by flowcytometry. Comparable ROS levels were observed in post-ASCT and normal bone marrow CD34+/CD38low cells while the CD34+/CD38+ post-ASCT cells demonstrated significantly higher ROS levels compared to normal bone marrow (p=0.01). In addition significant higher ROS levels were observed in CD34+/CD38low PBSC compared to normal bone marrow (p=0.043). To define pathways associated with altered quiescence and/or ROS production, gene expression analysis was performed comparing CD34+ cells from post-ASCT bone marrow (n=6), normal bone marrow (n=31) and PBSC (n=5). Preliminary analysis reveals an upregulation in post-ASCT CD34+ cells of genes involved in cell cycle compared to normal bone marrow. In contrast, cell cycle genes appeared to be downregulated in CD34+ PBSC cells. In conclusion, our data indicate that the diminished regenerative capacity of bone marrow post-ASCT is likely related to a loss of stem cell quiescence and enhanced ROS production by progenitor cells which will make the bone marrow cells more vulnerable for the effects of cytotoxic stress. Disclosures:No relevant conflicts of interest to declare.

Laminin Expression Is Necessary for Maintenance of the Vascular Hematopoietic Niche

Abstract 217Laminins are heterotrimeric proteins that are the backbone for all basal laminas. They provide structural support, points for focal adhesion, and biochemical feedback for nearby cells. Laminins are found in many tissues, including bone marrow and lymphoid organs, and are detected in the discontinuous basal lamina of sinusoidal blood vessels. Laminin g1 is the predominant gamma subunit found in the bone marrow and lymphoid tissues. Here we show that the continuous expression of laminin in adulthood is necessary for viability of the vascular hematopoietic niche (sinusoidal endothelial cells). In the absence of laminin, this niche fails and hematopoiesis is impaired.To determine if sinusoidal laminin expression correlates with bone marrow failure and recovery, we used a well-established model of bone marrow failure. Wild type mice were treated with a sublethal dose of 5-fluoruracil (5-FU), and bone marrow was analyzed for disruption of sinusoid architecture and endothelial cell receptor and laminin expression. As described in other studies, shortly after treatment with 5-FU, bone marrow sinusoids became disorganized and hemorrhagic, and endothelial cells lost expression of VEGFR2. In addition, sinusoids lost their laminin-containing basal lamina. These changes correlated with bone marrow failure and onset of peripheral blood pancytopenia. As the sinusoids regained normal cytoarchitecture and VEGFR2 expression was normalized, sinusoid expression of laminin also returned to basal levels. With 5-FU-induced bone marrow failure and recovery, hematopoiesis correlated with laminin expression in the vascular hematopoietic stem cell niche.To test whether laminin expression directly affects the quality of the niche and hematopoiesis, a mutant mouse line was generated in which laminin g1 expression could be deleted by brief exposure to tamoxifen (TM). Control mice were littermates that lacked the TM-responsive Cre transgene. Prior to TM treatment, adult mutant and control mice had normal peripheral blood cell counts, and there was no evidence of gene recombination in genomic DNA samples. Adult mutant and control mice were then injected with either TM or vehicle, and the peripheral blood, bone marrow, spleen, and liver were collected after three weeks. Laminin g1 genes were deleted and laminin expression was lost in bone marrow, spleen, and liver sinusoids of only the TM-treated mutant mice. These mice also became thrombocytopenic and leukopenic. Flow cytometry showed early arrest of B-lymphocyte development within the bone marrow, bone marrow sinusoids became hemorrhagic, and the normal cytoarchitecture of the sinusoids and endothelial expression of VEGFR2 were lost. All other mouse groups were unaffected.These results suggest that continuous laminin expression is necessary for support of sinusoidal endothelial cells and the vascular hematopoietic niche. Disruption of the hematopoietic microenvironment is sufficient to alter blood cell development and release into peripheral circulation. These studies support the critical role of the microenvironment and stroma in supporting hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Development of a highly sensitive method for detection of JAK2V617F

BackgroundPh- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage. JAK2V617F, an activation mutation form of tyrosine kinase JAK2, is found in the majority of patients with MPNs. Studies have demonstrated that JAK2V617F can cause MPNs, and various methods have been developed to detect JAK2V617F for diagnostic purposes. However, a highly sensitive method is still needed for the earliest possible detection and for disease prevention and treatment.MethodsIn the present study, we developed a method dubbed restriction fragment nested allele-specific PCR (RFN-AS-PCR). The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the JAK2V617F mutation site, 2) digestion of the PCR products with restriction enzyme BsaXI which only cleaves the wild type allele, and 3) detection of JAK2V617F by allele-specific PCR with nested primers.ResultsWe tested the sensitivity of the method by using purified plasmid DNAs and blood cell DNAs containing known proportions of JAK2V617F. We were able to detect JAK2V617F with a sensitivity of 0.001%. We further analyzed blood cell DNA samples from 105 healthy donors with normal blood cell counts and found three JAK2V617F-positive cases, which would have remained undetected using a less sensitive method.ConclusionsWe have developed a highly sensitive method that will allow for detection of JAK2V617F at a very early stage. This method may have major implications in diagnosis and prevention of MPNs and related diseases.

Acute Chikungunya and persistent musculoskeletal pain following the 2006 Indian epidemic: a 2-year prospective rural community study

Chikungunya virus (CHIKV) data from population studies are sparse. During the 2006 epidemic, 509 clinical cases (43% attack rate) were identified in a village survey (West India); laboratory investigations demonstrated normal blood cell counts, elevated acute-phase reactants [erythrocyte sedimentation rate, C-reactive protein and interleukin-6 (IL-6)] and excluded malaria and dengue. Acute CHIKV was characterized by high fever, severe peripheral polyarthralgias, axial myalgias and intense fatigue in over 90% of cases; skin rash (34%) and headache (19%) were uncommon. There were 49% and 62% of survey cases seropositive for IgM (rapid assay) and IgG (immunofluorescence) anti-CHIKV antibodies, respectively. Sixty-five percent of cases recovered within 4 weeks. None of the cases died. Of the population, 4·1% and 1·6% suffered from persistent rheumatic pains, predominantly non-specific, at 1 and 2 years, respectively. Chronic inflammatory arthritis was uncommon (0·3% at 1 year) although serum IL-6 often remained elevated in chronic cases. A larger population study is required to describe post-CHIKV rheumatism and its prognosis.

Sphingosine-1-phosphate Lyase Deficiency Produces a Pro-inflammatory Response While Impairing Neutrophil Trafficking

Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.

Liver Transplantation for Massive Hepatic Lymphangiomatosis in a Child

Liver Transplantation for Massive Hepatic Lymphangiomatosis in a Child

Vasculitis, Vitiligo, Thyroiditis, and Altered Hormone Levels After Anti-Tumor Necrosis Factor Therapy

To the Editor: A 55-year-old Israeli man with a history of psoriatic arthritis presented with vitiligo (Figure 1). He received infliximab infusions at a dose of 5 mg/kg every 6 weeks for 2 years to manage his psoriatic arthritis. Twelve weeks prior to presentation in our clinic, his physician doubled the dose of infliximab from 400 to 800 mg. After this, vitiligo appeared on the face, groin, hands, and feet over a 2-week period. The patient related that 2 years before this he received 500 mg of infliximab instead of the 400 mg dose and he then experienced areas of depigmentation, but to a lesser degree. Figure 1. A 55-year-old man with vitiligo after several months of therapy with an anti-TNF drug. Laboratory tests showed normal blood cell count, biochemistry, urine test, and complement levels. Prior to the increase of infliximab, his primary care physician noted elevated thyroid-stimulating hormone (TSH) of 5.62, suggesting early hypothyroidism. Rheumatoid factor, cardiolipin antibody, native DNA antibody, SM/RNP antibody, Scl-70 antibody, and antibodies to Ro/La were all negative. His total IgG was elevated at 2238 mg/dl. He had early evidence of gynecomastia and decreased libido, and sex steroid levels were investigated. On Day 700 several weeks after discontinuation … Address correspondence to Dr. Lahita; E-mail: r.lahita{at}att.net

Endothelium in polycythemia secondary to obstructive lung disease

The object of this editorial is the report of Boyer et al in this issue of the Journal of Applied Physiology .It has two merits. One, endothelial function was studied in individuals with polycythemia secondary to obstructive lung disease, a condition not commonly addressed by cardiovascular investigators, although its presence is associated with a clearcut increase in cardiovascular risk .Two, the authors made use of virtually all techniques available in man to determine endothelial-dependent modulation of vascular tone, an unusual effort that differentiates this study from most other studies so far published. The results provide evidence that in polycythemic patients with obstructive lung disease the forearm vasoconstriction induced by L-NMMA administration was not reduced as compared to controls, while the brachial artery dilatation in response to post-ischemic hyperemia was increased.

Picture of the Month—Quiz Case

A PREVIOUSLY HEALTHY 6-YEAR-OLD BOY PREsented to the emergency department (ED) with 2 weeks of progressive right leg pain and a limp. He reported pain only when bearing weight or with extension of his right knee, although his parents noted he had been waking from sleep in pain. He denied a history of trauma and had not experienced joint swelling or erythema. He had not had fever, weight loss, or recent infections and confirmed normal voiding patterns. Our patient saw his pediatrician for this same concern 3 days before our evaluation and was found to have a minimally elevated erythrocyte sedimentation rate and normal lower extremity plain radiographs. In the ED, he was wellappearing and afebrile with normal vital signs for age. He held his right knee in mild flexion and refused to ambulate secondary to pain. The findings of a physical examination confirmed a full range of motion of the right hip and ankle. No joint instability, deformity, or swelling of his leg was noted, although he had mild swelling and tenderness over his right posterior iliac crest. Laboratory results were significant for C-reactive protein, 2.2 mg/L (to convert to nanomoles per liter, multiply by 9.524), erythrocyte sedimentation rate, 30 mm/h, and normal complete blood cell count with a normal manual differential cell count. Plain radiographs of the hip and right femur were obtained. (Figure 1) What is your diagnosis?

STAT3 Deletion In Hematopoietic Tissue Causes Mitochondrial Dysfunction with Increased ROS Leading to LT-Repopulating Stem Cell Depletion, Erythroid Dysplasia, Peripheral Neutrophilia, and a Rapid Aging-Like Phenotype.

AbstractAbstract 2621The essential transcription factor, Stat3, has been extensively studied in the context of cytokine signal transduction. Tissue-specific targeted deletion of STAT3 has been studied in heart, liver, and other tissues. In mouse hematopoietic tissue, STAT3 deletion (STAT3-KO mice) results in uniform post-natal death about 6–8 weeks after birth, although we show here that the pups are born with normal peripheral lymphoid and myeloid blood cell counts and pups appear normal until 4–5 weeks old. Then an aging-like shift in the lymphoid/myeloid ratio quickly occurs and the animals die within about 2 weeks of this shift. These animals suffer from intestinal inflammation, leukocyte infiltration in various tissues/organs, osteoporosis, dysfunctional innate immunity, myeloid cell overproduction and dendritic cell depletion. These findings are believed to be due, primarily, to abnormal cytokine signaling, however, the exact cause of death remains unknown. Stat3 has recently been implicated as a positive regulator of mitochondrial respiration and function. Mitochondrial function has been linked to aging and aging-related diseases like myelodysplastic syndromes (MDS), anemia, and erythroid dysplasia. It is therefore possible that Stat3 deletion in hematopoietic tissue results in disruption of mitochondrial regulation, which could also contribute to the observed phenotype of STAT3-KO mice. We have now investigated the function of mitochondria in the bone marrow stem/progenitor compartment of these mice as well as hematopoietic stem cell (HSC) repopulating ability and other cytokine responses. We show, for the first time, that STAT3 deletion causes pronounced erythroid dysplasia and anemia with erythrocyte hypochromasia and codocytes (target cells), aging-like lymphoid/myeloid ratio shift, hypersegmented neutrophilia, pronounced bone marrow accumulation of immature myeloid cells (bands), erythroblast depletion, and splenic hypertrophy and enlargement that appears to be due to erythroid and myeloid cell infiltration suggesting a shift in the primary site of erythropoiesis from the bone marrow to the spleen. Colony formation from bone marrow myeloid progenitors was unaffected when stimulated with single cytokines, but synergistic cytokine combinations failed to demonstrate proliferative synergy. HSC competitive repopulating ability was significantly diminished, especially in female STAT3 KO donors, but secondary transplants show that HSC self-renewal potential may not be affected, thus suggesting a reduced HSC number/function in STAT3 KO mouse bone marrow. This is consistent with the finding of significantly decreased numbers of CD34−SLAM+LSK cells. Mitochondrial mass was significantly increased in CD34+LSK subpopulation, but mitochondrial function (membrane potential, ΔΨm) was significantly diminished in STAT3 KO mouse LSK cells, which is consistent with the proposed positive role of Stat3 in mitochondrial function and also consistent with the finding of significantly increased ROS levels in bone marrow LSK subpopulations. Graded loss of Sca-1 surface marker (differentiation) in these cells was associated a with a graded increase in ΔΨm (activation); STAT3 KO mouse LSK cells displayed premature mitochondrial activation during differentiation to Sca-1− progenitors. These data show, for the first time, that in-vivo deletion of STAT3 in mouse hematopoietic stem/progenitor cells results in defective mitochondrial function with increased ROS production along with disrupted differentiation-driven mitochondrial biogenesis and activation in these cells. This likely contributes to an aging-like phenotype of the hematopoietic system with lymphoid/myeloid shift, anemia, non-sideroblastic erythroid dysplasia, myeloproliferative dysfunction, and diminished HSC number/function and raises the possibility that aberrant Stat3/mitochondria regulation could contribute to aging-associated diseases like MDS and progressive anemias. Disclosures:Broxmeyer:Corduse: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Combination of the histone deacetylase inhibitor valproic acid with oral hydroxyurea or 6-mercaptopurin can be safe and effective in patients with advanced acute myeloid leukaemia – a report of five cases

Disease-stabilizing therapy with the histone deacetylase inhibitor valproic acid and all-trans retinoic acid (ATRA) has been investigated in acute myelogenous leukemia (AML) in a number of trials. Experimental studies suggest that valproic acid induces a broad chemoresistant phenotype in human AML cells; however, clinical observations combining valproic acid with conventional therapy in a disease-stabilizing setting have not been reported that would confirm this as a clinical issue. We describe five patients receiving oral treatment with low-dose oral 6-mercaptopurin and/or hydroxyurea together with ATRA+valproric acid+theophylline. Hyperleukocytosis was controlled by low doses of the cytotoxic drugs, no unexpected toxicity appeared and the increases in normal peripheral blood cell counts induced by ATRA+valproic acid+theophylline were maintained during therapy. In two patients increasing blast counts later occurred during chemotherapy; a change to the alternative cytotoxic drug was then effective in controlling hyperleukocytosis. We conclude that valproic acid+ATRA+theophylline combined with 6-mercaptopurin or hydroxyurea can be safe and effective in palliative treatment of human AML.

Normal Red Blood Cell Count Reference Values in Chinese Presenile Women Given by Geographical Area

We aimed to standardize the normal reference value of red blood cell (RBC) counts in Chinese presenile women using an underlying scientific basis. This research was conducted to study the relationship between the normal reference value of 31,405 RBC samples from presenile women in eight different geographical areas in China. RBC counts were determined using a microscopic counting method. There was a significant correlation between geographical factors and the normal reference RBC value in presenile women (F = 187.82, p = 0.000). Using stepwise regression analysis, one regression equation was obtained. If geographical values are obtained in a certain area, the normal RBC reference value in presenile women in this area can be obtained using the regression equation.

Juvenile xanthogranuloma with hematological dysfunction treated with 2CDA‐AraC

Juvenile xanthogranuloma was diagnosed in two infants aged 8 and 2 months with skin lesions, hepatosplenomegaly, and pancytopenia. Disease control was not achieved with first-line vinblastine-steroid-VP16 combination therapy. Two courses of 2-chlorodeoxyadenosine (2CDA) (0.3 mg/kg) and cytosine arabinoside (AraC) (1 g/m(2)) were then administered for 5 days and were followed, after hematological recovery, by maintenance therapy. Both patients had normal complete blood cell counts and no signs of JXG, respectively, 31 and 24 months after diagnosis.