Bone Marrow Hyperplasia Research Articles

Anaplasma phagocytophilum and Ehrlichia muris induce cytopenias and global defects in hematopoiesis

Peripheral blood cytopenias result from a wide variety of infectious diseases and may significantly complicate the prognosis and outcome of those diseases. Infection with Gram-negative, LPS-negative members of the Anaplasmataceae family, including Anaplasma phagocytophilum and Ehrlichia muris, results in bi- or pancytopenia. The specific mechanisms responsible for the cytopenias are unknown. Viruses and bacteria have profound yet often discrepant effects on bone marrow (BM) and circulating numbers of blood cells. Multiple line-age cytopenias (bi- and pancytopenia) are a frequent sequel of viral infections [1]. Different viruses can directly infect BM stromal cells or haematopoietic stem cells and/or modulate cytokine production by accessory cells. Infections with Gram-negative, LPS-positive bacteria result in an endotoxin-mediated, acute phase, proinflammatory cascade of events that result in granulocyte efflux from the BM, peripheral neutrophilia and BM hyperplasia [2]. In contrast, gram-negative, LPS-negative bacteria, including members of the Anaplasmataceae family, elicit a very different, poorly-characterised, cascade of events. Haematologic alterations in both natural and experimental infection include a marked thrombocytopenia, moderate leukopenia (lymphopenia and neutropenia) and mild to moderate, non-regenerative anaemia [3,4]. Cytopenias vary from profound and irreversible (for example, dogs with chronic E. canis infection) to moderate and transient. Paradoxically, although Anaplasma family organisms display cell tropism for specific haematopoietic cell lineages, infection, regardless of aetiologic agent, host species or cell target, is characterised by bicytopenia or, occasionally, pancytopenia. This suggests the potential for more global haematopoietic disturbances. We hypothesised that the cytopenias may be due in part to bacteria-induced haematopoietic suppression. C57/Bl6 mice were infected intraperitoneally with E. muris and samples were collected on days 0–60 post-infection. C3H/HeN mice were infested with A. phagocytophilum-infected Ixodes scapularis nymphal ticks and sampled on days 0–21 post-infection. Blood samples were collected for a complete blood cell analysis and quantitative PCR for pathogen detection (A. phagocytophilum p44 DNA). BM was collected for haematopoietic colony-forming assays, cytology, histology and quantitative PCR. Cultured BM supernatants from A. phagocytophilum infected mice were collected for cytokine analysis. Thrombocytopenia and anaemia (bicytopenia) developed in both E. muris and A. phagocytophilum-infected mice. Leukopenia was variable. A. phagocytophilum infection induced significant decreases in BM proliferation and differentiation from day 4 to day 21 post-infection, concurrent with the cytopenias (Fig. 1). E. muris infection resulted in a similarly dramatic decrease in BM proliferation and differentiation on days 8 to 14 post-infection. Decreases in BM haematopoietic production included all lineages. Haematopoietic colony forming assays are supplemented with cytokines and assess haematopoietic stem cell/progenitor cell activity in the absence of stromal cells. As such, findings support infection-induced alterations in stem cell differentiation, proliferation or mobilisation. Both thrombocytopenia and myelosuppression were more severe in mice infected with A. phagocytophilum via tick bite compared with mice infected intraperitoneally in previous studies. Thrombocytopenia did not correlate with pathogen burden in blood, as assessed by quantitative PCR, but was strongly correlated with BM colony numbers in A. phagocytophilum-infected mice. This temporal association suggests that rapid changes in BM may be contributing to alterations in circulating platelet numbers. Cytologic evaluation of BM revealed infection-induced lymphocyte depletion and a reciprocal granulocytic hyperplasia in both infection models, similar to kinetics noted in other models of inflammation [5]. In addition, A. phagocytophilum infection resulted in BM production of myelosuppressive chemokines, including the murine interleukin-8 homologues, macrophage inflammatory protein-2 and KC. Findings are compatible with previous work that demonstrated a predominance of myelosuppressive chemokines in human BM after A. phagocytophilum infection [6]. Anaplasma family pathogens appear to perturb normal haematopoiesis, resulting in shifts in BM cell populations and transient failure of haematopoietic colony formation, which may contribute to peripheral blood cytopenias. Fig. 1 Bone marrow was collected from Anaplasma phagocytophilum-infected and uninfected mice on days 0–21 after tick-borne infection. An aliquot of BM was plated in duplicate and cultured in complete MethoCult media for 7–10 days (StemCell Technologies) ...

The Erlenmeyer flask bone deformity in the skeletal dysplasias

Erlenmeyer flask bone deformity (EFD) is a long-standing term used to describe a specific abnormality of the distal femora. The deformity consists of lack of modeling of the di-metaphysis with abnormal cortical thinning and lack of the concave di-metaphyseal curve resulting in an Erlenmeyer flask-like appearance. Utilizing a literature review and cohort study of 12 disorders we found 20 distinct disorders were associated with EFD. We interrogated the International Skeletal Dysplasia Registry (ISDR) radiographic database (1988-2007) to determine which skeletal dysplasias or syndromes were highly associated with EFD, whether it was a uniform finding in these disorders, and if forms of EFD could be differentiated. EFD was classified into three groups. The first catogory was the typical EFD shaped bone (EFD-T) resultant from absent normal di-metaphyseal modeling with relatively normal appearing radiographic trabecular bone. EFD-T was identified in: frontometaphyseal dysplasia, craniometaphyseal dysplasia, craniodiaphyseal dysplasia, diaphyseal dysplasia-Engelmann type, metaphyseal dysplasia-Pyle type, Melnick-Needles osteodysplasty, and otopalatodigital syndrome type I. The second group was the atypical type (EFD-A) due to absence of normal di-metaphyseal modeling with abnormal radiographic appearance of trabecular bone and was seen in dysosteosclerosis and osteopetrosis. The third group was EFD-marrow expansion type (EFD-ME) in which bone marrow hyperplasia or infiltration leads to abnormal modeling (e.g., Gaucher disease). Further, radiographic review determined that it was not always a consistent finding and that there was variability in both appearance and location within the skeleton. This analysis and classification aided in differentiating disorders with the finding of EFD.

Measurement of immature platelet fraction using the automatic hematology analyzer and its clinical utility

Objective The immature platelet fraction (IPF) could be detected quantificationally in Sysmex XE-2100 with the software of XE-pro and IPF master.The study aimed to perform the methodological evaluation of IPF detection and investigate the clinical significance for the monitoring for bone marrow hyperplasia in cancer chemotherapy patients.Methods The high-level, middle-level and low-level whole blood samples were randomly chosen for detection repeatly 20 times to obtain interrun coefficient of variation (CV) for evaluation of the precision and reproducibility. Integrated quality controls were determined for continuous 20 days to obtain intrarun CV, and the stability and carryover was investigated.Furthermore, the correlation between results from Sysmex XE-2100 and results from flow cytometry was assessed.182 healthy subjects and 130 cancer patients undergoing chemotherapy were selected and the latter were divided into two groups according to platelet counts after therapy, one was normal PLT group, the other was decreased PLT group.The IPF of either group was measured and was compared with each other.Results The precision of IPF for high-level, middle-level and low-level were 4.71%, 4.33% and 4.95%, respectively, they were less than 5%.The interrun CV of IPF detection for middle-level and low-level were less than 5%.The interrun CV of IPF detection for high-level were less than 10%.The carryover ranged from 0.6% to 2.7%,and the average rate was 1.2%.A good correlation for IPF detection was shown between results from Sysmex XE-2100 and flow cytometry(r = 0.880 9,P ＜ 0.01).Regarding clinical utility of IPF detection in treatment monitoring for chemotherapy effect, the median of IPF levels in decreased PLT group, normal PLT group,control group were 14.45% ,7.35% and 15.68%, respectively.There was significant difference among the three groups (H =49.032,P ＜0.01 ).The IPF level was higher in decreased PLT group than normal PLT group (t = -5.681, P ＜ 0.O1 ), and was lower in normal PLT group after chemotherapy than the control group (t = -6.662 ,P ＜0.01 ).Conclusions The determination of IPF by the Sysmex XE-2100 owns high precision and good stability. IPF is an effective marker for evaluation of thrombopoietic condition in the cancer chemotherapy patients. Key words: Platelet count; Blood cell count; Flow cytometry; Thrombopoiesis

Androgen ablation augments prostate cancer vaccine immunogenicity only when applied after immunization

Androgen ablation (AA) causes apoptosis of normal and neoplastic prostate cells. It is a standard treatment for advanced prostate cancer. Androgen ablation-mediated immunological effects include bone marrow hyperplasia, thymic regeneration, T and B cell lymphopoeisis and restoration of age-related peripheral T cell dysfunction. Androgens also regulate the transcription of several cytokines. Dendritic cells (DC) are the most potent antigen presenting cells that can activate antigen-specific naïve T cells. Despite myriad clinical trials involving DC-based prostate cancer immunotherapies, the effects of AA on DC function remain largely uncharacterized. Therefore, we investigated the effects of AA on DC and whether it could improve the efficacy of prostate cancer immunotherapy. Cytokine expression changes due to AA were quantified by multiplex ELISA. Flow cytometry was used to assess AA-mediated effects on DC maturation and expression of costimulatory markers. Mixed leukocyte reactions and cell-mediated lysis assays elucidated the role of androgens in DC function. The effect of AA on the efficacy of vaccination against a prostate tumor-associated antigen was tested using Elispot assays. Androgen ablation increased dendritic cell maturation and costimulatory marker expression, but had no effect on DC costimulatory function. However, DC isolated from castrated mice increased the expression of key cytokines by antigen-experienced T cells while decreasing their expression in naïve cells. Finally, androgen ablation improved immune responses to vaccination only when applied after immunization. Androgen ablation causes differential effects of DC on primary and secondary T cell responses, thus augmenting vaccine immunogenicity only when applied after immunization.

A modified canine model of portal hypertension with hypersplenism

Objective. The aim of this study was to develop and describe an experimental canine model of portal hypertension with hypersplenism. Material and methods. Twenty-five dogs were used randomly divided into three groups: group I (control group, n = 5), group II (PVS, n = 10) and group III (PVS + SVS, n = 10). Portal vein stenosis (PVS) was performed in dogs of group II; in group III dogs the model was first prepared by PVS and additional splenic vein stenosis 3 weeks later (PVS + SVS). Portal vein pressure (PVP), length of spleen and fluctuation of hematocyte counts were measured and recorded at the appointed times. Surgery permitted visual verification of portosystemic collateral circulation. Histopathological variation of the spleen and condition of the bone marrow hyperplasia were examined to confirm the development of hypersplenism. Result. Both group II and group III developed prehepatic portal hypertension; group III also presented satisfactory hypersplenism compared to the control group and group II, as documented at surgery and by hematologic and pathologic examination. Conclusions. Based on this study, the modified model of portal hypertension (by PVS + SVS) appears appropriate when studying the relationship between hypersplenism and hemodynamics in portal hypertension. It is also likely to be useful in studying the influence of diseased spleen in the treatment of portal hypertension.

Bone Mineral Density in Children with Sickle Cell Disease (SCD) Is Low and Not Related to Disease Severity, Vitamin D Status, or Bone Hyperresorption

Background: SCD is characterized by HbS polymerization, chronic hemolytic anemia, and recurrent acute painful episodes. Recent data have established that SCD patients have chronic inflammation, with endothelium activation related to hypoxia/reoxygenation cycles and reperfusion injury, increased inflammatory markers, and enhanced endothelium adhesion of red cells and leukocytes. The bone is a key target of SCD, being the site of both acute events (painful crises and infections) and chronic changes. Osteopenia and osteoporosis are reported in 30 to 40% of adults with SCD. By analogy with thalassemia, bone marrow hyperplasia secondary to chronic anemia has been hypothesized to be a causative factor. The prevalence of low bone mineral density (BMD) is not known in children.Aim: We assessed BMD in a cohort of SCD children and looked for correlations between BMD and age, gender, SCD severity, growth and pubertal development, serum vitamin D, calcium intake, and markers of bone turnover.Methods: Fifty-three children (45 SS, 4 SC, 4 Sb-thalassemia), 27 females, and 26 males, with a mean age of 12.8±2.4 y (9–19 y) were enrolled between 2002 and 2006. They originated from: West and Central Africa (n=41), the Caribbean (n=8), North Africa (n=3), and Brazil (n=1). We assessed height; weight; sexual maturation (Tanner); number of hospitalizations and of painful crises and of transfusions in the last 3 years; calcium intake; steady-state hemoglobin (Hb) and leukocyte count; calcemia, phosphatemia, calciuria/creatinuria; 25-OH D and PTH concentrations; osteocalcin, urinary deoxypyridinolin (DPD) and C-terminal component of pro-collagen type I (CTX). BMD was assessed using a dual X-ray absorptiometry (DXA).Results: In the overall population, mean lumbar spine z-score was −1.1±1.3 (−3.9–1.8), females had non-significantly lower values (−1.42±0.29) than males (−0.77±0.28). In prepubertal children, lumbar spine z-score was lower in females (−1.74±0.27) than in males (−0.53±0.31) (p=0.01). BMD did not correlate with hospitalization and transfusion episodes, Hb, or leukocytes counts. Hb and leukocyte count were inversely correlated (p=0.02) and leukocytosis was correlated to the number of hospitalizations (p<0.0001). Vitamin D deficiency (25-OH D<10 ng/ml) was found in 72% of patients and secondary hyperparathyroidism (PTH>46 pg/ml) in 38%. BMD was not related to calcium intake, vitamin D status, osteocalcin, or bone resorption markers. The normal or low osteocalcin and CTX values argue against increased bone resorption as the cause of the low BMD. Mean serum IGF concentrations measured in 7 children were low (134±98 ng/ml)Conclusion: A slight decrease of BMD was observed in SCD children, starting before puberty and being more marked in females. This low BMD was not related to the disease severity or to bone hyperresorption, suggesting that biphosphonates would be unhelpful. Neither was it related to vitamin D deficiency. The most likely cause is abnormal bone formation. Future research should focus on the mechanisms involved, with the goal of preventing osteoporosis in adults with SCD.

Mast Cells Are Involved by the Malignant Process and Play An Important Role in the Pruritogenesis in Patients with Myeloproliferative Disorders

The Philadelphia chromosome negative (Ph−) myeloproliferative disorders (MPD), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are thought to result from the transformation of a multipotent hematopoietic stem cell (HSC). These Ph− MPDs are characterized by trilineage bone marrow hyperplasia with increased production of red cells, granulocytes and platelets which largely determines their clinical manifestations. Pruritus is common in Ph− MPDs occurring in approximately 50% of cases. The itching occurs spontaneously or appears when taking a hot shower or following other sudden environmental changes. In the present study, we explored the hypothesis that mast cells (MCs) play an important role in pruritogenesis in Ph− MPDs. PB CD34+ cells from 18 PV, 11 PMF, and 7 G-CSF mobilized (Gmob) volunteers were cultured in the presence of SCF (100ng/ml) and IL-6 (50ng/ml) for 7–8 weeks. After 49 days of culture, Gmob PB CD34+ cells generated significantly greater numbers of MCs (range: 5.6–20.1×106; mean±SD: 11.3±5.2×106) than PV (0.5–3.8×106; 1.2±0.9×106; p<0.005) or PMF (0.5–18.5×106; 4.9±5.5×106; p<0.05) CD34+ cells. When MC colony forming assays were conducted with PB CD34+ cells from 13 PV, 7 PMF, 4 Gmob volunteers, the average number of MC colonies formed by PV (36.1±30.3/1×103 CD34+ cells), PMF (28.9±20.8) or Gmob (37.6±4.5) PB CD34+ cells were similar. Further analysis showed that cultured PV or PMF MCs underwent increased apoptosis in vitro. By using JAK2V617F, MplW515L and chromosomal abnormalities as markers of involvement by the malignant process, we demonstrated that MPD MCs were involved by the malignant clone in Ph− MPDs. In addition, JAK2V617F+ MC colonies were assayable from all of the 17 JAK2V617F+ MPD patients examined. Cultured MPD MCs exhibited an increased ability to migrate toward SCF and IL-8. To evaluate the role of MPD MCs in the pruritogenesis, we analyzed the production of pruritogenic MC mediators and cytokines including histamine, leukotrienes (LTs), prostaglandins D2 (PGD2), IL-4 and IL-31 by the cultured MPD and normal MCs. The release and/or production of histamine and leukotrienes (LTs) by cultured MPD MCs was significantly elevated as compared to those of normal MCs (p<0.05), while the level of an anti-pruritogenic factor, prostaglandins D2 (PGD2) was shown to be decreased following incubation at 42°C. The production of IL-31, a pruritogenic mediator known to be produced by activated T cells, was significantly higher in cultured MPD MCs and CD3+ T cells than MCs and T cells from healthy volunteers. However, the release of tryptase and IL-4 by MPD and normal MCs was shown to be similar. When the data obtained from MPD MCs were correlated with the degree of pruritus in the cohort of MPD patients studied, we found that pruritus was associated withthe generation of a greater number of MCs by CD34+ cells;a greater number of MC colonies formed by CD34+ cells;a lower percentage of apoptotic cells;a higher plasma levels of IL-31; anda lower levels of PGD2 released by cultured MCs.Erlotinib, a JAK2 inhibitor, was able to inhibit the ability of JAK2V617F+ PV CD34+ cells to form MC colonies, indicating that abnormal MC progenitors are a potential cellular target for JAK2 inhibitors. These studies not only provide direct evidence for the clonal involvement of MCs in MPDs, but also supply evidence suggesting that abnormal MCs play a crucial role in pruritogenesis in patients with MPDs by producing higher levels of pruritogenic and/or decreased levels of anti-pruritogenic factors. These studies might lead to the identification of cellular and molecular targets for the development anti-pruritus drugs for patients with Ph− MPDs.

랫드를 이용한 Acetanilide의 반복투여 및 생식/발생독성 병행시험

The study was conducted to assess the repeated dose and reproduction and developmental toxicities of acetanilide, an intermediate for drug production, as a part of OECD Screening Information Data Set (SIDS) program. The test agent was administered by gavage at dose levels of 0, 22, 67, 200 and 600 mg/kg to Sprague-Dawley rats (12/group/sex) during pre-mating and mating period for males(up to 30 days) and females and pregnancy and early lactation period for females (up to 39-50 days). At 22 mg/kg, decreases in HGB, HCT (males) and MCHC (females), hyperplasia of spleen red pulp, hyperplasia of femur bone marrow (both sexes) were observed. At 67 mg/kg, salivation (males), reduced food consumption (both sexes), decreases in RBC, HGB, HCT and MCHC (males), increases in MCV (males) and spleen weight (males), hyperplasia of spleen red pulp and femur bone marrow (both sexes) were observed. At 200 mg/kg, decreases in locomotor activity and salivation (both sexes), reduced food consumption (both sexes), decreases in RBC, HGB, HCT and increases in MCV, MCH, BUN, T-BIL (males), enlargement of spleen (both sexes), increased weight of spleen (males), hyperplasia of spleen red pulp and femur bone marrow and extramedullary hematopoiesis of liver (both sexes), atrophy of thymus and corpus luteum hyperplasia of ovary (females) were observed. At 600 mg/kg, decreases in locomotor activity, cyanosis (both sexes), reddish tear, and salivation (males), mortality (4 out of 12 females), decreased body weight (females), reduced food consumption (both sexes), decreases in RBC, HGB, HCT and MCHC and increases in WBC, MCV, MCH, reticulocyte, neutrophil, lymphocyte, monocyte, AST, ALT, BUN, T-BIL, ALB, Ca and A/G ratio (males), enlargement of spleen, increased weights of spleen (both sexes), liver (males), kidney and ovary, decreased weights of thymus (females), hyperplasia of spleen red pulp, hyperplasia of femur bone marrow and extramedullary hematopoiesis of liver (both sexes), and atrophy of thymus and corpus luteum hyperplasia of ovary (females) were observed. Regarding the reproduction and development toxicities, there were no treatment-related changes in precoital time, mating index, fertility index and pregnancy index at all doses tested. At 22 and 67 mg/kg, there were no adverse effects on all the parameters observed. At 200 mg/ kg, decreased body weight of pups (day 4 p.p.) were observed. At 600 mg/kg, decreased body weight of pups (day 0 and 4 p.p.) and viability index (day 4 p.p.), increased incidence of newborns dead or with abnormal clinical signs were observed. The results suggest that the NOAELs for general toxicity are < 22 mg/kg, LOAELs are 22 mg/kg and the NOAELs for reproductive toxicity are 67 mg/kg.

Magnetic resonance imaging of the lumbar spine and blood volume in professional cyclists

Stimulated hematopoiesis is observed in the bone marrow of endurance-trained athletes by magnetic resonance imaging (MRI) independent of the hemoglobin concentration (hematocrit or erythrocyte count) or circulating reticulocytes, but may be related to blood volume (BV). This study determined whether hyperplasia of hematopoietic bone marrow of professional cyclists correlates with their red cell volume (RCV). Twelve male professional cyclists (mean +/- SD; 20.2 +/- 1.4 years, 69.0 +/- 4.5 kg, VO2max 64.4 +/- 4.6 ml/min/kg, BV 7257 +/- 884 ml, RCV 2990 +/- 299 g) completed an MRI of the lumbar spine, a total BV determination using a CO-rebreathing method and a graded exercise testing within two consecutive days. Significant correlations were found between performance and BV data. A significant correlation existed also between the signal intensity of the Turbo inversion-recovery sequence with short inversion time (Turbo-STIR) and BV (r (2) = 0.47, P < 0.05), RCV (r (2) = 0.56, P < 0.05) and plasma volume (r (2) = 0.39, P < 0.05) per kilogram body mass. The present study provides evidence of stimulated erythropoiesis with hyperplasia of the hematopoietic bone marrow of endurance athletes explaining their large RCV.

Induction of thyroid and liver tumors by chronic exposure to 2-methylimidazole in F344/N rats and B6C3F1 mice

2-Methylimidazole (2MI) has been identified as a by-product of fermentation and is detected in foods and mainstream and side-stream tobacco smoke. It is used in the manufacture of pharmaceuticals, photographic chemicals, dyes and pigments, agricultural chemicals, and rubber. Carcinogenicity studies of 2MI were conducted because of its high potential for human exposure and a lack of carcinogenicity data. Groups of male and female Fischer 344/N rats were fed diets containing 0, 300, 1,000, or 3,000 ppm (males) or 0, 1,000, 2,500, or 5,000 ppm (females) 2MI for 106 weeks and groups of male and female B6C3F1 mice were fed 0, 625, 1,250, or 2,500 ppm 2MI for 105 weeks. Animals in each group were sacrificed at 8 days, 14 weeks, and 6 months for determinations of serum thyroid hormone and liver enzyme levels and histopathological examinations and at 2 years for evaluations of neoplastic lesions. In rats, 2MI administration reduced serum thyroxine and triiodothyronine and increased thyroid stimulating hormone levels. 2MI administration also increased total hepatic UDP-glucuronosyltransferase levels. At 2 years, the incidences of thyroid follicular cell hyperplasia, adenoma or carcinoma (combined), as well as follicular mineralization were increased. The incidences of hepatocellular adenoma or carcinoma (combined) in the two highest dose groups of males and females were also increased. The incidences of mixed cell focus in males and females were also significantly increased. In mice, the incidences of thyroid follicular cell hypertrophy and hyperplasia were significantly increased in the high dose males and females. The incidence of thyroid follicular cell adenoma in the 2,500 ppm males was significantly greater than that in the control group. The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in all exposed groups of males and in the 2,500 ppm females. Significant increases in incidences were also observed in spleen hematopoietic cell proliferation in both sexes and bone marrow hyperplasia, chronic active inflammation of the epididymis, sperm granuloma, and germinal epithelial atrophy of the testis in males. Under these experimental conditions, carcinogenic activity of 2MI was demonstrated in male and female rats and mice.

Verification of Extensive Bone Marrow Hyperplasia Using Magnetic Resonance Imaging

Bone marrow hyperplasia of the rib, lumbar spine, knee, and iliac crest is frequently encountered in patients undergoing magnetic resonance imaging (MRI) examination. In this study, we investigated extensive areas of bone marrow hyperplasia of the ascending mandibular ramus, posterior to the inferior alveolar nerve, the size of the lesions ranging from 5 to 7 cm. In a prospective study, an orthopantomography (OPT) was performed in 15,412 patients. Seven patients in the study group (0.05%) showed extensive bone marrow hyperplasia in the ascending mandibular ramus. The diagnosis derived by means of the OPT was confirmed by T1-weighted MRI with and without fat signal suppression and was finally proved by means of the histological findings from a bone biopsy. This investigation shows that a T1-weighted MR image with and without fat signal suppression shows a typical heterogeneous distribution of signal intensity with a unique pattern not found in other lesions. The MRI-based diagnosis in our study group was always confirmed by means of a bone biopsy performed via a transbuccal approach. We conclude that properly selected MRI studies are by themselves adequate to diagnose these lesions and that these imaging studies supplant the need for a bone biopsy.

Myofasciitis in the Domestic Ferret

Since late 2003, an inflammatory disease of muscle and fascia has been diagnosed in several ferrets at Northwest ZooPath, and this report describes the condition in 17 ferrets. It is a disease of young ferrets, characterized by rapid onset of clinical signs, high fever, neutrophilic leukocytosis, treatment failure, and death (or euthanasia). Gross lesions include atrophy of skeletal muscle; red and white mottling and dilatation of the esophagus; and splenomegaly. Histologically, moderate to severe suppurative to pyogranulomatous inflammation is in the skeletal muscle and the fascia at multiple sites, including esophagus, heart, limbs, body wall, head, and lumbar regions. Myeloid hyperplasia of spleen and/or bone marrow also is a prominent feature. Ultrastructural lesions include mitochondrial swelling, intracellular edema, disruption of myofibrils and Z bands. Bacterial and viral cultures, electron microscopy, immunohistochemistry, and polymerase chain reaction were negative for a variety of infectious agents. The clinical presentation and distribution of lesions suggests that polymyositis in domestic ferrets is likely a distinct entity. The etiopathogenesis if this condition is not known.

JAK2 V617F Mutation Is Infrequent in “5q-Syndrome” and 5q-Associated MDS.

Background: V617F mutation in Janus Kinase 2 (JAK2) gene has been found in chronic myeloproliferative disorders (MPD) including polycythemia vera (90%), essential thrombocythemia and chronic idiopathic myelofibrosis (30–50%), and occasionally in myelodysplastic syndromes (MDS). “5q- Syndrome” is a MDS that shares features with MPD and characterized by an atypical megakaryocytic hyperplasia in bone marrow and usually thrombocytosis in peripheral blood. The most common deleted region for this syndrome is 5q13.3q33.1. An interstitial deletion with variable proximal (5q12-14) and distal (5q31-33) breakpoints has been found in other MDS with/without additional chromosomal abnormalities beyond “5q- Syndrome”. To date, JAK2 mutation was detected in 6/97(6.2%) of patients having diagnosis of MDS with “5q- Syndrome”.Design: In our study 21 MDS patients (10 with “5q- Syndrome” and 11 MDS with isolated or complex 5q-) whose diagnosis by both bone marrow aspiration/biopsy and conventional chromosomal analysis were confirmed.Materials and Method: Primers were created to amplify a 460 bp fragment containing the site of JAK2 V617F mutation. Forty-five cycles of PCR were performed at an annealing temperature of 57°C. Resulting PCR product was digested with 2 U BsaXI for 16 hours and with an additional 2 U BsaXI for another 16 hours at 37°C, then analyzed on a 2% agarose gel. The mutant allele remained undigested whereas the wild-type allele was digested into 241 bp, 189 bp and 30 bp fragments. All experiments included a positive (HEL cells) and negative (K562 cells) control.Results: PCR results showed clear wild type PCR patterns in all 21 cases.Conclusion: No JAK2 mutations were detected in 21 patients either with “5q- Syndrome” or other 5q- associated MDS suggesting that JAK2 mutations are infrequent in these MDS patients.

Detection and Characterization of Naturally Acquired West Nile Virus Infection in a Female Wild Turkey

An adult female wild turkey exhibiting disorientation and failure to flee when approached was submitted to the Mississippi Veterinary Research and Diagnostic Laboratory. Gross pathologic examination revealed evidence of dehydration and the presence of modest numbers of adult nematodes in the small intestine. Histologic examination revealed extensive multifocal perivascular lymphocytic infiltration in brain, marked heterophilic hyperplasia in bone marrow, and multifocal interstitial lymphocytic infiltration in heart, pancreas, ventriculus, and skeletal muscles. West Nile virus (WNV) was isolated from the brain, lung, and kidney tissues using cultured Vero cells. Higher copies of viral RNA were detected from brain, lung, and kidney than from heart, liver, or spleen by quantitative real-time reverse transcription-polymerase chain reaction (RRT-PCR) analysis. Immunohistochemical (IHC) analysis detected WNV antigen in various tissues including neurons, kidney, respiratory tract epithelium, heart, and bone marrow. On the basis of the data from this investigation, it is concluded that WNV caused encephalitis along with many other pathologic changes in the affected wild turkey.

Stroke induced by splenectomy in hemoglobin Madrid: autopsy clues to the underlying mechanism

Thrombo-embolic events, which can be increased after splenectomy in hemoglobin disorders, can cause potentially lethal complications. Although venous thrombosis has been reported, arterial strokes are rarely reported. A case of stroke in a 52-year-old patient with a previously performed splenectomy for known hemoglobin Madrid, an extremely unstable hemoglobinopathy, led us to investigate the possible causal role of splenectomy. The patient had no history of hypertension, diabetes mellitus, smoking, or other vascular risk factors--but upon autopsy, thrombotic angiopathy was observed in multiple organs, including the lung, liver, kidney, coronary artery and brain. Bone marrow hyperplasia was also observed. A thrombotic middle cerebral artery territory infarction appears to have been caused by chronic recurrent thrombosis, which may have been a result of the splenectomy for unstable hemoglobinopathy. This case supports that splenectomy be strongly considered as an uncommon risk factor for stroke.

Genetic toxicity and carcinogenicity studies of glutaraldehyde?a review

Glutaraldehyde is a high production volume chemical that is highly reactive and has many medical and industrial uses. The majority of human exposures are via inhalation, but the exposure is not widespread. It has been extensively tested for genetic activity in vitro and in vivo, and there is disagreement in the literature with regard to glutaraldehyde's genetic activity. Glutaraldehyde produced DNA damage in bacteria and some cultured mammalian cell systems. In vitro, it was mutagenic in Salmonella and E. coli, produced inconsistent positive responses in mammalian cells, weak and inconsistent responses in chromosome aberration and SCE studies, and did not induce transformation in cultured SHE cells. In vivo, inhalation of glutaraldehyde induced cell proliferation in nasal tissue in rats and mice, but DNA damage and UDS were not induced at these sites in rats. Chromosome aberrations in bone marrow cells were reported in only one of eight studies using rats and mice, micronuclei were not induced in bone marrow cells of mice, and dominant lethal mutations were not induced in mice. Glutaraldehyde did not induce cell transformation in SHE cells in vitro. Bone marrow hyperplasia and low, but statistically significant, levels of leukemia were seen in one chronic drinking water study in rats, but not in a chronic inhalation study in rats or two chronic inhalation studies in mice.

Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis

AbstractTo develop murine models of leukemogenesis, a series of transgenic mice expressing BCR-ABL in different hematopoietic cell subsets was generated. Here we describe targeted expression of P210 BCR-ABL in stem and progenitor cells of murine bone marrow using the tet-off system. The transactivator protein tTA was placed under the control of the murine stem cell leukemia (SCL) gene 3′ enhancer. Induction of BCR-ABL resulted in neutrophilia and leukocytosis, and the mice became moribund within 29 to 122 days. Autopsy of sick mice demonstrated splenomegaly, myeloid bone marrow hyperplasia, and extramedullary myeloid cell infiltration of multiple organs. BCR-ABL mRNA and protein were detectable in the affected organs. Fluorescence-activated cell sorter (FACS) analysis demonstrated a significant increase in mature and immature myeloid cells in bone marrow and spleen, together with increased bilineal B220+/Mac-1+ cells in the bone marrow. tTA mRNA was expressed in FACS-sorted hematopoietic stem cells expanded 26-fold after BCR-ABL induction. Thirty-one percent of the animals demonstrated a biphasic phenotype, consisting of neutrophilia and subsequent B-cell lymphoblastic disease, reminiscent of blast crisis. In summary, this mouse model recapitulates many characteristics of human chronic myeloid leukemia (CML) and may help elucidate basic leukemogenic mechanisms in CML stem cells during disease initiation and progression. (Blood. 2005;105:324-334)

A Case of Transition of Polycythemia Vera to Chronic Neutrophilic Leukemia

Chronic Neutrophilic Leukemia (CNL) is a rare myeloproliferative disorder characterized by a persistent increase of mature peripheral neutrophils, myeloid hyperplasia in bone marrow, hepatosplenomegaly, elevated neutrophil alkaline phosphatase (NAP) and absence of Philadelphia chromosome, with no evidence of infection or malignancy sufficient to mimic a leukemoid reaction. CNL has been associated with multiple myelomas in many reported cases, but transition of Polycythemia Vera (PV) to CNL is very rare.An 81-year-old female patient, who had undergone intermittent phlebotomy following the diagnosis of PV 8 years previously, was admitted to our hospital due to lower back pain. A physical examination showed a splenomegaly 2cm below the costal margin, with tenderness of the thoracic and lumbar spine area. A peripheral blood examination showed a WBC count of 91,800/μL (neutrophil 88%) with a rare immature form, hemoglobin of 9.1 g/dL and a platelet count of 1,661,000/μL. Her NAP score was 58. The bone marrow examination showed 95% cellularity, with an M:E ratio of 10:1, increased megakaryocytes with normal morphology and the absence of myelofibrosis. Chromosomal studies showed no Philadelphia chromosome. A radiological examination showed compression fractures of the vertebrae and spinal cord compression. No underlying disease causing a leukemoid reaction was detected. With iron replacement, the hemoglobin level failed to increase over 12 g/dL. Therefore, it was concluded to be a transition of PV to CNL. After administration of hydroxyurea and vertebroplasty, the symptom improved and the WBC count was sustained below 40,000/μL.

Chlorpropham-induced splenotoxicity and its recovery in rats

To determine the reversibility of hematological and pathological changes in spleen induced by sub-chronic administration of chlorpropham (CIPC), male F344 rats were given CIPC in the diet at 0, 600, 3000 or 15,000 ppm for 13 weeks (administration period) and then were given standard (0 ppm) diet for 10 weeks (recovery period). At 0, 1, 2, 4 or 10 weeks in the recovery period, 5 rats in each groups were examined for hematology and pathology. At the end of CIPC administration, dose-dependent and significant methemoglobinemia, anemia, splenomegaly and pathological lesions indicating hemolytic anemia were observed in all the treated groups. The hematological changes, congestion of red pulp, lymphoid atrophy, increased extramedullary hematopoiesis in spleen and hematopoietic cell hyperplasia in bone marrow were diminished during the 10 weeks recovery period. However, increased hemosiderin deposition and capsular fibrosis in spleen of the treated groups remained at the end of recovery period. The results indicated that hematological changes induced by sub-chronic administration of CIPC were reversible but hemosiderin deposition and fibrosis in spleen were not reversible in the recovery period examined, suggesting the significance of splenic lesion in CIPC-toxicity.

Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease.

Oncogenic ras alleles are among the most common mutations found in patients with acute myeloid leukemia (AML). Previously, the role of oncogenic ras in cancer was assessed in model systems overexpressing oncogenic ras from heterologous promoters. However, there is increasing evidence that subtle differences in gene dosage and regulation of gene expression from endogenous promoters play critical roles in cancer pathogenesis. We characterized the role of oncogenic K-ras expressed from its endogenous promoter in the hematopoietic system using a conditional allele and IFN-inducible, Cre-mediated recombination. Mice developed a completely penetrant myeloproliferative syndrome characterized by leukocytosis with normal maturation of myeloid lineage cells; myeloid hyperplasia in bone marrow; and extramedullary hematopoiesis in the spleen and liver. Flow cytometry confirmed the myeloproliferative phenotype. Genotypic and Western blot analysis demonstrated Cre-mediated excision and expression, respectively, of the oncogenic K-ras allele. Bone marrow cells formed growth factor-independent colonies in methylcellulose cultures, but the myeloproliferative disease was not transplantable into secondary recipients. Thus, oncogenic K-ras induces a myeloproliferative disorder but not AML, indicating that additional mutations are required for AML development. This model system will be useful for assessing the contribution of cooperating mutations in AML and testing ras inhibitors in vivo.