Spleen Histology Research Articles

Dragon’s blood and its extracts attenuate radiation-induced oxidative stress in mice

Dragon's blood (DB) possesses great medicinal values due to the presence of several phenolic compounds. This study was designed to investigate the effects of DB and its extracts (DBEs) on oxidative stress in mice exposed to whole body 60Co-γ irradiation (4 Gy). DB and DBEs were intragastrically administered to mice for 5 d prior to radiation. The antioxidant activities, including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels in liver and spleen were measured using kits. Furthermore, DB and DBE effects were determined by organ indices and histology of liver and spleen. Our results indicated that the DB and DBE-treated groups showed a significant decrease (P < 0.05) in levels of MDA in liver and spleen compared with the irradiation-only group. Moreover, the activity of SOD, CAT and the level of GSH in liver and spleen tissue were enhanced significantly (P < 0.05) in the DB and DBE groups. DB and DBE also had a significant effect on the recovery of thymus indices. The histological observations of groups having treatment with DB and DBE indicated significant reduction in the radiation-induced damage to the liver and spleen, together with improvement in the morphology of the liver and spleen. These results suggest that DB and DBE treatment prevents radiation-induced oxidative stress injury and restores antioxidant status and histopathological changes in the liver and spleen, but there is need for further study to explore the precise molecular mechanism and strategy for optimal practical application of DB and DBE.

Effects of saffron (Crocus sativus) petal ethanolic extract on hematology, antibody response, and spleen histology in rats.

Saffron petal is a by-product that contains flavonoids and anthocyanins. In order to study the effects of saffron petal extract (SPE) on blood parameters, immune system, and spleen histology, five treatments (n=6) were used in a completely randomized design. The treatments were 0, 75, 150, 225, and 450 mg/kg body weight of SPE. The SPE was injected intraperitoneally to 30 rats (10-week old, weighing 225±15 g) for 14 days. Immunization was performed using 1×10(8) sheep red blood cells (SRBC) on days 0 and 7 subcutaneously in all treatment groups. On day 15, blood was collected from the heart of rats after anesthesia. One part of samples were poured in heparinized tubes for counting whole blood cells (CBC) and different white blood cells (WBC) and the other part was used to measure IgG using ELISA technique. The spleen was stained by hematoxylin- eosin for histological study. The data were statistically analyzed using ANOVA program and the means evaluation was done using Tukey's test. RESULTS are presented as mean±SD. RESULTS showed no significant difference between treatments and control group regarding the amount of RBC, HGB, HCT, and PLT. The level of IgG at 75 mg/kg was significantly increased in comparison with other groups. No changes were observed in spleen histology. The results indicate that use of SPE at dose of 75 mg/kg causes an increase in antibody response without any change in hematological parameters and spleen histology.

Evaluation of Ethanolic Extracts of Mullaca (Physalis Angulata L.) Herbs for Treatment of Lupus Disease in Mice Induced Pristane

Systemic lupus erythematosus is a systemic autoimmune disease characterized by multisystem inflammation due to immune complex deposits in the organs like kidneys, joints, pleura, skin, and so on. Physalisangulata known as mullaca, has been widely studied for its pharmacological activities such as antiinflammatory, immunosuppressive, cytotoxic, and also inhibition of organ rejection in transplantation. This study was directed to investigate the activities of P. angulata extract as an immunomodulatory agent. P. angulata powder was extracted by maceration with 70% of ethanol. An animal model of lupus was obtained by an injection 0.7mL of pristane, i.p. Successful induction obtained in two weeks after injection which can be monitored by measuring total leukocyte count. For ensuring successful induction, another test was done four weeks and eight weeks after injection by detecting the presence of specific antinuclear antibodies using SDS PAGE method. Other measured parameters were including nonspecific immune response (measurement of total leukocyte count and differential leukocyte count), specific humoral immune response (hemagglutination test), specific cellular immune responses (delayed type hypersensitivity test), organ index and histology of kidney and spleen. As the results shown, ethanolic extract of Physalisangulata at dose of 1000mg/kg BW orally, gave immunomodulatory effect in Lupus rat model. The extract worked primarily on specific immune response by lowering immune response near to the normal value, it was not suppressing immune response as prednisone.

Malay

Disturbances in immune system contribute to chronic infection among diabetic patients. Hibiscus sabdariffa L. (roselle) fruit extract has been scientifi cally proven to possess antioxidant, antidiabetic and antiinfl ammatory properties. The aim of this study was to investigate the effects of H. sabdariffa fruit extract against oxidative stress parameter and T lymphocyte population in spleen of streptozotocin (STZ) induced diabetic rats. Male Sprague-Dawley rats were injected with 45 mg/kg STZ to induce diabetic condition and further treated with 100 mg/kg H. sabdariffa fruit aquoeus extract daily for 28 days. Spleen was harvested to determine the oxidative stress indicators and quantifi cation of T lymphocytes. The results showed a signifi cant decreased in the number of spleen cells and spleen weight in the diabetic rats compared with control rats. However, there were no signifi cant changes in the level of malondialdehyde (MDA), protein carbonyl and superoxide dismutase (SOD) activity the percentage of spleen CD3+ CD4+ and CD3+ CD8+ T lymphocytes amongst groups of study. In addition, histology observation showed no pathological alteration in spleen histology of diabetic rats. The fi ndings suggested that aqueous extract of H. Sabdariffa fruit supplementation has no effect on the oxidative stress and the percentage of CD3+ CD4+ and CD3+ CD8+ T lymphocytes in spleen of diabetic rats. DOI: http://dx.doi.org/10.17576/jskm-1201-2014-03

Splenectomy Is Still a Valid Option For Splenic Marginal Zone Lymphoma

IntroductionSplenic marginal zone lymphoma (SMZL) is an indolent B-cell lymphoma characterized by splenomegaly, frequent moderate lymphocytosis with or without villous morphology and possible involvement of various organs, especially the bone marrow (BM). Diagnosis is classically based on the spleen histology, but it can be made on the BM biopsy, based on the typical intrasinusoidal cell infiltration pattern and immunohistochemistry. Different therapeutic options are available, but to date there are no conclusive comparative data. Patients and methodsWe retrospectively analyzed 83 consecutive patients with a diagnosis of SMZL observed at our Institution between 1999 and 2013. The diagnosis was based on the BM biopsy in 79 patients; the BM was negative in 4 patients. Diagnosis was histologically confirmed on the spleen in 27 patients who underwent splenectomy. Patients presented a median age of 72.5 years (range 38-84); 43 were males. The median spleen size at diagnosis was 145 mm, ranging from 100 to 300 mm. The majority of patients were stage IV at diagnosis for BM infiltration (95%); B symptoms were present in 4 of them (4.8%). Forty-two patients (50.6%) had a lymphocytosis at diagnosis and 13 (15.6%) presented an IPI score higher than 3. Thirty-five of them (42%) had a MZL BM infiltration superior to 30% of the total bone cellularity. Forty-two patients (50.6%) underwent a watch and wait policy (WW), while 41 (49.4%) were treated within 6 months from diagnosis, mainly because of symptomatic splenomegaly; in these patients, treatment consisted of splenectomy, chemotherapy or chemotherapy plus immunotherapy with Rituximab. The features of patients submitted to WW with respect to patients treated at diagnosis were comparable for the various parameters mentioned above, except for spleen size (higher in patients treated at presentation) and lymphocyte count (higher in patients who were observed). After a median follow-up of 64 months, the overall median survival was 96%. Among the 42 WW patients, 18 (42.8%) are still untreated after a median follow-up of 57.5 months, while 24 (57.2%) have required therapy; the median treatment-free interval in these patients was 25.5 months. Concerning the 41 patients who underwent treatment at diagnosis, after a median follow-up of 50 months, 13 (31.7%) have required a subsequent second-line treatment. The interval between first-line approach and re-treatment in patients treated at diagnosis was 30 months. Overall, 27 patients were treated with splenectomy only (either at diagnosis or after a WW period): only 6 of them (22%) had a subsequent progression after a median latency of 42 months; 26 were treated with chemotherapy alone (alkylating agents in the majority of them, combination therapy in a minority): 15 of them (60%) had a subsequent relapse or progression after a median of 9 months; 12 patients received a Rituximab-containing regimen: of these, only 2 (16%) have so far required a second-line therapy after 10 and 26 months respectively. ConclusionsThe WW policy is a valid option for asymptomatic patients: in these patients, after 4.5 years from diagnosis more than 40% is still untreated. In patients requiring treatment, splenectomy alone is followed in the majority of patients by a long period of good disease control: only 22% required a second-line therapy after 3.5 years. The addiction of Rituximab to chemotherapy seems to reduce the probability of relapse and to prolong the response duration. However, these preliminary data need to be confirmed by larger studies. Disclosures:No relevant conflicts of interest to declare.

Extramedullary Hematopoiesis and Splenic Vein Thrombosis, a Unique Presentation Of Pre-Clinical Essential Thrombocythemia

Essential thrombocythemia (ET) is of the BCR-ABL-Negative myeloproliferative neoplasms (MPN). The incidence of ET is approximately 2.5 in every 100,000 person per year. However, given the good prognosis, associated long life expectancy, and increasing detection in younger populations,ET is associated with a higher prevalence rate estimated to be 24 in every 100,000 person per year. ET is characterized by thrombocytosis, vasomotor symptoms, and a variable but increased risk of thrombosis and bleeding. Half of all ET patients will have a positive JAK2 and/or MPL mutation(s).Extramedullary hematopoiesis (EMH) is not a common finding in ET. Nonetheless, ET and other MPNs are associated with the mobilization of CD34+ cells into the peripheral blood. This process can ultimately lead to the seeding of extramedullary sites with primitive hematopoietic capacity, resulting in EMH within the spleen and liver, as well as a variety of other organs. Herein we describe a case that presented with life-threatening thrombosis and was found to have hepatic EMH several months prior to a clinical and pathologic diagnosis of ET. Case descriptionA 22 year-old woman presented 10 days post Cesarean section with abdominal pain and hematemesis. Abdominal imaging showed hepatomegaly, splenomegaly, along with splenic and portal vein thrombosis. The patient underwent an emergency surgical splenectomy due to severe portal hypertension and endoscopic evidence of gastric variceal bleeding. A random liver biopsy was also performed intra-operatively. The splenectomy resulted in resolution of the GI bleeding and the varices normalized on follow up. Her platelet count was normal at the time of operation, but post-splenectomy her platelet count peaked at 1,217 K/ µL. Extensive testing did not unravel any identifiable inherited and/or acquired hypercoaguable factors. Subsequently anticoagulation therapy was recommended for 6 months.On pathology review, the spleen histology showed congestion, but otherwise no diagnostic abnormalities were noted. The liver biopsy showed evidence of EMH but did not identify any liver parenchymal disease.On subsequent follow up, the patient had persistent and marked thrombocytosis for over a year. A bone marrow biopsy was performed which showed a hypercellular bone marrow and megakaryocytic hyperplasia with a few large forms. There was no dysplasia or significant reticulin fibrosis. JAK2 mutation and BCR-ABL translocation were negative. Hydroxyurea and aspirin were started due to high risk of thrombosis. DiscussionWe report this unique case in which there was evidence of extramedullary hematopoiesis, along with pathologic and life threatening visceral thrombosis several months before the patient met criteria for diagnosis of ET. This supports the notion that neoplastic cells can mobilize and seed other organs early in the course of MPNs, including ET. Thrombotic risk in MPNs can also occur in the preclinical phase of MPNs as has been suggested in other reports. We also conclude that the demonstration of EMH in individuals with no preexisting hematologic neoplasm should warrant close follow up and assessment. Disclosures:No relevant conflicts of interest to declare.

JAK1/2 and Pan-Deacetylase Inhibitor Combination Therapy Yields Improved Efficacy in Preclinical Mouse Models of JAK2V617F-Driven Disease

The myeloproliferative neoplasm myelofibrosis is characterized by frequent deregulation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, and JAK inhibitors were shown to reduce splenomegaly and ameliorate disease-related symptoms. However, the mutant clone and bone marrow fibrosis persist in the majority of patients. Using preclinical models, we explored whether JAK and pan-deacetylase inhibitor combination yielded additional benefits. The combination of the JAK1/2 inhibitor ruxolitinib and panobinostat was investigated using two different mouse models of JAK2(V617F)-driven disease. A Ba/F3 JAK2(V617F) cell-driven leukemic disease model was used to identify tolerated and efficacious doses. The drugs were then evaluated alone and in combination in a mouse model of myeloproliferative neoplasm-like disease based on transplantation of bone marrow transduced with a retrovirus expressing JAK2(V617F). Exposures were determined in blood and tissues, and phosphorylated STAT5 and acetylated histone H3 pharmacodynamic readouts were assessed in spleen and bone marrow. Histologic analysis was conducted on spleen and bone marrow, including staining of reticulin fibers in the latter organ. The combination of ruxolitinib and panobinostat was found to have a more profound effect on splenomegaly, as well as on bone marrow and spleen histology, compared with either agent alone, and the analysis of pharmacodynamic readouts showed that ruxolitinib and panobinostat have nonoverlapping and complementary effects. Combining JAK1/2 and pan-deacetylase inhibitors was fairly well tolerated and resulted in improved efficacy in mouse models of JAK2(V617F)-driven disease compared with the single agents. Thus, the combination of ruxolitinib and panobinostat may represent a promising novel therapeutic modality for myeloproliferative neoplasms.

Establishment of the retrovirus-mediated murine model with MLL-AF9 leukemia

This study was purposed to establish a retrovirus-mediated murine model with MLL-AF9 leukemia, so as to provide a basis for further investigation of the pathogenesis and therapeutic strategy of MLL associated leukemia. Murine (CD45.2) primary hematopoietic precursor positively selected for expression of the progenitor marker c-Kit by means of MACS were transduced with a retrovirus carrying MLL-AF9 fusion gene. After cultured in vitro, the transduced cells were injected intravenously through the tail vein into the lethally irradiated mice (CD45.1). PCR, flow cytometry and morphological observation were employed to evaluate the murine leukemia model system. The results showed that MLL-AF9 fusion gene was expressed in the infected cells, and the cells had a dramatically enhanced potential to generate myeloid colonies with primitive and immature morphology. Flow cytometric analysis revealed that the immortalized cells highly expressed myeloid lineage surface markers Gr-1 and Mac-1. Moreover, the expression levels of Hoxa9 and Meis1 mRNA were significantly higher in the MLL-AF9 cells than that in control. The mice transplanted with MLL-AF9 cells displayed typical signs of leukemia within 6-12 weeks. Extensive infiltration leukemic cells was observed in the Wright-Giemsa stained peripheral blood smear and bone marrow, and also in the histology of liver and spleen. Flow cytometric analysis of the bone marrow and spleen cells demonstrated that the CD45.2 populations expressed highly myeloid markers Gr-1 and Mac-1. The leukemic mice died within 12 weeks. It is concluded that the retrovirus-mediated murine model with MLL-AF9 leukemia is successfully established, which can be applied in the subsequent researches.

Increased Thickness of Abdominal Subcutaneous Adipose Tissue Occurs More Frequently in Steatohepatitis Than in Simple Steatosis

The incidence of obesity is increasing and contributes to the rising incidence of fatty liver. Body mass index (BMI) is used to assess the degree of obesity but does not take into account the pattern of body fat distribution. To confirm the increasing incidence of fatty liver in an autopsy study. We hypothesized that a standardized measurement of abdominal subcutaneous adipose tissue (ASAT) might be a good noninvasive method for differentiating steatohepatitis from steatosis. Consecutive complete adult postmortem cases were studied and liver sections were assessed with a steatohepatitis scoring system. Spleen weight, ASAT, and clinical information were obtained. Spleen histology was assessed in a subset of patients having splenomegaly in the absence of cirrhosis. Patients with human immunodeficiency virus, hepatitis C virus, and appreciable alcohol use were excluded. Of 306 cases, the frequency of fatty liver was 51.6% with 33.3% having simple steatosis and 18.3%, having steatohepatitis. Mean ASAT was 3.7 cm in the steatohepatitis group versus 2.6 cm in the steatosis group (P < .001); this difference was greater in patients with a BMI less than 25 kg/m(2) (P = .05). Fibrocongestive splenomegaly was noted in 9 of 38 patients with nonalcoholic steatohepatitis (24%) in the absence of cirrhosis. In this series of autopsy cases, a dramatic increase in the prevalence of fatty liver disease is demonstrated. Thicker ASAT is associated more with steatohepatitis than with simple steatosis, especially in patients with BMI below 25 kg/m(2). Fibrocongestive splenomegaly may occur in the absence of cirrhosis in the presence of steatohepatitis.

A toll-like receptor 9 antagonist reduces pain hypersensitivity and the inflammatory response in spinal cord injury

Toll-like receptors (TLRs) are mediators of the innate immune response to exogenous pathogens. They have also been implicated in sterile inflammation associated with systemic injury and non-infectious diseases via binding of endogenous ligands, possibly released by damaged cells. Emerging evidence indicates that some TLRs play a role in nervous system injury and especially in injury-elicited pain and sterile inflammation. However, no information is available about the contribution of TLR9, a member of the TLR family, to traumatic spinal cord injury (SCI). Moreover, the therapeutic potential of TLR9 ligands in the functional outcomes of SCI, including pain, has not been explored. We report, for the first time, that the intrathecal administration of a TLR9 antagonist, cytidine–phosphate–guanosine oligodeoxynucleotide 2088 (CpG ODN 2088), to mice sustaining a severe contusion SCI, diminishes injury-induced heat hypersensitivity. Investigations on the potential mechanisms underlying the reduction in pain sensitivity indicated an attenuation of the inflammatory reaction manifested by a decrease in the number of CD11b-, CD45- and CD3-immunoreactive cells and a reduction in tumor necrosis factor-α (TNF-α) expression at the epicenter. Conversely, intrathecal delivery of a TLR9 agonist, CpG ODN 1826, increased inflammatory cell numbers and TNF-α expression in the epicenter. The CpG ODN 2088 treatment did not appear to induce systemic adverse effects as shown by spleen histology and serum cytokine levels. We propose that CpG ODN 2088 dampens injury-induced heat hypersensitivity by suppressing the inflammatory response and TNF-α expression. This investigation defines a previously unreported therapeutic role for CpG ODN 2088 in SCI-induced pain.

A Functional Thrombopoietin Receptor Is Required for Full Expression of Phenotype in a JAK2 V617F Transgenic Mouse Model of Polycythemia Vera

Abstract 427Polycythemia vera (PV) is a clonal stem cell disorder in which there is overproduction of red cells, white cells and platelets, extramedullary hematopoiesis and transformation to myelofibrosis. Enhanced hematopoiesis in PV is due to constitutive activation of JAK2, the cognate tyrosine kinase of type I cytokine receptors such as the erythropoietin and thrombopoietin receptors. Hematopoietic stem cells express the thrombopoietin receptor (Mpl) but paradoxically mice lacking the MPL gene exhibit only thrombocytopenia while maintaining the capacity to not only substantially elevate their platelet and leukocyte counts during marrow reproductive stress but also the number of multipotent and committed hematopoietic progenitor cells as well. Equally paradoxical, Mpl expression is impaired in PV, but how impaired expression of a master regulator of stem cell function could be associated with a global increase in hematopoiesis has been a conundrum. Recently, data were presented (Blood 119: 4625, 2012) suggesting that Mpl down regulation in PV was due to constitutive JAK2 activation, which abrogated the antiproliferative effects of the high plasma thrombopoietin (TPO) levels in PV, thus sustaining the PV phenotype. We have used a JAK2 V617F transgenic mouse model (V617Ftg) (Blood 111:5109, 2009) to examine the role of Mpl in the pathogenesis of the JAK2 V617F-positive PV phenotype and have observed that deletion of the MPL gene ameliorated the PV phenotype in this model, reducing the hemoglobin level, leukocyte and platelet counts and splenomegaly and reversing marrow fibrosis, while the presence of a single MPL gene was sufficient to restore the PV phenotype. However, total deletion of the MPL gene does not perfectly model human PV where Mpl is present but quantitatively reduced. Therefore, we examined the effect of manipulating Mpl behavior in vivo by modifying Mpl signaling while maintaining Mpl expression, first, by eliminating TPO production, and second, by expressing a functionally impaired Mpl receptor, and as a control, specifically impairing megakaryocyte differentiation alone. To this end, we crossbred the V617Ftg PV mouse with a TPO gene knockout mouse (TPO ko), with a mouse homozygous for the MPL mutation, C40R (C40R hom), which in the homozygous state causes thrombocytopenia, and finally, with an MKL1 knockout mouse (MKL1ko) that is thrombocytopenic due to impaired megakaryocyte maturation. Crossbreeding produced the three expected genotypes (JAK2 V617F/TPO ko, JAK2 V617F/C40R hom and JAK2 V617F/MKL1ko) without unexplained mortality or impoverished growth. We compared the blood counts, spleen weights and bone marrow and spleen histology of these three genotypes with each other and with JAK2 V617F/TPO heterozygous (het) mice over a 33 week period. The natural history of the V617Ftg PV mouse is one of initial erythrocytosis, thrombocytosis, leukocytosis, splenomegaly, and expansion of short term marrow repopulating cells (ST-HSC), common myeloid progenitor cells (CMP) and marrow and spleen committed hematopoietic progenitor cells (HPC)(CFU-GEMM, CFU-GM, BFU-E, CFU-E and CFU-MK), and myelofibrosis with a subsequent decline in erythrocytosis. Abrogating TPO production in the V617Ftg PV mouse normalized the platelet and leukocyte counts, delayed the development of erythrocytosis, reduced spleen size and ST-HSC, CMP, and all marrow and spleen committed HPC compartments and reversed marrow fibrosis. The V617Ftg PV - C40R MPL cross prevented thrombocytosis, erythrocytosis, leukocytosis and splenomegaly, and reduced all the marrow and spleen committed HPC compartments. By contrast, the V617Ftg PV-MKL1 ko cross slowed the rate of rise of the platelet count and hemoglobin level but had no effect on splenomegaly or marrow fibrosis, nor was there a reduction in any of the marrow or spleen committed HPC. Expression of one TPO gene by crossing the V617Ftg PV mouse with mice heterozygous for the TPO gene (JAK2 V617F/TPO het) restored the PV phenotype. In conclusion, while it is unquestioned that JAK2 V617F is necessary for expression of the PV phenotype, our data indicate that it alone is insufficient and requires not only the expression of a functional TPO receptor but also receptor activation by its cognate ligand TPO. This latter observation suggests that interfering with the TPO-Mpl interaction could be of therapeutic value in the treatment of human PV. Disclosures:No relevant conflicts of interest to declare.

Amelioration of hyperglycemia, hyperlipidemia, oxidative stress and inflammation in steptozotocin-induced diabetic rats fed a high fat diet by riceberry supplement

Dark purple riceberry bran contains a higher dietary fiber and antioxidant compounds than unpigmented rice bran. Riceberry supplement (RB) was used to evaluate the effects on biochemical parameters, skeletal muscle glucose transporter 4 (GLUT4), oxidative stress and inflammation in a streptozotocin (STZ)-induced diabetes rat. To elucidate the effects were due to dietary fiber supplementation and/or bioactive components, equivalent amounts of dietary fiber present in RB were also fed to STZ-induced diabetic rats. Diabetes Sprague–Dawley rats (non-FBG⩾16.65mM) were randomly divided into five groups: DM fed a high fat (HF) diet, DM-RB1 fed 5% RB, DM-RB2 fed 41% RB, DM-F1 fed 0.6% fiber and DM-F2 fed 5% fiber. After 12weeks, significant improvement of BG, insulin, HbA1C, IPGTT and GLUT4 levels were observed in DM-RB1 and DM-RB2 groups. Hyperlipidemia was significantly improved in DM-RB2 and DM-F2 groups. Oxidative stress (TBARS), antioxidant enzymes (SOD, CAT, and GPx), antioxidant capacity (ORAC), pro-inflammation cytokine (TNF-α and IL-6) were improved in DM-RB1 and DM-RB2 groups. Improvement of pancreas and spleen histology was found in DM-RB1 and DM-RB2 groups. These indicate the potential of RB to improve hyperglycemia and hyperlipidemia conditions as well as alleviate oxidative stress and inflammation.

Pulmonary exposure to single-walled carbon nanotubes does not affect the early immune response against Toxoplasma gondii

BackgroundSingle-walled carbon nanotubes (SWCNT) trigger pronounced inflammation and fibrosis in the lungs of mice following administration via pharyngeal aspiration or inhalation. Human exposure to SWCNT in an occupational setting may occur in conjunction with infections and this could yield enhanced or suppressed responses to the offending agent. Here, we studied whether the sequential exposure to SWCNT via pharyngeal aspiration and infection of mice with the ubiquitous intracellular parasite Toxoplasma gondii would impact on the immune response of the host against the parasite.MethodsC57BL/6 mice were pre-exposed by pharyngeal administration of SWCNT (80 + 80 μg/mouse) for two consecutive days followed by intravenous injection with either 1x103 or 1x104 green fluorescence protein and luciferase-expressing T. gondii tachyzoites. The dissemination of T. gondii was monitored by in vivo bioluminescence imaging in real time for 7 days and by plaque formation. The inflammatory response was analysed in bronchoalveolar lavage (BAL) fluid, and by assessment of morphological changes and immune responses in lung and spleen.ResultsThere were no differences in parasite distribution between mice only inoculated with T. gondii or those mice pre-exposed for 2 days to SWCNT before parasite inoculum. Lung and spleen histology and inflammation markers in BAL fluid reflected the effects of SWCNT exposure and T. gondii injection, respectively. We also noted that CD11c positive dendritic cells but not F4/80 positive macrophages retained SWCNT in the lungs 9 days after pharyngeal aspiration. However, co-localization of T. gondii with CD11c or F4/80 positive cells could not be observed in lungs or spleen. Pre-exposure to SWCNT did not affect the splenocyte response to T. gondii.ConclusionsTaken together, our data indicate that pre-exposure to SWCNT does not enhance or suppress the early immune response to T. gondii in mice.

Abstract 1850: The role of Thoc1 in tissue homeostasis in the adult mouse

Abstract Thoc1 encodes a protein (Thoc1) that interacts with the retinoblastoma protein (Rb). Thoc1 functions in an evolutionary conserved protein complex called THO that is involved in nuclear ribonucleoprotein particle (RNP) formation. Loss of THO function compromises the packaging of nascent RNA, which causes defects in transcription, RNA processing and mRNA export. In mice, Thoc1 is required for embryonic development. It is currently unknown whether Thoc1 is required in adult mice for normal tissue homeostasis. We hypothesize that different tissues will vary in their dependence on Thoc1, possibly based on their proliferative potential. To test this hypothesis, we conditionally deleted Thoc1 in adult mice in a wide variety of tissues using the Tamoxifen/CreER recombination system, and assessed the consequences on tissue homeostasis. Thoc1F/F:Rosa26CreER+/− (test) and Thoc1+/+:Rosa26CreER+/− (control) adult mice were treated with 2 mg/day Tamoxifen for 6 days. Slowly proliferating (liver, kidney, colon) and rapidly proliferating (small intestinal (S.I.)) tissue were collected 24 hours after the last treatment. Histological analysis shows Thoc1 loss disrupts the architecture of the small intestine (S.I.) but does not affect the other tissues examined. The functional unit of the S.I. is the crypt-villus axis. S.I. crypts contain rapidly proliferating stem and progenitor cells that continuously feed the differentiated, non-proliferative villus epithelium. The crypts proliferate so rapidly that the entire S.I. epithelium is replaced every three to five days. Interestingly, Thoc1 loss only causes apoptosis in the S.I. crypts, suggesting disruption of villi architecture following Thoc1 loss is a result of altered crypt function, not a direct effect of Thoc1 loss. Supporting this, the crypts of mice in which Thoc1 has been deleted also have a loss of cell proliferation. While the colon and S.I. have similar tissue architecture, the colon's rate of turnover is much slower. Therefore, it is not surprising the colon is not affected by Thoc1 loss. These findings support the hypothesis that tissues with a high cell proliferative potential require Thoc1. To explore this further, we examined how Thoc1 deletion would affect the highly proliferative hematopoietic system using the same test and control mice as previously listed. Further supporting the hypothesis, Thoc1 loss decreases the number of lymphocytes in the blood, but does not affect spleen and thymus (slowly proliferating tissues) histology. The results of this study suggest highly proliferative tissues that turnover rapidly require Thoc1, most likely to support the gene expression and cell growth necessary for rapid cell division. This hypothesis is significant as it predicts cancer cells will be more sensitive to Thoc1 loss than normal cells, a prediction verified by preliminary results in our lab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1850. doi:1538-7445.AM2012-1850

Rituximab, Either As Single Agent or in Combination, Is Superior to Other Treatments in Terms of Response Rate and Progression-Free Survival (PFS) in Patients with Splenic Marginal Zone Lymphoma (SMZL)

Abstract 4986 Background.Splenic marginal zone lymphoma (SMZL) is an uncommon indolent B-cell non-Hodgkin's lymphoma usually presenting with marked splenomegaly and bone marrow (BM) and/or peripheral blood (PB) involvement. Although splenectomy has traditionally been the treatment of choice in symptomatic patients, systemic treatment is required in cases of widespread disease, high risk from surgery, or relapse after splenectomy. There is no standard first line treatment in SMZL, but the anti-CD20 monoclonal antibody Rituximab has shown encouraging results in SMZL, with sustained responses. Aims.To assess, retrospectively, response to treatment, toxicity profile, progression-free survival (PFS) and overall survival (OS) after treatment with rituximab in SMZL. Methods.Twenty-nine patients from two different centers, diagnosed with SMZL between 1982 and 2011, received one or more treatments with rituximab. Eighteen patients (62%) received rituximab alone and 20 (69%) combined with chemotherapy; thus 9 patients received each of these treatments sequentially, to improve response, with or without interim relapse. The median age at diagnosis was 62 years (range 37–89 years); the male:female ratio was 2:3; B symptoms were present in 12 patients; ECOG performance status was 0–2 in 28/29 patients. All presented with splenomegaly, with involvement of the BM in 27 patients, PB in 24, lymph nodes in 11 and extranodal involvement in 7 patients. Diagnosis was made according to the WHO 2008 classification by spleen histology (n=12), BM histology (n=19), PB morphology (n=12) and immunophenotype (n=13). Rituximab monotherapy was administered at 375 mg/m2/weekly x4 weeks. In combination with fludarabine-based regimens (n=14), or other regimens including CHOP (n= 6), rituximab was administered on day 1 of each cycle. Responses were assessed according to the Response Criteria Guidelines for SMZL (Matutes, Leukemia 2008). Toxicity was graded according to the CTCAE v3.0. The Fisher exact test was used to compare best responses between groups. Survival was estimated using the Kaplan-Meier method. Results.All patients responded to rituximab monotherapy and/or to a combination treatment. At least one complete response (CR) was achieved in 20/29 patients (69%). This compares with a CR in only 4/17 (24%) of these same patients after any prior therapy (p=0.005): 11 patients had received splenectomy, with or without chemotherapy and 6 had received chemotherapy only. There was no difference in the CR rate between rituximab monotherapy (71%) versus rituximab combinations (68%). The most common adverse event was grade 3–4 neutropenia (n=7, 24%). Two patients had grade 3–4 infections. Anemia (n=3) and thrombocytopenia (n=2) were grade 1–2 only. Four cases presented with histological transformation prior to rituximab, all achieving CR with rituximab combination therapy. After a median follow-up of 24 months from rituximab treatment (range 4–102 months), progression-free survival (PFS) has been evaluated in 28/29 patients (1 patient not assessed yet at the time of presenting these data). PFS was longer after rituximab (n=32 evaluable treatments), either alone or in combination, than it had been in these same patients after prior therapy (n=17 prior treatments in total) (p=0.002) (Fig 1). When we compared PFS between rituximab monotherapy (n=17) and rituximab combined with chemotherapy (n=15 combined treatments), single agent rituximab was superior to rituximab in combination (p=0.02) (Fig 2). The median overall survival was not yet reached. Survival at 2 years after rituximab was 100%. Only one death, due to lung cancer, occurred at 48 months follow-up. Conclusions.In patients with SMZL, the CR rate after rituximab, either alone or in combination with chemotherapy, was significantly better than the CR rate after other prior treatments in the same patients, with manageable toxicity. Although we observed no difference in the CR rate between rituximab monotherapy or combination therapy, PFS was longer in patients receiving rituximab as single agent. Therefore, the addition of chemotherapy did not appear to add any advantage over rituximab alone in our cohort. Rituximab, with or without splenectomy, should be considered in the therapeutic scenario of SMZL. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

JAK2-V617F Expressing Stem Cells Display a Competitive Advantage At Low Limiting Dilution and Are Capable of Initiating MPN Phenotype

Abstract 615JAK2-V617F is the most common genetic alteration in myeloproliferative neoplasms (MPN). Forced expression of JAK2-V617F in animal models induced a MPN phenotype. However, MPN in these models was initiated by polyclonal expansion of multiple hematopoietic progenitor and stem cells. In contrast, patients with MPN display clonal hematopoiesis. Frequently, additional somatic mutations precede the acquisition of JAK2-V617F, suggesting that a pre-JAK2 mutation could be required for clonal expansion. Compatible with this hypothesis, in one JAK2-V617F knockin model the MPN phenotype was not transplantable and the stem cells showed signs of exhaustion. To address the question of whether JAK2-V617F alone is sufficient to initiate MPN we performed competitive repopulation assays and serial transplantations in a transgenic JAK2-V617F mouse model.Our transgenic mice express the human JAK2-V617F under the endogenous JAK2 promoter and display a polycythemia vera (PV) or essential thrombocythemia (ET) phenotype. The transgene can be activated by Cre recombinase and the donors were induced 5–6 weeks prior to transplantation. To allow monitoring of the reconstitution, a GFP expressing transgenic strain was used as the source of competitor cells. Bone marrow transplantations were performed into lethally irradiated mice in the inbred BL/6 background. A total of 2×106 bone marrow cells per recipient were transplanted with ratios of mutant (V617F) to wild type (WT) set as 1:1, 1:10 or 1:100. Results:At 1:1 and 1:10 ratios, the mice displayed a PV phenotype beginning at 4 weeks post transplantation (Hb ∼200-220 g/L and neutrophil count of 5–20×109 /L) that remained stable for ∼26 weeks. An increase in platelet counts (4-6×1012/L) followed at later time points. V617F cells rapidly outcompeted WT cells reaching >95% chimerisms in peripheral blood. The MPN phenotype was transplantable into secondary and tertiary recipients and no signs of exhaustion were observed. An increase in the frequencies of lineage-negative, Sca-1+ c-kit+ (LSK) cell numbers in bone marrow was noted (4.1% in V617F vs 1.2% for WT, p<0.05). To assess their cycling rate, LSK cells were sorted, labeled with carboxyfluorescein succinimidyl ester and transplanted into non-conditioned recipients. After 8 weeks, the number of LSK cells was 62-fold increased and V617F LSK cells outcompeted host cells (52% vs. 3% for WT, p<0.01). The number of slow-dividing (0-2 divisions) LSK cells was substantially reduced in the V617F transplanted mice (0.095% vs. 7.54% for WT, p<0.01). Thus, V617F increases the cycling of LSK cells and provides a competitive advantage.To allow detecting low numbers of donor cells in 1:100 transplantations, the GFP transgene was bred into the V617F background and a mixture of 2×104 V617F/GFP bone marrow cells plus 1.98×106 WT competitors was used. Based on the expected frequency of 3 long term hematopoietic stem cells (HSC) per 105 bone marrow cells, the recipients should receive on average 0.6 GFP-positive long term HSC. In mice transplanted with V617F/GFP and WT competitors at 1:100, we detected reconstitution defined as >0.1% GFP positive cells in 12 of 40 recipients (28%) that persisted beyond 20 weeks, whereas in mice transplanted with WT/GFP and WT competitors, only 5/40 (12%) showed reconstitution. The relative contribution in the WT group ranged from 0.1 – 3%, whereas in the V617F group we observed chimerism between 5 –100%. Four of the 12 mice that reconstituted with V617F/GFP cells developed a MPN phenotype (3PV, 1ET). Terminal workup of 3 mice (2PV and 1ET) at 29 weeks revealed that mice with erythrocytosis had enlarged spleens (∼150mg vs. 65mg for WT) and increased erythropoiesis in spleen histology. In contrast the mouse with ET phenotype showed only a slight increase in spleen size (90mg vs. 65mg for WT) and no increase in spleen erythropoiesis. Conclusions:Our results show that JAK2-V617F positive cells were able to rapidly outcompete WT cells. The MPN phenotype was transplantable into secondary and tertiary recipients. At low limiting dilution (1:100), V617F cells displayed > 2-fold higher engraftment rate (28%) than WT cells (12%) and a higher contribution to hematopoiesis (5-100% chimerism vs. 0.1–3%). Since 4/12 mice (30%) reconstituted with V617F cells also developed a MPN phenotype, our data demonstrate than JAK2-V617F as the sole genetic alteration can initiate MPN under conditions resembling monoclonal disease. Disclosures:No relevant conflicts of interest to declare.

Role of tumor suppressor p53 in megakaryopoiesis and platelet function

The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on invivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53(-/-) megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets invitro. Culture of p53(-/-) marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53(+/+) megakaryocytes. This was due to 30% greater DNA synthesis in p53(-/-) megakaryocytes and 31% greater apoptosis in p53(+/+) megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53(+/+) and p53(-/-) mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53(-/-) mice. Although their platelet counts were normal, p53(-/-) mice exhibited significantly longer bleeding times and p53(-/-) platelets were less sensitive than p53(+/+) platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our invivo and exvivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.

The immunological and histopathological changes of Tramadol, Tramadol/Acetaminophen and Acetaminophen in male Albino rats

Background: Tramadol is a synthetic opioid analgesic. It is commonly prescribed for moderate to severe pain, becoming abused more popular among teens in most countries. Paracetamol as anti-inflammatory drugs (acetaminophen) (APAP) is widely used as an analgesic and antipyretic agent. Meanwhile, tramadol/acetaminophen (tramacet) is effective in acute or chronic moderate-to-moderately severe pain. In comparative study, the current investigation threw the light on the effect of over doses of tramadol and/or APAP on the immune function and hepatocytes in adult maleSprague-Dawley rats. Material and methods: Treated rats received oral doses of each drug for 15 consecutive days and after last treatment, they kept three days later for withdrawal studies. The rats were divided into four treatment groups, in the first group, rats received saline and used as control. The second, third and fourth groups treated with tramadol (45 mg/kg), tramadol/APAP (45/450 mg/kg), APAP (450 mg/kg) respectively, once a-day at the first week and ending with 90, 90/900, 900 mg/kg at the second week. Rats were sacrificed at the end of the first, second weeks and three days of last treatment. Results: Daily doses of tramadol and /or APAP exposure in rats decreased the cellularity of spleen. Moreover, phagocytic and killing of S. aureus by PMN and macrophage cells caused a highly significant decrease in treated groups. IFN-γ was reduced in a statistically different treated group of rats. Serum IL-10 was unaffected by any of the treatment regimens but increased only in tramadol/APAP treated rats. Spleen histology exhibited mild pathological alteration with different injures between treated groups. Splenic white pulp accompanied by ill deformed which reflected the reduction of white pulp zones, thickened vasculature in the splenic net work, fibrous trabeculae become prominent feature, where splenic red pulp occupied large areas of the splenic network with predominant edema and megakaryocytes. On the other hand, the effect of tramadol and/or APAP induced DNA alterations of hepatocytes in dose dependent pattern as elucidated by dendrogramatic analysis. Liver histopathological changes of treated groups included vacuolated hepatocytes, dilated sinusoid with proliferated Kupffer cells; atrophied hepatocytes with nuclei reduced in size and darkly stained. Many areas of hepatocytes showed loss of architecture, congested central vein, expanded portal area with edema and inflammatory reaction. Conclusion: It could be concluded that the effect of tramadol/APAP induced anti-inflammatory cytokines than tramadol and APAP alone. Tramadol and/or APAP may display severe pathological consequences of hepatocytes. These hepatic lesions may be caused impairment of the liver function.

The immunological and histopathological changes of Tramadol, Tramadol/Acetaminophen and Acetaminophen in male Albino rats “Comparative study”

Background: Tramadol is a synthetic opioid analgesic. It is commonly prescribed for moderate to severe pain, becoming abused more popular among teens in most countries. Paracetamol as anti-inflammatory drugs (acetaminophen) (APAP) is widely used as an analgesic and antipyretic agent. Meanwhile, tramadol/acetaminophen (tramacet) is effective in acute or chronic moderate-to-moderately severe pain. In comparative study, the current investigation threw the light on the effect of over doses of tramadol and/or APAP on the immune function and hepatocytes in adult maleSprague-Dawley rats. Material and methods: Treated rats received oral doses of each drug for 15 consecutive days and after last treatment, they kept three days later for withdrawal studies. The rats were divided into four treatment groups, in the first group, rats received saline and used as control. The second, third and fourth groups treated with tramadol (45 mg/kg), tramadol/APAP (45/450 mg/kg), APAP (450 mg/kg) respectively, once a-day at the first week and ending with 90, 90/900, 900 mg/kg at the second week. Rats were sacrificed at the end of the first, second weeks and three days of last treatment. Results: Daily doses of tramadol and /or APAP exposure in rats decreased the cellularity of spleen. Moreover, phagocytic and killing of S. aureus by PMN and macrophage cells caused a highly significant decrease in treated groups. IFN-γ was reduced in a statistically different treated group of rats. Serum IL-10 was unaffected by any of the treatment regimens but increased only in tramadol/APAP treated rats. Spleen histology exhibited mild pathological alteration with different injures between treated groups. Splenic white pulp accompanied by ill deformed which reflected the reduction of white pulp zones, thickened vasculature in the splenic net work, fibrous trabeculae become prominent feature, where splenic red pulp occupied large areas of the splenic network with predominant edema and megakaryocytes. On the other hand, the effect of tramadol and/or APAP induced DNA alterations of hepatocytes in dose dependent pattern as elucidated by dendrogramatic analysis. Liver histopathological changes of treated groups included vacuolated hepatocytes, dilated sinusoid with proliferated Kupffer cells; atrophied hepatocytes with nuclei reduced in size and darkly stained. Many areas of hepatocytes showed loss of architecture, congested central vein, expanded portal area with edema and inflammatory reaction. Conclusion: It could be concluded that the effect of tramadol/APAP induced anti-inflammatory cytokines than tramadol and APAP alone. Tramadol and/or APAP may display severe pathological consequences of hepatocytes. These hepatic lesions may be caused impairment of the liver function.

A Novel Canine Model of Portal Vein Stenosis Plus Thioacetamide Administration-Induced Cirrhotic Portal Hypertension With Hypersplenism

Current large animal models that could closely resemble the typical features of cirrhotic portal hypertension in human have not been well established. Thus, we aimed to develop and describe a reliable and reproducible canine cirrhosis model of portal hypertension. A total of 30 mongrel dogs were randomly divided into four groups: 1 (control; n = 5), 2 (portal vein stenosis [PVS]; n = 5], 3 (thioacetamide [TAA]; n = 5), and 4 (PVS plus TAA; n = 15). After 4-months modeling period, liver and spleen CT perfusion, abdominal CT scans, portal hemodynamics, gastroscopy, hepatic function, blood routine, the bone marrow, liver, and spleen histology were studied. The animals in group 2 (PVS) developed extrahepatic portosystemic collateral circulation, particularly esophageal varices, without hepatic cirrhosis and portal hypertension. Animals from group 3 (TAA) presented mild cirrhosis and portal hypertension without significant symptoms of esophageal varices and hypersplenism. In contrast, animals from group 4 (PVS + TAA) showed well-developed micronodular and macronodular cirrhosis, associated with significant portal hypertension and hypersplenism. The combination of PVS and TAA represents a novel, reliable, and reproducible canine cirrhosis model of portal hypertension, which is associated with the typical characteristics of portal hypertension, including hypersplenism.