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1.  Meta-Modeling of Methylprednisolone Effects on Glucose Regulation in Rats 
PLoS ONE  2013;8(12):e81679.
A retrospective meta-modeling analysis was performed to integrate previously reported data of glucocorticoid (GC) effects on glucose regulation following a single intramuscular dose (50 mg/kg), single intravenous doses (10, 50 mg/kg), and intravenous infusions (0.1, 0.2, 0.3 and 0.4 mg/kg/h) of methylprednisolone (MPL) in normal and adrenalectomized (ADX) male Wistar rats. A mechanistic pharmacodynamic (PD) model was developed based on the receptor/gene/protein-mediated GC effects on glucose regulation. Three major target organs (liver, white adipose tissue and skeletal muscle) together with some selected intermediate controlling factors were designated as important regulators involved in the pathogenesis of GC-induced glucose dysregulation. Assessed were dynamic changes of food intake and systemic factors (plasma glucose, insulin, free fatty acids (FFA) and leptin) and tissue-specific biomarkers (cAMP, phosphoenolpyruvate carboxykinase (PEPCK) mRNA and enzyme activity, leptin mRNA, interleukin 6 receptor type 1 (IL6R1) mRNA and Insulin receptor substrate-1 (IRS-1) mRNA) after acute and chronic dosing with MPL along with the GC receptor (GR) dynamics in each target organ. Upon binding to GR in liver, MPL dosing caused increased glucose production by stimulating hepatic cAMP and PEPCK activity. In adipose tissue, the rise in leptin mRNA and plasma leptin caused reduction of food intake, the exogenous source of glucose input. Down-regulation of IRS-1 mRNA expression in skeletal muscle inhibited the stimulatory effect of insulin on glucose utilization further contributing to hyperglycemia. The nuclear drug-receptor complex served as the driving force for stimulation or inhibition of downstream target gene expression within different tissues. Incorporating information such as receptor dynamics, as well as the gene and protein induction, allowed us to describe the receptor-mediated effects of MPL on glucose regulation in each important tissue. This advanced mechanistic model provides unique insights into the contributions of major tissues and quantitative hypotheses for the multi-factor control of a complex metabolic system.
doi:10.1371/journal.pone.0081679
PMCID: PMC3847111  PMID: 24312573
2.  Pharmacodynamics of Glucose Regulation by Methylprednisolone. I. Adrenalectomized Rats 
Mechanisms related to the adverse effects of corticosteroids on glucose homeostasis were studied. Five groups of adrenalectomized (ADX) rats were given methylprednisolone (MPL) intravenously at 10 and 50 mg/kg, or a continuous 7 day infusion at rates of 0, 0.1, 0.3 mg/kg/h via subcutaneously implanted Alzet mini-pumps. Plasma concentrations of MPL, glucose and insulin were determined at various time points up to 72 h after injection or 336 h after infusion. The pharmacokinetics of MPL was captured with a two-compartment model. The Adapt II software was used in modeling. Injection of MPL caused a temporary glucose increase over 6 h by stimulating gluconeogenesis. The glucose changes stimulated pancreatic β-cell secretion yielding a later insulin peak at around 10 h. In turn, insulin can stimulate glucose disposition. However, long-term MPL treatment caused continuous hyperglycemia during and after infusion. Insulin was increased during infusion, and immediately returned to baseline after the infusion was terminated, despite the almost doubled glucose concentration. A disease progression model incorporating the reduced endogenous glucose disposition was included to capture glucose homeostasis under different treatments. The results exemplify the importance of the steroid dosing regimen in mediating pharmacological and adverse metabolic effects. This mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model quantitatively describes the induction of hyperglycemia and provides additional insights into metabolic disorders such as diabetes.
doi:10.1002/bdd.643
PMCID: PMC3712292  PMID: 19156931
corticosteroids; methylprednisolone; pharmacodynamics; pharmacokinetics; glucose; insulin
3.  Pharmacodynamic/Pharmacogenomic Modeling of Insulin Resistance Genes in Rat Muscle After Methylprednisolone Treatment: Exploring Regulatory Signaling Cascades 
Corticosteroids (CS) effects on insulin resistance related genes in rat skeletal muscle were studied. In our acute study, adrenalectomized (ADX) rats were given single doses of 50 mg/kg methylprednisolone (MPL) intravenously. In our chronic study, ADX rats were implanted with Alzet mini-pumps giving zero-order release rates of 0.3 mg/kg/h MPL and sacrificed at various times up to 7 days. Total RNA was extracted from gastrocnemius muscles and hybridized to Affymetrix GeneChips. Data mining and literature searches identified 6 insulin resistance related genes which exhibited complex regulatory pathways. Insulin receptor substrate-1 (IRS-1), uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), fatty acid translocase (FAT) and glycerol-3-phosphate acyltransferase (GPAT) dynamic profiles were modeled with mutual effects by calculated nuclear drug-receptor complex (DR(N)) and transcription factors. The oscillatory feature of endothelin-1 (ET-1) expression was depicted by a negative feedback loop. These integrated models provide testable quantitative hypotheses for these regulatory cascades.
PMCID: PMC2733097  PMID: 19787081
corticosteroid; glucocorticoid; microarrays; mathematical modeling; insulin resistance
4.  Dynamic modeling of methylprednisolone effects on body weight and glucose regulation in rats 
Influences of methylprednisolone (MPL) and food consumption on body weight (BW), and the effects of MPL on glycemic control including food consumption and the dynamic interactions among glucose, insulin, and free fatty acids (FFA) were evaluated in normal male Wistar rats. Six groups of animals received either saline or MPL via subcutaneous infusions at the rate of 0.03, 0.1, 0.2, 0.3 and 0.4 mg/kg/h for different treatment periods. BW and food consumption were measured twice a week. Plasma concentrations of MPL and corticosterone (CST) were determined at animal sacrifice. Plasma glucose, insulin, and FFA were measured at various times after infusion. Plasma MPL concentrations were simulated by a two-compartment model and used as the driving force in the pharmacodynamic (PD) analysis. All data were modeled using ADAPT 5. The MPL treatments caused reduction of food consumption and body weights in all dosing groups. The steroid also caused changes in plasma glucose, insulin, and FFA concentrations. Hyper-insulinemia was achieved rapidly at the first sampling time of 6 h; significant elevations of FFA were observed in all drug treatment groups; whereas only modest increases in plasma glucose were observed in the low dosing groups (0.03 and 0.1 mg/kg/h). Body weight changes were modeled by dual actions of MPL: inhibition of food consumption and stimulation of weight loss, with food consumption accounting for the input of energy for body weight. Dynamic models of glucose and insulin feedback interactions were extended to capture the major metabolic effects of FFA: stimulation of insulin secretion and inhibition of insulin-stimulated glucose utilization. These models of body weight and glucose regulation adequately captured the experimental data and reflect significant physiological interactions among glucose, insulin, and FFA. These mechanism-based PD models provide further insights into the multi-factor control of this essential metabolic system.
doi:10.1007/s10928-011-9194-4
PMCID: PMC3407886  PMID: 21394487
Glucocorticoids; Methylprednisolone; Pharmacodynamics; Food intake; Body weight; Glucose; Insulin; Free fatty acids
5.  Pharmacodynamics of Glucose Regulation by Methylprednisolone. II. Normal Rats 
A physiologic pharmacodynamic model was developed to jointly describe the effects of methylprednisolone (MPL) on adrenal suppression and glycemic control in normal rats. Six groups of animals were given MPL intravenously at 0, 10 and 50 mg/kg, or by subcutaneous 7 day infusion at rates of 0, 0.1 and 0.3 mg/kg/h. Plasma concentrations of MPL, corticosterone (CST), glucose and insulin were determined at various times up to 72 h after injection and 336 h after infusion. The pharmacokinetics of MPL was described by a two-compartment model. A circadian rhythm for CST was found in untreated rats with a stress-altered baseline caused by handling, which was captured by a circadian harmonic secretion rate with an increasing mesor. All drug treatments caused CST suppression. Injection of MPL caused temporary increases in glucose over 4 h. Insulin secretion was thereby stimulated yielding a later peak around 6 h. In turn, insulin can normalize glucose. However, long-term dosing caused continuous hyperglycemia during and after infusion. Hyperinsulinemia was achieved during infusion, but diminished immediately after dosing despite the high glucose concentration. The effects of CST and MPL on glucose production were described with a competitive stimulation function. A disease progression model incorporating reduced endogenous glucose uptake/utilization was used to describe glucose metabolism under different treatments. The results exemplify the roles of endogenous and exogenous hormones in mediating glucose dynamics. The pharmacokinetic/pharmacodynamic model is valuable for quantitating diabetogenic effects of corticosteroid treatments and provides mechanistic insights into the hormonal control of the metabolic system.
doi:10.1002/bdd.642
PMCID: PMC3712293  PMID: 19156669
corticosterone; methylprednisolone; pharmacodynamics; pharmacokinetics; glucose; insulin
6.  Pharmacokinetic/Pharmacodynamic Modeling of Methylprednisolone Effects on iNOS mRNA Expression and Nitric Oxide During LPS-Induced Inflammation in Rats 
Pharmaceutical Research  2012;29(8):2060-2069.
Purpose
Increased expression of inducible nitric oxide synthase (iNOS) resulting in nitric oxide elevation represents an important component of inflammatory responses. We assess the effects of methylprednisolone (MPL) on these processes during endotoxin-induced acute inflammation and provide a mechanism-based model to quantitatively describe them.
Methods
Male Lewis rats were dosed with lipopolysaccharide (50 μg/kg LPS) alone or with methylprednisolone (10 and 50 mg/kg) and sacrificed at different time points. Plasma MPL, lung iNOS mRNA expression, plasma nitric oxide (NO) and other physiological factors were measured. Sodium nitrate (750 μmole/kg) was given to a separate cohort of rats to assess NO disposition kinetics. PK-PD modeling was performed with ADAPT 5.
Results
Disposition kinetics of plasma MPL and NO showed bi-exponential decline and were described by two-compartment models. LPS increased expression of iNOS mRNA in lung and increased plasma NO, while MPL dosing palliated this increase in a dose-dependent manner. These effects were well captured using tandem indirect response and precursor-pool models.
Conclusion
The model provides a quantitative assessment of the suppression of NO production by MPL and shows that the major effects are at the transcriptional level by reducing expression of iNOS mRNA.
doi:10.1007/s11095-012-0733-5
PMCID: PMC3400266  PMID: 22422321
corticosteroids; inflammation; iNOS; nitric oxide; PK-PD modeling
7.  MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia  
PLoS Medicine  2006;3(7):e270.
Background
The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR).
Methods and Findings
DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9–4.0 × 10 12/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis.
Conclusions
Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.
Editors' Summary
Background.
Myelofibrosis with myeloid metaplasia (MF) is one of a group of chronic blood disorders, known as chronic myeloproliferative disorders. These disorders sometimes turn into acute leukemia. The main abnormality in myelofibrosis is for the bone marrow to become filled with fibrous (scar) tissue (hence the name myelofibrosis), which stops it from producing normal blood cells efficiently. In addition, the white blood cells that remain are abnormal (that is, metaplastic). The clinical effect of these abnormalities are that patients are anemic (they have low numbers of red cells), are more likely to get infections because of the abnormal white cells which cannot fight infections normally, and may bleed more easily because of a lack of the platelets that help the blood to clot. Scientists who study this disorder believe that the disease starts from just one abnormal cell, which divides to replace all the other cells—that is, all the abnormal cells are part of one clone.
Why Was This Study Done?
In two similar diseases, polycythemia vera (in which the bone marrow produces too many red blood cells) and essential thrombocytosis (in which the bone marrow produces too many platelets), and in some patients with MF, scientists have found genetic changes which seem to trigger these diseases. However, there are some patients with MF in which no abnormal gene has been found. The scientists here wanted to look at other genes to see if they could find any changes that might trigger MF.
What Did the Researchers Do and Find?
They decoded the DNA sequence of three genes that are known to be involved in how blood cells develop for 45 patients with MF. They looked at DNA from white blood cells, and also from normal cheek cells for comparison. They found that in four of the 45 patients the DNA in the bone marrow, but not the cheek, carried a mutation in a gene for the thrombopoietin receptor (also called MPL). This gene is necessary for the cells that make platelets to grow correctly. The mutation was not present in any samples from patients with diseases related to MF, nor in 270 normal samples. The mutation that was identified was at position 515 in the MPL gene sequence, hence the name MPLW515L—the W and the L are the shorthand way of indicating exactly which change occurred. The change meant that the gene became abnormally active. The researchers tested the effect of the abnormal gene by putting it into cells grown in culture in the laboratory; they found that it made the cells grow more than was normal. In addition, when cells with the abnormal gene were put into mice, the mice developed a blood disorder similar to that seen in humans with MF.
What Do These Findings Mean?
It seems likely that the genetic change that has been identified here is responsible for the MF that develops in some patients. The MPL gene is known to be part of a pathway of genes that control how certain blood cells develop. However, it is not yet clear exactly how the genetic change found here causes the blood cells to grow abnormally, or how it causes the other clinical effects of MF. Further work will also need to be done to see if it is possible to develop drugs that can act on this gene mutation, or on the other genes that it affects so as to return the cells to normal.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030270.
• MedlinePlus, a Web site of the US National Library of Health, has pages of information on myelofibrosis and related diseases
• The National Cancer Institute, which funds research into many cancers, has information for patients on myelofibrosis, including information on clinical trials
• The MPD Foundation has information for patients with myelofibrosis and related diseases
Activation of JAK-STAT signaling via a mutation - MPLW515L- in the thrombopoietin receptor seems to have a role in the pathogenesis of some patients with myelofibrosis.
doi:10.1371/journal.pmed.0030270
PMCID: PMC1502153  PMID: 16834459
8.  Molecular detection of c-mpl thrombopoietin receptor gene expression in chronic myeloproliferative disorders. 
Molecular Pathology  1999;52(3):146-150.
BACKGROUND: Chronic myeloproliferative disorders (CMPD) originate from a pluripotent haematopoietic progenitor cell but show a marked degree of heterogeneity, especially between Philadelphia chromosome positive and negative disease entities. Abnormal megakaryopoiesis is a frequent finding in CMPD, often associated with thrombocythaemic cell counts. Recent experimental data have suggested that the c-Mpl thrombopoietin receptor, together with its ligand thrombopoietin, are not only the major physiological regulators of megakaryopoiesis and platelet production, but also play a crucial role in chronic myeloproliferation. METHODS: A total of 18 peripheral blood mononuclear cell samples obtained from patients with CMPD (chronic myelocytic leukaemia (CML), n = 10; polycythaemia vera (PV), n = 6; and primary thrombocythaemia (PTH), n = 2) were analysed for c-mpl mRNA using the reverse transcriptase polymerase chain reaction (RTPCR). In another 20 patients (CML, n = 10; chronic megakaryocytic granulocytic myelosis (CMGM), n = 3; PV, n = 3; PTH, n = 4), we compared the number of haematopoietic progenitors expressing c-Mpl, as characterised by coexpression with the CD34 antigen, in the bone marrow using double immunofluorescence staining. RESULTS: c-mpl mRNA was detected in all samples from patients with CML analysed, whereas only two of six PV and one of two PTH samples were positive (p < or = 0.008; chi 2 test). Expression of the c-mpl receptor gene was absent in healthy subjects used as controls. Similarly, an increase of c-Mpl expressing CD34 positive haematopoietic cells was detected in seven of 10 bone marrow aspirates obtained from patients with CML. Increased numbers of c-Mpl positive CD34 positive cells were found in only one of four patients with PTH, whereas in PV and CMGM the numbers of c-Mpl positive CD34 positive cells did not exceed normal values, despite thrombocythaemic cell counts. CONCLUSIONS: These data confirm recent findings showing an impaired expression of the c-mpl thrombopoietin receptor gene in Philadelphia chromosome negative CMPD when compared with patients with Philadelphia chromosome positive CML. The relevance of this observation to the functional and morphological characteristics of abnormal megakaryopoiesis remains unclear. Thrombocythaemic cell counts and a mature phenotype in megakaryocytes occur frequently in Philadelphia chromosome negative CMPD but require an intact c-Mpl receptor under physiological conditions. Therefore, further studies are warranted to elucidate the mechanisms contributing to megakaryopoiesis in CMPD disease entities with decreased c-mpl gene expression.
PMCID: PMC395689  PMID: 10621836
9.  Expression pattern of the thrombopoietin receptor (Mpl) in the murine central nervous system 
Background
Thrombopoietin (Thpo) and its receptor (Mpl), which regulate megakaryopoiesis, are expressed in the central nervous system (CNS), where Thpo is thought to exert pro-apoptotic effects on newly generated neurons. Mpl expression has been analysed in brain tissue on transcript level and in cultured primary rat neurons and astrocytes on protein level. Herein, we analysed Mpl expression in the developing and adult murine CNS by immunohistochemistry and investigated the brain of mice with homozygous Mpl deficiency (Mpl-/-) by MRI.
Results
Mpl was not detectable at developmental stages E12 to E15 in any resident cells of the CNS. From E18 onwards, robust Mpl expression was found in various brain areas, including cerebral cortex, olfactory bulb, thalamus, hypothalamus, medulla, pons, and the grey matter of spinal cord. However, major developmental changes became obvious: In the subventricular zone of the cerebral cortex Mpl expression occurred only during late gestation, while in the hippocampus Mpl expression was detectable for first time at stage P4. In the white matter of the cerebellum Mpl expression was restricted to the perinatal period. In the adult cerebellum, Mpl expression switched to Purkinje cell. The majority of other Mpl-positive cells were NeuN-positive neurons. None of the cells could be double-labelled with astrocyte marker GFAP. Mpl-/- mice showed no gross abnormalities of the brain.
Conclusions
Our data locate Mpl expression to neurons at different subdivisions of the spinal cord, rhombencephalon, midbrain and prosencephalon. Besides neuronal cells Mpl protein is also expressed in Purkinje cells of the adult cerebellum.
doi:10.1186/1471-213X-10-77
PMCID: PMC2921376  PMID: 20667107
10.  Pharmacokinetic/Pharmacodynamic Modeling of Corticosterone Suppression and Lymphocytopenia by Methylprednisolone in Rats 
Journal of pharmaceutical sciences  2008;97(7):2820-2832.
Adrenal suppression and lymphocytopenia are commonly monitored pharmacological responses during systemic exposure to exogenously administered corticosteroids. The pharmacodynamics of plasma corticosterone (CS) and blood lymphocytes were investigated in 60 normal rats which received either 50 mg/kg methylprednisolone (MPL) or vehicle intramuscularly. Blood samples were collected between 0.5 and 96 h following treatment. Plasma CS displayed a transient suppression with re-establishment of a normal circadian rhythm 24 h following drug treatment. An indirect response model with suppression of production well captured plasma CS profiles. An early stress-induced rise in CS was also factored into the model. Blood lymphocyte numbers exhibited a sharp decline and then returned to a new circadian rhythm which was half of the original baseline level. An integrated pharmacodynamic (PD) model with inhibition of lymphocyte trafficking from tissue to blood by both MPL and CS and induction of cell apoptosis by MPL reasonably captured this lymphocytopenia. Rats and humans differ in lymphocyte responses with humans showing full recovery of baselines. Modeling provides a valuable tool in quantitative assessment of dual, complex drug responses.
doi:10.1002/jps.21167
PMCID: PMC3726057  PMID: 17828751
pharmacokinetics; pharmacodynamics; hormones; mathematical model; pharmacokinetic/pharmacodynamic models; corticosteroid; lymphocyte; cell trafficking; indirect response model; circadian rhythm
11.  Lipopolysaccharide and monophosphoryl lipid A differentially regulate interleukin-12, gamma interferon, and interleukin-10 mRNA production in murine macrophages. 
Infection and Immunity  1997;65(8):3239-3247.
Monophosphoryl lipid A (MPL) is a nontoxic derivative of the lipid A region of lipopolysaccharide (LPS) that is being developed as both an adjuvant and prophylactic drug for septic shock. We compared the ability of LPS and MPL to induce interleukin-10 (IL-10), IL-12 p35, IL-12 p40, gamma interferon (IFN-gamma), glucocorticoid receptor (GR), IL-1 receptor antagonist (IL-1ra), and inducible nitric oxide synthase mRNA expression in murine peritoneal macrophages. These genes were chosen for their ability to positively or negatively regulate the host immune response and thus for their potential involvement in MPL-induced adjuvanticity or in its ability to protect against sepsis. LPS was a more potent inducer of IL-12 p35, IL-12 p40, and IFN-gamma mRNA, as well as of IL-12 protein, than MPL. In contrast, MPL induced higher levels of IL-10 mRNA than did LPS from 1 to 1,000 ng/ml. In general, MPL was not a more potent inducer of negative regulatory genes, since MPL and LPS induced similar levels of GR and IL-1ra mRNA. Addition of anti-IL-10 antibody to cultures increased the induction of MPL-induced IL-12 p35, IL-12 p40, and IFN-gamma mRNA, suggesting that the enhanced production of IL-10 by MPL-stimulated macrophages contributes to decreased production of mRNA for IL-12 (p35 and p40) and IFN-gamma. Conversely, the addition of exogenous IL-10 to LPS-treated macrophages reduced the mRNA expression of these cytokine genes. These studies suggest that enhanced production of IL-10 by MPL-stimulated macrophages may contribute to the reduced toxicity of MPL through its negative action on induction of cytokines shown to enhance endotoxicity.
PMCID: PMC175458  PMID: 9234781
12.  The thrombopoietin receptor, c-Mpl, is a selective surface marker for human hematopoietic stem cells 
Background
Thrombopoietin (TPO), the primary cytokine regulating megakaryocyte proliferation and differentiation, exerts significant influence on other hematopoietic lineages as well, including erythroid, granulocytic and lymphoid lineages. We previously demonstrated that the receptor for TPO, c-mpl, is expressed by a subset of human adult bone marrow hematopoietic stem/progenitor cells (HSC/PC) that are enriched for long-term multilineage repopulating ability in the SCID-hu Bone in vivo model of human hematopoiesis.
Methods
Here, we employ flow cytometry and an anti-c-mpl monoclonal antibody to comprehensively define the surface expression pattern of c-mpl in four differentiation stages of human CD34+ HSC/PC (I: CD34+38--, II: CD34+38dim, III: CD34+38+, IV: CD34dim38+) for the major sources of human HSC: fetal liver (FL), umbilical cord blood (UCB), adult bone marrow (ABM), and cytokine-mobilized peripheral blood stem cells (mPBSC). We use a surrogate in vivo model of human thymopoiesis, SCID-hu Thy/Liv, to compare the capacity of c-mpl+ vs. c-mpl-- CD34+38--/dim HSC/PC for thymocyte reconstitution.
Results
For all tissue sources, the percentage of c-mpl+ cells was significantly highest in stage I HSC/PC (FL 72 ± 10%, UCB 67 ± 19%, ABM 82 ± 16%, mPBSC 71 ± 15%), and decreased significantly through stages II, III, and IV ((FL 3 ± 3%, UCB 8 ± 13%, ABM 0.6 ± 0.6%, mPBSC 0.2 ± 0.1%) [ANOVA: P < 0.0001]. The relative median fluorescence intensity of c-mpl expression was similarly highest in stage I, decreasing through stage IV [ANOVA: P < 0.0001]. No significant differences between tissue sources were observed for either % c-mpl+ cells [P = 0.89] or intensity of c-mpl expression [P = 0.21]. Primary Thy/Liv grafts injected with CD34+38--/dimc-mpl+ cells showed slightly higher levels of donor HLA+ thymocyte reconstitution vs. CD34+38--/dimc-mpl---injected grafts and non-injected controls (c-mpl+ vs. c-mpl--: CD2+ 6.8 ± 4.5% vs. 2.8 ± 3.3%, CD4+8-- 54 ± 35% vs. 31 ± 29%, CD4--8+ 29 ± 19% vs. 18 ± 14%).
Conclusion
These findings support the hypothesis that the TPO receptor, c-mpl, participates in the regulation of primitive human HSC from mid-fetal through adult life. This study extends our previous work documenting human B-lineage, myeloid and CD34+ cell repopulation by c-mpl+ progenitors to show that c-mpl+ HSC/PC are also capable of significant T-lineage reconstitution in vivo. These results suggest that c-mpl merits consideration as a selective surface marker for the identification and isolation of human HSC in both basic research and clinical settings.
doi:10.1186/1479-5876-4-9
PMCID: PMC1402332  PMID: 16480521
13.  Tissue-Specific Gene Expression and Regulation in Liver and Muscle Following Chronic Corticosteroid Administration 
Although corticosteroids (CSs) affect gene expression in multiple tissues, the array of genes that are regulated by these catabolic steroids is diverse, highly tissue specific, and depends on their functions in the tissue. Liver has many important functions in performing and regulating diverse metabolic processes. Muscle, in addition to its mechanical role, is critical in maintaining systemic energy homeostasis and accounts for about 80% of insulin-directed glucose disposal. Consequently, a better understanding of CS pharmacogenomic effects in these tissues would provide valuable information regarding the tissue-specificity of transcriptional dynamics, and would provide insights into the underlying molecular mechanisms of action for both beneficial and detrimental effects.
We performed an integrated analysis of transcriptional data from liver and muscle in response to methylprednisolone (MPL) infusion, which included clustering and functional annotation of clustered gene groups, promoter extraction and putative transcription factor (TF) identification, and finally, regulatory closeness (RC) identification.
This analysis allowed the identification of critical transcriptional responses and CS-responsive functions in liver and muscle during chronic MPL administration, the prediction of putative transcriptional regulators relevant to transcriptional responses of CS-affected genes which are also potential secondary bio-signals altering expression levels of target-genes, and the exploration of the tissue-specificity and biological significance of gene expression patterns, CS-responsive functions, and transcriptional regulation.
The analysis provided an integrated description of the genomic and functional effects of chronic MPL infusion in liver and muscle.
doi:10.4137/GRSB.S13134
PMCID: PMC3956809  PMID: 24653645
liver; muscle; glucocorticoids; corticosteroids; gene expression; gene regulation; promoter analysis
14.  Effects of Clinically Relevant MPL Mutations in the Transmembrane Domain Revealed at the Atomic Level through Computational Modeling 
PLoS ONE  2011;6(8):e23396.
Background
Mutations in the thrombopoietin receptor (MPL) may activate relevant pathways and lead to chronic myeloproliferative neoplasms (MPNs). The mechanisms of MPL activation remain elusive because of a lack of experimental structures. Modern computational biology techniques were utilized to explore the mechanisms of MPL protein activation due to various mutations.
Results
Transmembrane (TM) domain predictions, homology modeling, ab initio protein structure prediction, and molecular dynamics (MD) simulations were used to build structural dynamic models of wild-type and four clinically observed mutants of MPL. The simulation results suggest that S505 and W515 are important in keeping the TM domain in its correct position within the membrane. Mutations at either of these two positions cause movement of the TM domain, altering the conformation of the nearby intracellular domain in unexpected ways, and may cause the unwanted constitutive activation of MPL's kinase partner, JAK2.
Conclusions
Our findings represent the first full-scale molecular dynamics simulations of the wild-type and clinically observed mutants of the MPL protein, a critical element of the MPL-JAK2-STAT signaling pathway. In contrast to usual explanations for the activation mechanism that are based on the relative translational movement between rigid domains of MPL, our results suggest that mutations within the TM region could result in conformational changes including tilt and rotation (azimuthal) angles along the membrane axis. Such changes may significantly alter the conformation of the adjacent and intrinsically flexible intracellular domain. Hence, caution should be exercised when interpreting experimental evidence based on rigid models of cytokine receptors or similar systems.
doi:10.1371/journal.pone.0023396
PMCID: PMC3157383  PMID: 21858098
15.  Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway. 
Molecular and Cellular Biology  1997;17(9):4991-5000.
Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation.
PMCID: PMC232350  PMID: 9271377
16.  Utility of cleavable isotope coded affinity tagged reagents for quantification of low-copy proteins induced by methylprednisolone using liquid chromatography/tandem mass spectrometry 
Analytical chemistry  2006;78(13):4543-4552.
Gene expression changes underlie important biological and pharmacological responses. Although mRNA expression profiling is routine, quantification of low abundance proteins, which typically represent key effectors of responses, remains challenging. A novel strategy was developed for sensitive and accurate quantification of low abundance proteins in highly complex biological matrices. First, the cysteine specificity of cleavable isotope-coded affinity tags (cICAT) was employed to reduce the complexity of the digested proteome of tissue homogenates, and to improve the quantification of low abundance proteins. Second, cICAT treated tissue samples were analyzed on a capillary LC coupled to an ion-trap MS to screen for the subset of cICAT-peptides, derived from target proteins of interest, that was successfully labeled and retrieved. Third, putatively identified peptides derived from target proteins were synthesized, cICAT-labeled, and used both to optimize multiple reactions monitoring (MRM) analysis and to confirm chromatographic retention time and fragmentation pattern. Finally, batch quantification of target peptides was performed using MRM on a LC/triple-quad MS/MS using 12C- (control) and 13C (experimental) -cICAT labeled tissue mixtures. The utility of this method was demonstrated by elucidating the time course of tyrosine aminotransferase (TAT) induction in the liver of rats following treatment with the corticosteroid methylprednisolone (MPL). This approach significantly improved quantitative sensitivity, and the linear range was 10-fold greater than published previously. An additional advantage is that archived samples may be re-interrogated to investigate the regulation of additional targets that become of interest. Stored samples were sucessfully re-interrogated to monitor the induction of ornithine decarboxylase (ODC), which is also an MPL-induced protein. To our knowledge, this is the first report of an ICAT-based method capable of quantifying low abundance proteins in highly complex samples such as tissue homogenates. The approach enables simultaneous quantification of multiple effector proteins induced by biological or pharmacological stimuli, and the processed samples can be interrogated repeatedly as additional targets of interest arise.
doi:10.1021/ac0521697
PMCID: PMC2516203  PMID: 16808464
17.  Dissociation of lipopolysaccharide (LPS)-inducible gene expression in murine macrophages pretreated with smooth LPS versus monophosphoryl lipid A. 
Infection and Immunity  1993;61(6):2325-2333.
Lipopolysaccharide (LPS) and the nontoxic derivative of lipid A, monophosphoryl lipid A (MPL), were employed to assess the relationship between expression of LPS-inducible inflammatory genes and the induction of tolerance to LPS in murine macrophages. Both LPS and MPL induced expression (as assessed by increased steady-state mRNA levels) of a panel of seven "early" inflammatory genes including the tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, type 2 TNF receptor (TNFR-2), IP-10, D3, D8, and D2 genes (the last four represent LPS-inducible early genes whose functions remain unknown). In addition, LPS and MPL were both capable of inducing tolerance to LPS. The two stimuli differed in the relative concentration required to induce various outcome measures, with LPS being 100- to 1,000-fold more potent on a mass concentration basis. Characterization of the tolerant state identified three distinct categories of responsiveness. Two genes (IP-10 and D8) exhibited strong desensitization in macrophages pretreated with tolerance-inducing concentrations of either LPS or MPL. In macrophages rendered tolerant by pretreatment with LPS or MPL, a second group of inducible mRNAs (TNF-alpha, interleukin-1 beta, and D3) showed moderate suppression of response to secondary stimulation by LPS. The third category of inducible genes (TNFR-2 and D2) showed increased expression in macrophages pretreated with tolerance-inducing concentrations of either LPS or MPL. All of the LPS-inducible genes examined exhibited modest superinduction with less than tolerance-inducing concentrations of either stimulus, suggesting a priming effect of these adjuvants at low concentration. The differential behavior of the members of this panel of endotoxin-responsive genes thus offers insight into molecular events associated with acquisition of transient tolerance to LPS.
PMCID: PMC280852  PMID: 8388859
18.  Pulmonary and Hepatic Gene Expression following Cecal Ligation and Puncture: Monophosphoryl Lipid A Prophylaxis Attenuates Sepsis-Induced Cytokine and Chemokine Expression and Neutrophil Infiltration 
Infection and Immunity  1998;66(8):3569-3578.
Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1β, IL-6, gamma interferon (IFN-γ), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-γ-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-α, MCP-1, MIP-1α, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.
PMCID: PMC108388  PMID: 9673235
19.  Live-Cell Visualization of Intracellular Interaction between a Nuclear Migration Protein (hNUDC) and the Thrombopoietin Receptor (Mpl) 
PLoS ONE  2012;7(12):e51849.
We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.
doi:10.1371/journal.pone.0051849
PMCID: PMC3524126  PMID: 23284788
20.  Gene Expression Profiles of Chlamydophila pneumoniae during the Developmental Cycle and Iron Depletion–Mediated Persistence 
PLoS Pathogens  2007;3(6):e83.
The obligate intracellular, gram-negative bacterium Chlamydophila pneumoniae (Cpn) has impact as a human pathogen. Little is known about changes in the Cpn transcriptome during its biphasic developmental cycle (the acute infection) and persistence. The latter stage has been linked to chronic diseases. To analyze Cpn CWL029 gene expression, we designed a pathogen-specific oligo microarray and optimized the extraction method for pathogen RNA. Throughout the acute infection, ratio expression profiles for each gene were generated using 48 h post infection as a reference. Based on these profiles, significantly expressed genes were separated into 12 expression clusters using self-organizing map clustering and manual sorting into the “early”, “mid”, “late”, and “tardy” cluster classes. The latter two were differentiated because the “tardy” class showed steadily increasing expression at the end of the cycle. The transcriptome of the Cpn elementary body (EB) and published EB proteomics data were compared to the cluster profile of the acute infection. We found an intriguing association between “late” genes and genes coding for EB proteins, whereas “tardy” genes were mainly associated with genes coding for EB mRNA. It has been published that iron depletion leads to Cpn persistence. We compared the gene expression profiles during iron depletion–mediated persistence with the expression clusters of the acute infection. This led to the finding that establishment of iron depletion–mediated persistence is more likely a mid-cycle arrest in development rather than a completely distinct gene expression pattern. Here, we describe the Cpn transcriptome during the acute infection, differentiating “late” genes, which correlate to EB proteins, and “tardy” genes, which lead to EB mRNA. Expression profiles during iron mediated–persistence led us to propose the hypothesis that the transcriptomic “clock” is arrested during acute mid-cycle.
Author Summary
Chlamydophila (Chlamydia) pneumoniae (Cpn) accounts for approximately one-tenth of the cases of community-acquired pneumonia worldwide, and persistent Cpn infections are thought to be associated with a variety of chronic diseases. Little is known about Cpn transcriptome changes during its biphasic developmental cycle (the acute infection) and persistence stages. Iron limitation, among several other treatments, has recently been shown to lead to persistent Cpn infection. How this pathogen reacts to iron-limiting host defense mechanisms is of great interest, as iron is an important factor affecting virulence. This article reports on the Cpn transcriptome during the developmental cycle and iron depletion–mediated persistence and reveals that genes coding for proteins of the infectious particle (the elementary body [EB]) were expressed constantly at the end of the cycle. In contrast, genes contributing to EB mRNA but not to EB protein showed an increasing expression at the end of the cycle. This suggested that most EB proteins are made in mid-cycle, and the redifferentiation process is initiated only by a limited number of genes. During iron depletion–mediated persistence, the Cpn transcriptome was altered in such a way that an arrest in Cpn gene expression can be proposed.
doi:10.1371/journal.ppat.0030083
PMCID: PMC1894823  PMID: 17590080
21.  The Propeptide of the Metalloprotease of Listeria monocytogenes Controls Compartmentalization of the Zymogen during Intracellular Infection▿  
Journal of Bacteriology  2009;191(11):3594-3603.
Integral to the virulence of the intracellular bacterial pathogen Listeria monocytogenes is its metalloprotease (Mpl). Mpl regulates the activity and compartmentalization of the bacterial broad-range phospholipase C (PC-PLC). Mpl is secreted as a proprotein that undergoes intramolecular autocatalysis to release its catalytic domain. In related proteases, the propeptide serves as a folding catalyst and can act either in cis or in trans. Propeptides can also influence protein compartmentalization and intracellular trafficking or decrease folding kinetics. In this study, we aimed to determine the role of the Mpl propeptide by monitoring the behavior of Mpl synthesized in the absence of its propeptide (MplΔpro) and of two Mpl single-site mutants with unstable propeptides: Mpl(H75V) and Mpl(H95L). We observed that all three Mpl mutants mediate PC-PLC activation when bacteria are grown on semisolid medium, but to a lesser extent than wild-type Mpl, indicating that, although not essential, the propeptide enhances the production of active Mpl. However, the mutant proteins were not functional in infected cells, as determined by monitoring PC-PLC maturation and compartmentalization. This defect could not be rescued by providing the propeptide in trans to the mplΔpro mutant. We tested the compartmentalization of Mpl during intracellular infection and observed that the mutant Mpl species were aberrantly secreted in the cytosol of infected cells. These data indicated that the propeptide of Mpl serves to maintain bacterium-associated Mpl and that this localization is essential to the function of Mpl during intracellular infection.
doi:10.1128/JB.01168-08
PMCID: PMC2681923  PMID: 19346305
22.  Mechanisms of Monophosphoryl Lipid A Augmentation of Host Responses to Recombinant HagB from Porphyromonas gingivalis  
Infection and Immunity  2002;70(7):3557-3565.
Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection to P. gingivalis infection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 μg) alone or with MPL (25 μg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P < 0.05) and serum IgG (P < 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P < 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was ≈1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.
doi:10.1128/IAI.70.7.3557-3565.2002
PMCID: PMC128110  PMID: 12065496
23.  Evaluation of Leptospiral Recombinant Antigens MPL17 and MPL21 for Serological Diagnosis of Leptospirosis by Enzyme-Linked Immunosorbent Assays ▿  
Clinical and Vaccine Immunology : CVI  2008;15(11):1715-1722.
Leptospirosis is a zoonosis of multisystem involvement caused by pathogenic strains of the genus Leptospira. In the last few years, intensive studies aimed at the development of a vaccine have provided important knowledge about the nature of the immunological mechanisms of the host. The purpose of this study was to analyze the immune responses to two recombinant proteins, MPL17 and MPL21 (encoded by the genes LIC10765 and LIC13131, respectively) of Leptospira interrogans serovar Copenhageni in individuals during infection. The recombinant proteins were expressed in Escherichia coli as six-His tag fusion proteins and were purified from the soluble bacterial fraction by affinity chromatography with Ni2+-charged resin. The recombinant proteins were used to evaluate their ability to bind to immunoglobulin G (IgG) (and IgG subclass) or IgM antibodies in serum samples from patients in the early and convalescent phases of leptospirosis (n = 52) by enzyme-linked immunosorbent assays. The prevalences of total IgG antibodies against MPL17 and MPL21 were 38.5% and 21.2%, respectively. The titers achieved with MPL17 were statistically significantly higher than those obtained by the reference microscopic agglutination test. The specificity of the assay was estimated to be 95.5% for MPL17 and 80.6% for MPL21 when serum samples from individuals with unrelated febrile diseases and control healthy donors were tested. The proteins are conserved among Leptospira strains that cause human and animal diseases. MPL17 and MPL21 are most likely new surface proteins of leptospires, as revealed by liquid-phase immunofluorescence assays with living organisms. Our results demonstrate that these recombinant proteins are highly immunogenic and, when they are used together, might be useful as a means of diagnosing leptospirosis.
doi:10.1128/CVI.00214-08
PMCID: PMC2583518  PMID: 18799647
24.  Protective effects on myelosuppression mice treated by three different classic Chinese medicine formulae 
Pharmacognosy Magazine  2011;7(26):133-140.
Background:
In order to observe the protective therapeutic action and mechanism of Liuwei Dihuang Decoction, Buzhong Yiqi Decoction, and Compound Danshen Decoction on Myelosuppression induced by cyclophosphamide.
Materials and Methods:
The mice model was established by intraperitoneal injected with 100 mg/kg cyclophosphamide by human and mice dose conversion on the 9th, 11th, 13th days during the experiment. Flow cytometry (FCM) was used for detecting the number of cells and investigating bone marrow cell cycles. Spleen was taken out and the mRNA expression level of thrombopoietin (TPO) and c-Mpl were detected by Q-PCR, and c-Mpl in spleen in order to discuss the mechanism of myelosuppression and the protective effects of traditional Chinese medicine.
Results:
Both Liuwei Dihuang Decoction Group and Buzhong Yiqi Decoction Group can accelerate bone marrow hematopoietic stem progenitor cells (HSPCs) in marrow-suppressed mice and enhance cell proliferation by promoting cell cycles from G0/G1 phase to access into S, G2/M phase. And at the same time these Chinese decoctions can increase the mRNA expression level of TPO and c-Mpl in spleen.
Conclusion:
Researched showed that Chinese formula take effect by affecting these genes on myelosuppressed mice.
doi:10.4103/0973-1296.80671
PMCID: PMC3113352  PMID: 21716623
Buzhong Yiqi decoction; cell cycle; compound Danshen decoction; Liuwei Dihuang decoction; myelosuppression; thrombopoietin; thrombopoietin receptor
25.  Adjuvant Formulations Possess Differing Efficacy in the Potentiation of Antibody and Cell Mediated Responses to a Human Malaria Vaccine under Selective Immune Genes Knockout Environment 
International immunopharmacology  2008;8(7):1012-1022.
Infections and chronic diseases can alter the host’s immunological balance or result in immuno-deficiencies. We hypothesize that this may also affect the performance of vaccine adjuvants. Accordingly, the potency and adjuvanticity of eight adjuvant formulations based on Montanide ISA720, MF59, monophosphoryl lipid A (MPL), QS21 (saponin derivative), MPL-SE (stable emulsion of a MPL derivative), and MPL-AF (MPL in aqueous formulation) were studied in immune gene knockout mice, IFN-γ −/−, IL-4 −/−, and STAT6 −/−, using the P. falciparum MSP1 vaccine, P30P2MSP1-19 as a model immunogen. The adjuvants showed preferential requirements for the immune mediators to induce immune responses to MSP1-19, and the effects were formulation-specific. While emulsion-type adjuvants were highly effective in mice, their potency was more readily suppressed by immune knockouts; and additions of immunomodulators were required to restore efficacy. Formulated adjuvants had characteristics distinct from their individual components, and multi-components formulations were not necessarily superior. We conclude that perturbation of immune environments will have measurable impact on adjuvant’s potency. Evaluation of adjuvants in immune knockout models may be a supplementary approach to measure and compare adjuvants’ efficacy, and to further unveil their distinct biological activities.
doi:10.1016/j.intimp.2008.03.005
PMCID: PMC2464358  PMID: 18486913

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