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1.
Physiol Plant ; 166(2): 596-611, 2019 Jun.
Article in English | MEDLINE | ID: mdl-30175846

ABSTRACT

Understanding the molecular and physiological mechanisms of trait diversity is crucial for crop improvement to achieve drought adaptation. Root traits such as high biomass and/or deep rootedness are undoubtedly important drought adaptive traits. The major aim of this investigation was to functionally characterize a set of ethyl methane sulfonate-induced rice mutants for root traits. We report the identification of a high-root biomass mutant through a novel screening strategy for yield and Δ13 C measurements. The high-root mutant (392-9-1) thus identified, had a 66% higher root biomass compared to wild-type (Nagina-22). Better maintenance of leaf turgor and carbon assimilation rates resulted in lower drought susceptibility index in 392-9-1. Targeted resequencing revealed three non-synonymous single nucleotide variations in 392-9-1 for the genes HOX10, CITRATE SYNTHASE and ZEAXANTHIN EPOXIDASE. Segregation pattern of phenotype and mutant alleles in a single parent backcross F2 population revealed a typical 3:1 segregation for each of the mutant alleles. The number of F2 progeny with root biomass equal to or greater than that of 392-9-1 represented approximately one-third of the population indicating a major role played by HOX10 gene in regulating root growth in rice. Allele-specific Sanger sequencing in contrasting F2 progenies confirmed the co-segregation of HOX10 allele with the root biomass. The non-synonymous mutations in the other two genes did not reveal any specific pattern of co-segregation with root phenotype, indicating a strong role of HOX10, an upstream transcription factor, in regulating root biomass in rice.


Subject(s)
Oryza/growth & development , Oryza/metabolism , Plant Roots/growth & development , Plant Roots/metabolism , Transcription Factors/metabolism , Alleles , Biomass , Oryza/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Transcription Factors/genetics
2.
Cell Death Dis ; 5: e1145, 2014 Mar 27.
Article in English | MEDLINE | ID: mdl-24675463

ABSTRACT

Glioblastoma Multiforme (GBM) is an aggressive adult primary brain tumor with poor prognosis. GBM patients develop resistance to the frontline chemotherapy, temozolomide (TMZ). As the connexins (Cx) have been shown to have a complex role in GBM, we investigated the role of Cx43 in TMZ resistance. Cx43 was increased in the TMZ-resistant low passage and cell lines. This correlated with the data in The Cancer Genome Atlas. Cx43 knockdown, reporter gene assays, chromatin immunoprecipitation assay, real-time PCR and western blots verified a role for Cx43 in TMZ resistance. This occurred by TMZ-resistant GBM cells being able to activate epidermal growth factor receptor (EGFR). In turn, EGFR activated the JNK-ERK1/2-AP-1 axis to induce Cx43. The increased Cx43 was functional as indicated by gap junctional intercellular communication among the resistant GBM cells. Cell therapy could be a potential method to deliver drugs, such as anti-EGF to tumor cells. Similar strategies could be used to reverse the expression of Cx43 to sensitize GBM cells to TMZ. The studies showed the potential for targeting EGF in immune therapy. These agents can be used in conjunction with stem cell therapy to treat GBM.


Subject(s)
Connexin 43/metabolism , Dacarbazine/analogs & derivatives , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/metabolism , Glioblastoma/drug therapy , Glioblastoma/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/enzymology , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Survival/drug effects , Coloring Agents/metabolism , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Knockdown Techniques , Glioblastoma/enzymology , Glioblastoma/pathology , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Protein Binding , Signal Transduction/drug effects , Temozolomide , Transcription Factor AP-1/metabolism
3.
Curr Pharmacogenomics Person Med ; 8(1): 25-36, 2010 Mar 01.
Article in English | MEDLINE | ID: mdl-20563265

ABSTRACT

The euphoria of stem cell therapy has diminished, allowing scientists, clinicians and the general public to seriously re-examine how and what types of stem cells would effectively repair damaged tissue, prevent further tissue damage and/or replace lost cells. Importantly, there is a growing recognition that there are substantial person-to-person differences in the outcome of stem cell therapy. Even though the small molecule pharmaceuticals have long remained a primary focus of the personalized medicine research, individualized or targeted use of stem cells to suit a particular individual could help forecast potential failures of the therapy or identify, early on, the individuals who might benefit from stem cell interventions. This would however demand collaboration among several specialties such as pharmacology, immunology, genomics and transplantation medicine. Such transdisciplinary work could also inform how best to achieve efficient and predictable stem cell migration to sites of tissue damage, thereby facilitating tissue repair. This paper discusses the possibility of polarizing immune responses to rationalize and individualize therapy with stem cell interventions, since generalized "one-size-fits-all" therapy is difficult to achieve in the face of the diverse complexities posed by stem cell biology. We also present the challenges to stem cell delivery in the context of the host related factors. Although we focus on the mesenchymal stem cells in this paper, the overarching rationale can be extrapolated to other types of stem cells as well. Hence, the broader purpose of this paper is to initiate a dialogue within the personalized medicine community by expanding the scope of inquiry in the field from pharmaceuticals to stem cells and related cell-based health interventions.

4.
Curr Med Chem ; 17(20): 2159-67, 2010.
Article in English | MEDLINE | ID: mdl-20423304

ABSTRACT

Implantation of adult human mesenchymal stem cells (MSCs) to treat neural disorders shows promise. Depending on their microenvironment, MSCs could potentially be used for the repair and/or replacement of neurons in traumatic brain injury or the treatment of Parkinson's disease. This cross-disciplinary review incorporates aspects of neuroscience, stem cell biology, cancer biology and immunology to discuss interactions between inflammatory mediators and MSCs. We first discuss the role of microRNAs (miRNAs) in neurological development. Secondly, we discuss the ability of MSCs to transdifferentiate into functional neurons, which are regulated by miRNAs, and the implications of these cells for the therapy of neuropathological states. The administration of effective and safe MSC therapy must acknowledge immune mediators that may predispose the early differentiating MSCs to oncogenic insults. Thus, we discuss a key gene, RE-1 silencing transcription factor (REST), based on its dual role in neurogenesis and cancer development. Immune mediators could be central to MSC responses within a region of tissue injury and are also discussed in detail. Exploring the predisposition of MSCs to oncogenesis is critical for translational science since the implementation of safeguarding measures prior to therapy can lead to the successful delivery of stem cells to patients. The method by which MSCs could be applied for future therapies might require trans-disciplinary approaches for personalized treatments.


Subject(s)
Mesenchymal Stem Cells/cytology , MicroRNAs/metabolism , Neurogenesis/physiology , Brain Injuries/therapy , Humans , Inflammation Mediators/metabolism , Mesenchymal Stem Cell Transplantation , Neurons/cytology , Neurons/metabolism , Parkinson Disease/therapy , Repressor Proteins/metabolism , Repressor Proteins/physiology
5.
Minerva Cardioangiol ; 58(1): 127-46, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20145600

ABSTRACT

Despite advances in clinical interventions, drug therapy and preventative strategies for cardiovascular disease, heart disease remains the number one cause of death in the United States. A major cause of heart failure leading to death is myocardial ischemic disease. Terminal heart failure can be salvaged in some cases by cardiac transplants, but this therapeutic approach is limited by lack of supply, high cost, and problems with immunosuppression. An attractive alternative approach proposed over the last 1-2 decades is the replacement of myocardium at the level of the myocyte, which has focused on stem cell therapy. This form of therapy has been successful for hematopoietic replacement. Similar therapy has been proposed to treat hearts ravaged by ischemic necrosis and apoptosis. However, the experimental studies have not been effectively translated to patients with myocardial infarction or heart failure. This review discusses the current literature and points out key studies that are required for future directions, focusing on key roles for microenvironmental factors, such as cytokines, in stem cells responses when placed at sites of cardiac injuries. In the case of mesenchymal stem cells (MSCs), they exert both immune- enhancer and -suppressor functions, which are referred to as immune plasticity. This type of immune properties by MSCs is significant to therapeutic outcomes. Thus, the plasticity of MSCs, with regards to immune responses, has to be considered carefully in tissue repair and replacement and in gene delivery systems. The route by which cytokines are delivered as adjuvant to cell therapy, or as methods to mobilize stem cells, will show varied results, depending on the degree of injury, underlying clinical disorders and other diverse parameters, such as ethnicity, age and genomic profile. In addition to MSCs, roles exist for other stem cells, such as those from placenta, cord blood, hematopoietic stem/progenitor cells and cardiac stem cells.


Subject(s)
Heart Diseases/surgery , Stem Cell Transplantation , Cytokines/physiology , Heart Diseases/immunology , Humans , Inflammation/etiology
6.
J Prim Prev ; 26(5): 439-54, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16215693

ABSTRACT

The Family and Community Violence Prevention (FCVP) Program was established in 1994 to address the escalation of youth violence among ethnic minorities. This federally funded program adapted the public health model and organized Family Life Centers throughout the country to serve youth who were considered to be at risk for violence and other abusive behaviors. The purpose of this three-year study, 1999-2002, was to determine the effectiveness of the FCVP Program's six-component curriculum in reducing violence among participants. Results from posttest scores of 2,315 youth showed girls 12 and over to be most at risk for deviant behaviors; the program was most effective with boys under age 12. Academic performance and bonding to school were protective factors whereas exposure to violence was a risk factor for all four ethnic groups studied--African Americans, Hispanics, Native Americans, and Native Hawaiians. EDITORS' STRATEGIC IMPLICATIONS: Cultural anthropologists, public health specialists, and school officials should know that prevention programs can be designed to reflect the unique, culturally appropriate norms of specific ethnic minority groups, even as these programs address shared risk factors. The authors discuss the promising strategy of enhancing academic performance and school bonding to serve as protective factors against school violence, but they also describe age, gender, and cultural differences that must be addressed in future research.


Subject(s)
Child Behavior Disorders/prevention & control , Cultural Diversity , Curriculum , Minority Groups/psychology , School Health Services , Social Behavior Disorders/prevention & control , Students/psychology , Violence/prevention & control , Adolescent , Age Factors , Child , Child Behavior Disorders/ethnology , Female , Humans , Male , Minority Groups/education , Models, Psychological , Program Development , Program Evaluation , Risk Assessment , Risk Factors , Sex Factors , Social Behavior Disorders/ethnology , United States , Violence/ethnology
7.
Physiol Behav ; 82(2-3): 581-7, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15276825

ABSTRACT

The present study sought to determine the effects of kindled seizures generated from the left and right amygdala upon weight gain in rat. Seventy-five female Sprague-Dawley rats were implanted with electrodes in basal amygdala of the left and right hemispheres. A kindling paradigm was employed in which electrical stimulation was applied once per day for 30 days after Stage 5 seizures. An electrode was implanted into the basal amygdala of the control rats but no stimulation was applied. All rats were weighed daily during the course of the experiment and changes in weight during this period were recorded for all rats. The results demonstrated that kindling from either the left or right amygdala induced significant increases in weight gain relative to the control rats. However, kindling from the left basal amygdala induced increases in body weight that were four times greater than control rats and two times greater than the rats kindled from the right side of the basal amygdala. Likewise, serum leptin levels, which were highly correlated with weight gain, also showed significantly greater increases in left amygdaloid kindled rats relative to rats kindled from the right amygdala and control rats. These findings demonstrate that basal amygdaloid kindling induces significant increases in weight gain and that the magnitude of these effects is linked to the dominance of the left hemisphere.


Subject(s)
Amygdala/physiology , Dominance, Cerebral/physiology , Kindling, Neurologic/physiology , Leptin/blood , Weight Gain/physiology , Analysis of Variance , Animals , Electric Stimulation , Energy Metabolism/physiology , Female , Rats , Rats, Sprague-Dawley , Seizures/metabolism
8.
Acta Haematol ; 109(1): 1-10, 2003.
Article in English | MEDLINE | ID: mdl-12486316

ABSTRACT

Bone marrow (BM) fibrosis could occur secondarily to several clinical disorders: hematological and nonhematological. Clinical presentation of fibrosis could occur in myeloproliferative diseases, lymphoma, myelodysplastic syndrome and myeloma. The pathophysiology underlying BM fibrosis remains unclear despite intensive study, with a corresponding lack of specific therapy. This review discusses new insights in the role of substance P, cytokines and fibronectin in the development of BM fibrosis. Substance P is a neuropeptide that possesses pleiotropic properties, e.g. neurotransmission and immune/hematopoietic modulation and is linked to BM fibrosis. Cytokines and growth factors, in particular those associated with fibrogenic properties, e.g. TGF-beta, IL-1 and platelet-derived growth factor, are linked to BM fibrosis. Extracellular matrix proteins are increased in patients with BM fibrosis. Fibronectin in the sera of patients with BM fibrosis is complexed to substance P. Fibronectin appears to protect substance P from degradation by endogenous peptidases. This review describes the preliminary findings on the colocalization of substance P and fibronectin in the BM of patients with fibrosis. These data are reviewed in the context of published reports with particular focus on the relevant cytokines. A more detailed understanding of intra- and intercellular mechanisms in BM fibrosis may lead to effective therapy.


Subject(s)
Cytokines/metabolism , Fibronectins/metabolism , Myeloproliferative Disorders/metabolism , Primary Myelofibrosis/metabolism , Substance P/metabolism , Drug Interactions/physiology , Humans , Myeloproliferative Disorders/complications , Myeloproliferative Disorders/pathology , Primary Myelofibrosis/etiology , Primary Myelofibrosis/pathology , Protein Binding
9.
Neuropeptides ; 36(1): 13-21, 2002 Feb.
Article in English | MEDLINE | ID: mdl-12147210

ABSTRACT

The neurokinin-1 (NK-1) receptor interacts with peptides that belong to the tachykinin family. NK-1 is inducible in bone marrow (BM) stroma. In neural cells, its expression is high to constitutive. Screening of three cDNA libraries indicated that this different in NK-1 expression in neural and BM cells could not be explained by differences in the cDNA sequence. Analyses the 5' flanking sequence in BM stroma and three neural cell lines indicated that sequence +1/+358 relative to the transcription start (TS) site could account for the differences in NK-1 expression. Particular cytokines could reverse the repressive effects of region +1/+358 in BM stroma. The effects of NF-kappa B and cAMP activators were studied in stromal cells using a dominant negative inhibitor of NF-kappa B (I kappa B) or a repressor of CRE activators (ICERII gamma). The results showed that their effects of these transcription factors depended on the stimulating cytokine. This study provides insight into the tissue-specific differences in the expression of the NK-1 gene.


Subject(s)
Bone Marrow Cells/metabolism , Gene Expression Regulation/physiology , Mesoderm/metabolism , Neurons/metabolism , Receptors, Neurokinin-1/biosynthesis , 5' Untranslated Regions/genetics , Cell Line/metabolism , Cyclic AMP/metabolism , Cytokines/pharmacology , DNA, Complementary/genetics , Gene Expression Regulation/drug effects , Genes, Reporter , Humans , Mesoderm/cytology , NF-kappa B/metabolism , Neuroblastoma/pathology , Organ Specificity , Receptors, Neurokinin-1/genetics , Recombinant Fusion Proteins/biosynthesis , Second Messenger Systems/drug effects , Stromal Cells/metabolism , Transcription, Genetic/drug effects , Tumor Cells, Cultured/metabolism
10.
J Neuroimmunol ; 121(1-2): 22-31, 2001 Dec 03.
Article in English | MEDLINE | ID: mdl-11730936

ABSTRACT

Communication within the hematopoietic-neuroendocrine-immune axis is partly mediated by neurotransmitters (e.g. substance P, SP) and cytokines. SP mediates neuromodulation partly through the stimulation of bone marrow (BM) progenitors. This study shows that SP, through the neurokinin-1 receptor, stimulates the proliferation of primitive hematopoietic progenitors: cobblestone-forming cells (CAFC, CD34+). This effect is optimal when macrophage is included within the fibroblast support. Indirect induction of IL-1 could be important in the proliferation of CAFC colonies by SP. Phenotypic and functional studies suggest that SP might directly interact with the CD34+/CD45(dim) population. These studies indicate that SP can initiate a cascade of biological responses in the BM stroma and stem cells to stimulate hematopoiesis.


Subject(s)
Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Stromal Cells/cytology , Stromal Cells/immunology , Substance P/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cell Division/drug effects , Cell Division/immunology , Cells, Cultured , Drug Synergism , Fluorouracil/pharmacology , Gene Expression/immunology , Humans , Immunosuppressive Agents/pharmacology , Interleukin-1/immunology , Interleukin-3/pharmacology , Interleukin-6/pharmacology , Macrophages/cytology , Macrophages/immunology , Neuroimmunomodulation/physiology , RNA, Messenger/analysis , Receptors, Interleukin-1/genetics , Receptors, Neurokinin-1/genetics , Receptors, Neurokinin-2/genetics , Stem Cell Factor/pharmacology
11.
Blood ; 98(9): 2697-706, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11675340

ABSTRACT

Hematopoietic regulation is a complex but dynamic process regulated by intercellular and intracellular interactions within the bone marrow (BM) microenvironment. Through neurokinin-1 (NK-1) and NK-2 receptors, peptides (eg, substance P [SP]) encoded by the preprotachykinin-I gene mediate distinct hematopoietic effects. Cytokines, associated with hematopoietic stimulation, and SP regulate the expression of each other in BM mesenchymal and immune cells. Neutral endopeptidase (NEP) uses SP as a substrate to produce SP(1-4), which inhibits the proliferation of matured myeloid progenitor. This study determines whether the degradation of SP to SP(1-4) by endogenous NEP in BM stroma could be a feedback on hematopoietic stimulation by stem cell factor (SCF). SP(1-4) induced the production of transforming growth factor (TGF)-beta and tumor necrosis factor-alpha in BM stroma. TGF-beta production accounted for part of the inhibitory effects by SP(1-4) on the proliferation of early (granulocyte-macrophage colony-forming units) and late (long-term culture-initiating cells) hematopoietic progenitors. Enzyme-linked immunosorbent assays and/or protein-chip arrays indicated a timeline change of SP to SP(1-4) in BM stroma stimulated with SCF, which correlated with increase in NEP messenger RNA. Since SP and its fragment, SP(1-4), interact with the same receptor to mediate opposing hematopoietic effects, 2 interactive studies were done to understand the dual responses of NK-1: (1) a 3-dimensional molecular model of NK-1 and SP and (2) screening of a random dodecapeptide library for SP(1-4) interacting sites. The effects of SP(1-4) on hematopoietic progenitors and the timeline change of SP to SP(1-4), together with the 3-dimensional model, provide a partial explanation for the feedback on the stimulatory effects of SCF and SP on hematopoiesis.


Subject(s)
Feedback, Physiological , Hematopoiesis/drug effects , Neprilysin/metabolism , Stem Cell Factor/pharmacology , Substance P/metabolism , Binding Sites , Bone Marrow Cells , Discoidin Domain Receptor 1 , Hematopoietic Stem Cells/drug effects , Humans , Membrane Proteins , Models, Molecular , Neprilysin/pharmacology , Peptide Library , RNA, Messenger/drug effects , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Neurokinin-1/drug effects , Receptors, Neurokinin-1/metabolism , Receptors, Neurokinin-2/drug effects , Receptors, Neurokinin-2/metabolism , Stem Cell Factor/physiology , Substance P/pharmacology , Substance P/physiology , Transforming Growth Factor beta/drug effects , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor-alpha/drug effects , Tumor Necrosis Factor-alpha/metabolism
12.
J Immunol ; 167(8): 4600-8, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11591789

ABSTRACT

The bone marrow (BM), which is the major site of immune cell development in the adult, responds to different stimuli such as inflammation and hemorrhagic shock. Substance P (SP) is the major peptide encoded by the immune/hemopoietic modulator gene, preprotachykinin-1 (PPT-I). Differential gene expression using a microarray showed that SP reduced hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA levels in BM stroma. Because long-term hypoxia induced the expression of PPT-I in BM mononuclear cells, we used timeline studies to determine whether PPT-I is central to the biologic responses of BM stroma subjected to 30-min hypoxia (pO(2) = 35 mm Hg) followed by reoxygenation. HIF-1alpha mRNA and protein levels were increased up to 12 h. At this time, beta-PPT-I mRNA was detected with the release of SP at 16 h. SP release correlated with down-regulation of HIF-1alpha to baseline. A direct role for SP in HIF-1alpha expression was demonstrated as follows: 1) transient knockout of beta-PPT-I showed an increase in HIF-1alpha expression up to 48 h of reoxygenation; and 2) HIF-1alpha expression remained baseline during reoxygenation when stroma was subjected to hypoxia in the presence of SP. Reoxygenation activated the PPT-I promoter with concomitant nuclear translocation of HIF-1alpha that can bind to the respective consensus sequences within the PPT-I promoter. SP reversed active caspase-3, an indicator of apoptosis and erythropoiesis, to homeostasis level after reoxygenation of hypoxic stroma. The results show that during reoxgenation the PPT-I gene acts as a negative regulator on the expression of HIF-1alpha and active caspase-3 in BM stroma subjected to reoxygenation.


Subject(s)
Bone Marrow/metabolism , Caspases/metabolism , Oxygen/pharmacology , Substance P/biosynthesis , Transcription Factors/biosynthesis , 5' Untranslated Regions , Adult , Caspase 3 , Cell Hypoxia , Enzyme Activation , Gene Expression Regulation , Hematopoiesis , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Promoter Regions, Genetic , Protein Precursors/genetics , Stromal Cells/metabolism , Tachykinins/genetics
13.
Crit Care Med ; 29(9): 1780-5, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11546985

ABSTRACT

OBJECTIVE: To determine whether hemorrhagic shock-induced bone marrow failure is mediated by the gut through the production of toxic mesenteric lymph and whether shock-induced bone marrow failure could be prevented by division of the mesenteric lymphatics. DESIGN: Prospective, controlled study. SETTING: University surgical research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Rats were divided into five groups: unmanipulated controls (n = 12), hemorrhagic shock with laparotomy (n = 8), hemorrhagic shock with mesenteric lymph duct ligation (n = 10), sham shock with laparotomy (n = 6), and sham shock with mesenteric lymph duct ligation (n = 7). At either 3 or 6 hrs after resuscitation, bone marrow was obtained for determination of early (cobblestone forming cells) and late (granulocyte-macrophage colony forming unit and erythroid burst forming unit) hematopoietic progenitor cell growth. Parallel cultures were plated with plasma (1% and 2% v/v) from all groups to determine the effect of lymphatic ligation on hematopoiesis. MEASUREMENTS AND MAIN RESULTS: Bone marrow cellularity, cobblestone forming cells, granulocyte-macrophage colony forming unit, and erythroid burst forming unit growth in rats subjected to hemorrhagic with lymph duct ligation were similar to those observed in sham-treated animals and significantly greater than in rats subjected to shock and laparotomy without lymphatic duct ligation. Plasma from rats subjected to shock without lymph ligation was inhibitory to hematopoietic progenitor cell growth. In contrast, this shock-induced inhibition was not observed with plasma obtained from shocked rats that underwent mesenteric lymph ligation. CONCLUSIONS: Hemorrhagic shock suppresses bone marrow hematopoiesis as measured by a decrease in early and late progenitor cell growth. This suppression appears mediated through mesenteric lymph as the effect is abrogated by mesenteric lymph duct ligation. These data clearly demonstrate a link between the gut and bone marrow failure after hemorrhagic shock


Subject(s)
Bone Marrow/metabolism , Hematopoietic Stem Cells/metabolism , Shock, Hemorrhagic/metabolism , Animals , Laparotomy , Ligation , Lymph/metabolism , Male , Mesentery/metabolism , Rats , Rats, Sprague-Dawley
14.
Ann Surg ; 234(2): 224-32, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11505069

ABSTRACT

OBJECTIVE: To examine the effect of trauma plasma on clonogenic progenitor cultures. SUMMARY BACKGROUND DATA: Severely injured trauma patients often experience altered hematopoietic functions, manifested by an increased susceptibility to infection and the development of a persistent anemia. Experimental and clinical data suggest that trauma results in the release of cytokines into the plasma that have hematopoietic regulatory function, but few studies have examined human bone marrow. METHODS: Plasma was obtained from 42 severely injured patients admitted to the surgical intensive care unit from days 1 to 15 after injury. Bone marrow and normal plasma were obtained from volunteers. Bone marrow mononuclear cells were isolated and plated for granulocyte-monocyte colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) growth. Parallel cultures were incubated with 2% (v/v) trauma or normal plasma. Additional cultures were plated with neutralizing concentrations of antibodies to transforming growth factor (TGF)-beta1 and MIP-1alpha. Circulating plasma TGF-beta1 was determined by bioassay. mRNA from bone marrow stromal cultures was extracted and probed for TGF-beta1 and macrophage inflammatory protein (MIP)-1alpha. RESULTS: Trauma plasma suppressed CFU-GM and BFU-E colony growth by 40% to 60% at all time periods after injury compared with cultures incubated with normal plasma. Using a noncontact culture system, the authors showed that this inhibition of BFU-E and CFU-GM colony growth was mediated by bone marrow stroma. The inhibition appeared to be due to soluble plasma-induced bone marrow stromal products that did not require direct cell-cell contact. The addition of anti-TGF-beta1 antibodies reversed the suppressive effect of trauma plasma on CFU-GM and BFU-E colony growth during the early but not late time points after injury. Trauma but not normal plasma induced TGF-beta1 mRNA in bone marrow stroma. CONCLUSIONS: Trauma plasma inhibits bone marrow BFU-E and CFU-GM colony growth for up to 2 weeks after injury. This inhibition is mediated through the interaction of trauma plasma with bone marrow stroma. TGF-beta1 production by bone marrow stroma appears to plays an important role in the early but not late bone marrow suppression after injury.


Subject(s)
Bone Marrow/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/blood , Hematopoietic Stem Cells/metabolism , Interleukin-3/blood , Transforming Growth Factor beta/metabolism , Wounds and Injuries/blood , Adolescent , Adult , Aged , Chemokine CCL3 , Chemokine CCL4 , Female , Hematopoiesis/physiology , Humans , Macrophage Inflammatory Proteins/blood , Male , Middle Aged , Prognosis , Transforming Growth Factor beta1 , Wounds and Injuries/surgery
15.
Blood ; 97(10): 3025-31, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11342427

ABSTRACT

Bone marrow (BM) fibrosis may occur in myeloproliferative diseases, lymphoma, myelodysplastic syndrome, myeloma, and infectious diseases. In this study, the role of substance P (SP), a peptide with pleiotropic functions, was examined. Some of its functions-angiogenesis, fibroblast proliferation, and stimulation of BM progenitors-are amenable to inducing BM fibrosis. Indeed, a significant increase was found in SP-immunoreactivity (SP-IR) in the sera of patients with BM fibrosis (n = 44) compared with the sera of patients with hematologic disorders and no histologic evidence of fibrosis (n = 46) (140 +/-12 vs 18 +/-3; P <.01). Immunoprecipitation of sera SP indicated that this peptide exists in the form of a complex with other molecule(s). It was, therefore, hypothesized that SP might be complexed with NK-1, its natural receptor, or with a molecule homologous to NK-1. To address this, 3 cDNA libraries were screened that were constructed from pooled BM stroma or mononuclear cells with an NK-1 cDNA probe. A partial clone (clone 1) was retrieved that was 97% homologous to the ED-A region of fibronectin (FN). Furthermore, sequence analyses indicated that clone 1 shared significant homology with exon 5 of NK-1. Immunoprecipitation and Western blot analysis indicated co-migration of SP and FN in 27 of 31 patients with BM fibrosis. Computer-assisted molecular modeling suggested that similar secondary structural features between FN and NK-1 and the relative electrostatic charge might explain a complex formed between FN (negative) and SP (positive). This study suggests that SP may be implicated in the pathophysiology of myelofibrosis, though its role would have to be substantiated in future research. (Blood. 2001;97:3025-3031)


Subject(s)
Fibronectins/blood , Primary Myelofibrosis/blood , Receptors, Neurokinin-1/blood , Substance P/blood , Adult , Aged , Amino Acid Sequence , Binding Sites , Biological Transport , DNA, Complementary/chemistry , Drug Stability , Fibronectins/chemistry , Fibronectins/genetics , Humans , Immunosorbent Techniques , Middle Aged , Models, Molecular , Molecular Weight , Myeloproliferative Disorders/complications , Primary Myelofibrosis/complications , Protein Folding , Receptors, Neurokinin-1/chemistry , Receptors, Neurokinin-1/genetics , Sequence Homology , Static Electricity , Substance P/chemistry
16.
Brain Res Brain Res Protoc ; 7(2): 154-61, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11356382

ABSTRACT

Previous studies had demonstrated that, in the cat, aggression is mediated by glutamatergic neurons in the anterior medial hypothalamus which project to the periaqueductal gray. Additionally, NK(1) receptor activation in the medial hypothalamus plays a role in the regulation of aggressive behavior by the medial amygdala. In the present study, in situ hybridization and immunohistochemistry were combined in order to provide neurochemical characterization of medial hypothalamic neurons containing NK(1)-receptor mRNA. In order to identify NK(1) receptors in cat brain, a 650-bp fragment of the cat NK(1) cDNA was cloned. This fragment was used to synthesize a riboprobe for in situ hybridization. Partial DNA sequence analysis of the fragment indicated a 90% homology with human cDNA. In situ hybridization revealed the presence of NK(1)-receptor mRNA in cat hypothalamic neurons. Tissue used to localize NK(1) receptors was also processed for glutamate immunopositivity. The results demonstrated that NK(1)-receptor mRNA is present in glutamate-immunopositive neurons in the anterior medial hypothalamus of cat, thus reinforcing the hypothesis that NK(1) receptors play an important role in this neural circuit.


Subject(s)
Glutamic Acid/analysis , Hypothalamus/chemistry , Immunohistochemistry/methods , In Situ Hybridization/methods , Receptors, Neurokinin-1/analysis , Receptors, Neurokinin-1/genetics , Aggression/physiology , Animals , Antibody Specificity , Cats , Cloning, Molecular , Female , Glutamic Acid/immunology , Hypothalamus/physiology , Male , RNA, Messenger/analysis , Receptors, Neurokinin-1/immunology
17.
J Immunol ; 166(4): 2553-61, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11160316

ABSTRACT

Preprotachykinin-I gene (PPT-I) encodes several peptides with organ-specific functions that link the neuroendocrine-immune-hemopoietic axis. We cloned upstream of the initiation site of human PPT-I promoter and identified consensus sequences for two cAMP response elements (CRE). PPT-I is induced by cytokines including those that signal through the cAMP pathway. Therefore, we studied the role of the two CRE in IL-1alpha and stem cell factor (SCF) stimulation of bone marrow stroma because both cytokines induce endogenous PPT-I in these cells and activate the cAMP pathway. Furthermore, bone marrow stroma expresses the transcription factors regulated by the cAMP pathways such as the repressor (ICERIIgamma) and activator (CREMtau). Mutagenesis of the two CRE and/or cotransfection with vectors that express ICERIIgamma or CREMtau indicated that the two CRE have major roles in PPT-I expression. The two CRE are also required for optimal promoter activity by SCF and IL-1alpha. A particular cytokine could concomitantly induce PPT-I and the high affinity G protein-coupled receptor for PPT-I peptides, NK-1R. We showed that SCF, a representative cytokine, induced PPT-I and NK-1R leading to autocrine and/or paracrine cell activation. Because NK-1R activates cAMP through the G protein, the results suggest that the presence of CRE sequences within PPT-I promoter could be important in the regulation of PPT-I expression by cytokines, irrespective of their ability to signal through cAMP. As PPT-I is implicated in hemopoietic regulation, immune responses, breast cancer, and other neural functions, these studies add to the basic biology of these processes and could provide targets for drug development.


Subject(s)
Cyclic AMP Response Element-Binding Protein/genetics , Gene Expression Regulation/immunology , Interleukin-1/physiology , Promoter Regions, Genetic , Protein Precursors/biosynthesis , Protein Precursors/genetics , Response Elements/immunology , Stem Cell Factor/physiology , Tachykinins/biosynthesis , Tachykinins/genetics , 5' Untranslated Regions/immunology , Autocrine Communication/immunology , Base Sequence , Cell Line , Fibroblasts/immunology , Fibroblasts/metabolism , Humans , Macrophages/immunology , Macrophages/metabolism , Models, Immunological , Molecular Sequence Data , Organ Specificity/genetics , Organ Specificity/immunology , Protein Precursors/isolation & purification , Protein Precursors/metabolism , Receptors, Neurokinin-1/biosynthesis , Tachykinins/isolation & purification , Tachykinins/metabolism , Tumor Cells, Cultured
18.
J Neuroimmunol ; 112(1-2): 188-96, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11108948

ABSTRACT

We studied the complex interactions within the neuroendocrine-immune-hematopoietic axis by determining a possible link among ACTH, PRL, PPT-I and the receptors for its peptides, NK-1 and NK-2. Indeed, ACTH and PRL induced the expression of PPT-I and NK-1 in human bone marrow stroma with no effect on NK-2. Consistent with a role for PPT-I in regulating the development of myeloid and erythroid progenitors, we found that ACTH and PRL, through NK-1 stimulated the proliferation of both types of progenitors. Induction of PPT-I was regulated at the transcriptional and post-transcriptional levels. The results showed that ACTH and PRL stimulated the proliferation of bone marrow progenitors, partly through PPT-I and NK-1 induction.


Subject(s)
Adrenocorticotropic Hormone/pharmacology , Gene Expression Regulation/drug effects , Hematopoietic Stem Cells/drug effects , Prolactin/pharmacology , Protein Precursors/biosynthesis , Tachykinins/biosynthesis , Bone Marrow/metabolism , Humans , Protein Precursors/genetics , RNA, Messenger/analysis , Stromal Cells/metabolism , Tachykinins/genetics , Transcription, Genetic
19.
J Immunol ; 165(4): 2271-7, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10925316

ABSTRACT

Immune-mediated mechanisms have been implicated in the etiology of idiopathic bone marrow fibrosis (IMF). However, the mechanism remains poorly defined. Compared with healthy controls, IMF monocytes are overactivated, with increased production of TGF-beta and IL-1. TGF-beta is central to the progression of fibrosis in different organs. In the lung, fibrosis is associated with up-regulation of TGF-beta-inducible genes. Because IL-1 and TGF-beta have pro- and antiinflammatory properties and neither appears to regulate the high levels of each other in IMF, we studied the mechanism of this paradigm. We focused on the role of RelA, a subunit of the transcription factor, NF-kappaB that is associated with inflammatory responses. We transiently knocked out RelA from IMF monocytes with antisense oligonucleotides and showed that RelA is central to IL-1 and TGF-beta production and to the adhesion of IMF monocytes. Because the NF-kappaB family comprises subunits other than RelA, we used aspirin and sodium salicylate to inhibit kinases that activate NF-kappaB and showed effects similar to those of the RelA knockout system. It is unlikely that RelA could be interacting directly with the TGF-beta gene. Therefore, we determined its role in TGF-beta production and showed that exogenous IL-1 could induce TGF-beta and adherence of IMF monocytes despite the depletion of NF-kappaB. The results indicate that IL-1 is necessary for TGF-beta production in IMF monocytes, but NF-kappaB activation is required for the production of endogenous IL-1. Initial adhesion activates NF-kappaB, which led to IL-1 production. Through autocrine means, IL-1 induces TGF-beta production. In total, these reactions maintain overactivation of IMF monocytes.


Subject(s)
Homeostasis/immunology , Monocytes/immunology , Monocytes/metabolism , NF-kappa B/physiology , Primary Myelofibrosis/immunology , Primary Myelofibrosis/pathology , Transforming Growth Factor beta/biosynthesis , Adult , Aged , Animals , Biological Transport/immunology , Bone Marrow/pathology , Cell Adhesion/immunology , Cell Line , Cell Nucleus/immunology , Cell Nucleus/metabolism , Cell Separation , Humans , Interleukin-1/physiology , Ligases/deficiency , Ligases/genetics , Ligases/metabolism , Macrophage Activation , Mice , Middle Aged , NF-kappa B/blood , Oligonucleotides, Antisense/pharmacology , Primary Myelofibrosis/blood , Rats
20.
Am J Hematol ; 64(1): 20-5, 2000 May.
Article in English | MEDLINE | ID: mdl-10815783

ABSTRACT

Hemorrhagic shock leads to hypoxia and is associated with bone marrow (BM) failure. Hemorrhagic shock is also a predisposing factor in immune dysregulation. Since the BM is the major organ of immune cells in the adult, its failure following hemorrhagic shock may explain the increased susceptibility to infection. The in vitro evidence indicates that hypoxia mediates altered functions in BM stroma. Since similar hematopoietic alterations are reported in hypoxia and hemorrhagic shock, hypoxia alone could be a representative model to study BM responses during hemorrhagic shock. In this study, we use an animal model to dissect the hematopoietic effects of hypoxia. We subjected rats to hypoxia, and at days 1 and 5 post-hypoxia we determined the numbers of granulocytic-monocytic progenitors (CFU-GM) in the BM. We found significant increase (P < 0.05) in CFU-GM at day 1 and a downward trend by day 5. Enhanced BM cellularity could not explain the increase in CFU-GM by day 1. BM stromal cells mediated most of the stimulatory effects by hypoxia. CFU-GM was inversely proportional to bioactive TGF-beta and directly proportional to IL-1. Compared to normoxic rats, IL-6 production was suppressed in BM cells from hypoxic rats. The results show that hypoxia alone initiate a stimulatory response in CFU-GM progenitors. These effects are at least partially mediated through the BM stroma. In the absence of a second insult, CFU-GM reverts to baseline. The data also suggest that hypoxia mediates complex responses that include cytokine production. These results add to the current understanding of hematopoietic responses by hypoxia and adds to the mechanisms of immune dysfunctions following hemorrhagic shock.


Subject(s)
Cell Communication/physiology , Leukopoiesis/physiology , Stromal Cells/physiology , Animals , Cell Differentiation/physiology , Female , Granulocytes/cytology , Granulocytes/physiology , Hypoxia , Monocytes/cytology , Monocytes/physiology , Rats , Rats, Sprague-Dawley , Stromal Cells/cytology
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