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1.
Nat Commun ; 8: 14208, 2017 01 27.
Article in English | MEDLINE | ID: mdl-28128208

ABSTRACT

Alternatively activated macrophages (M2) have an important function in innate immune responses to parasitic helminths, and emerging evidence also indicates these cells are regulators of systemic metabolism. Here we show a critical role for mTORC2 signalling in the generation of M2 macrophages. Abrogation of mTORC2 signalling in macrophages by selective conditional deletion of the adaptor molecule Rictor inhibits the generation of M2 macrophages while leaving the generation of classically activated macrophages (M1) intact. Selective deletion of Rictor in macrophages prevents M2 differentiation and clearance of a parasitic helminth infection in mice, and also abrogates the ability of mice to regulate brown fat and maintain core body temperature. Our findings define a role for mTORC2 in macrophages in integrating signals from the immune microenvironment to promote innate type 2 immunity, and also to integrate systemic metabolic and thermogenic responses.


Subject(s)
Macrophages/physiology , Mechanistic Target of Rapamycin Complex 2/immunology , Strongylida Infections/immunology , Thermogenesis/physiology , Animals , Cell Differentiation/immunology , Cells, Cultured , Disease Models, Animal , Female , Gene Knockout Techniques , Helminthiasis, Animal/immunology , Humans , Immunity, Innate/physiology , Male , Mechanistic Target of Rapamycin Complex 2/genetics , Mechanistic Target of Rapamycin Complex 2/metabolism , Mice , Mice, Inbred C57BL , Nippostrongylus/immunology , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Rapamycin-Insensitive Companion of mTOR Protein/immunology , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Signal Transduction/physiology , Strongylida Infections/parasitology
2.
J Appl Microbiol ; 119(3): 711-23, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26075586

ABSTRACT

AIMS: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. METHODS AND RESULTS: We evaluated the kinetics of uptake, germination and proliferation of Bacillus anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3 and A549 cell lines. We also analysed the influence of various cell culture medium formulations related to spore germination. CONCLUSIONS: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the serum-free extracellular environment was evident. Spore germination was appreciably higher in immortalized cell cultures than in primary epithelial cells. Additionally, spores still germinated apically at a mucus-secreting air-liquid interface lung barrier that was devoid of cell culture medium much earlier than medium-only controls. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of lung epithelial cells in B. anthracis spore dissemination after inhalation remains poorly defined and rather controversial. These results are novel as they show spore germination is appreciably enhanced in the presence of lung cells in vitro, however, the cell line and cell state (air-liquid interface vs submerged in medium) dictates the extent of germination and in some cases proliferation.


Subject(s)
Anthrax/microbiology , Bacillus anthracis/growth & development , Lung/microbiology , Spores, Bacterial/growth & development , Bacillus anthracis/metabolism , Cell Line , Cell Line, Tumor , Culture Media, Serum-Free/metabolism , Humans , Models, Biological , Spores, Bacterial/metabolism
3.
Bone Marrow Transplant ; 48(10): 1335-41, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23604009

ABSTRACT

Because of the toxicity associated with myeloablative conditioning, nonmyeloablative regimens are increasingly being used in vulnerable patient populations. For patients with sickle cell disease, stable mixed chimerism has proven sufficient to reverse the phenotype. Because the vast majority of patients do not have an HLA-matched sibling, a safe nonmyeloablative regimen that could be applied to the haploidentical setting would be ideal. We employed a mismatched mouse model using BALB/c donors and C57BL/6 recipients. Recipient mice were conditioned with 200 cGy TBI and sirolimus or CSA with or without post transplant Cy (PT-Cy). Our data show that when sirolimus or PT-Cy alone is given to C57BL/6 recipients, donor cells are not detected. However, when sirolimus is administered for 15 or 31 days starting 1 day before or up to 6 days after transplant with PT-Cy, all mice maintain stable mixed chimerism. In contrast, conventional therapy employing CSA with or without PT-Cy does not result in stable mixed chimerism. Lastly, mice with stable mixed chimerism after sirolimus display decreased reactivity to donor Ag both in vitro and in vivo. These data identify a novel strategy for inducing mixed chimerism for the treatment of nonmalignant hematologic diseases.


Subject(s)
Cyclophosphamide/pharmacology , Immunosuppressive Agents/pharmacology , Sirolimus/pharmacology , Skin Transplantation/methods , Animals , Disease Models, Animal , Drug Synergism , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Transplantation Chimera
4.
Int J Cancer ; 133(4): 997-1005, 2013 Aug 15.
Article in English | MEDLINE | ID: mdl-23382028

ABSTRACT

There are few effective therapies for high-risk sarcomas. Initial chemosensitivity is often followed by relapse. In vitro, mammalian target of rapamycin (mTOR) inhibition potentiates the efficacy of chemotherapy on resistant sarcoma cells. Although sarcoma trials using mTOR inhibitors have been disappointing, these drugs were used as maintenance. We conducted a Phase I/II clinical trial to test the ability of temsirolimus to potentiate the cytotoxic effect of liposomal doxorubicin and present here the dose-finding portion of this study. Adult and pediatric patients with recurrent or refractory sarcomas were treated with increasing doses of liposomal doxorubicin and temsirolimus using a continual reassessment method for escalation, targeting a dose-limiting toxicity rate of 20%. Blood samples were drawn before and after the first dose of temsirolimus in Cycles 1 and 2 for pharmacokinetic analysis. The maximally tolerated dose combination was liposomal doxorubicin 30 mg/m(2) monthly with temsirolimus 20 mg/m(2) weekly. Hematologic toxicity was common but manageable. Dose-limiting toxicities were primarily renal. Concurrent administration of liposomal doxorubicin resulted in increased exposure to sirolimus, the active metabolite of temsirolimus. Thus, the combination of liposomal doxorubicin and temsirolimus is safe for heavily pretreated sarcoma patients. Co-administration with liposomal doxorubicin did not alter temsirolimus pharmacokinetics, but increased exposure to its active metabolite.


Subject(s)
Antineoplastic Agents/therapeutic use , Bone Neoplasms/drug therapy , Doxorubicin/therapeutic use , Sarcoma/drug therapy , Sirolimus/analogs & derivatives , Adolescent , Adult , Aged , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Child , Dose-Response Relationship, Drug , Doxorubicin/administration & dosage , Doxorubicin/pharmacokinetics , Female , Humans , Male , Middle Aged , Recurrence , Sirolimus/administration & dosage , Sirolimus/pharmacokinetics , Sirolimus/therapeutic use , Young Adult
5.
Ann Oncol ; 21(6): 1203-1210, 2010 Jun.
Article in English | MEDLINE | ID: mdl-19880437

ABSTRACT

BACKGROUND: Rituximab may improve transplant outcomes but may delay immunologic recovery. PATIENTS AND METHODS: Seventy-seven patients with low-grade or mantle cell lymphoma received autologous stem-cell transplantation (ASCT) on a phase II study. Rituximab 375 mg/m(2) was administered 3 days before mobilization-dose cyclophosphamide, then weekly for four doses after count recovery from ASCT. Immune reconstitution was assessed. RESULTS: Sixty percent of transplants occurred in first remission. Actuarial event-free survival (EFS) and overall survival (OS) were 60% and 73%, respectively, at 5 years, with 7.2-year median follow-up for OS in surviving patients. Median EFS was 8.3 years. Older age and transformed lymphomas were independently associated with inferior EFS, whereas day 60 lymphocyte counts did not predict EFS or late infections. Early and late transplant-related mortality was 1% and 8%, with secondary leukemia in two patients. B-cell counts recovered by 1-2 years; however, the median IgG level remained low at 2 years. Late-onset idiopathic neutropenia, generally inconsequential, was noted in 43%. CONCLUSION: ASCT with rituximab can produce durable remissions on follow-up out to 10 years. Major infections do not appear to be significantly increased or to be predicted by immune monitoring.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Immune System/physiology , Lymphoma, Mantle-Cell , Lymphoma , Recovery of Function/immunology , Stem Cell Transplantation/methods , Adult , Aged , Antibodies, Monoclonal, Murine-Derived , Antineoplastic Agents/administration & dosage , Combined Modality Therapy , Drug Administration Schedule , Female , Humans , Immunotherapy , Lymphoma/immunology , Lymphoma/pathology , Lymphoma/rehabilitation , Lymphoma/therapy , Lymphoma, Mantle-Cell/immunology , Lymphoma, Mantle-Cell/pathology , Lymphoma, Mantle-Cell/rehabilitation , Lymphoma, Mantle-Cell/therapy , Male , Middle Aged , Neoplasm Staging , Rituximab , Transplantation Immunology , Transplantation, Autologous
6.
Handb Exp Pharmacol ; (193): 399-441, 2009.
Article in English | MEDLINE | ID: mdl-19639290

ABSTRACT

The A(1), A(2A), A(2B) and A(3) G-protein-coupled cell surface adenosine receptors (ARs) are found to be upregulated in various tumor cells. Activation of the receptors by specific ligands, agonists or antagonists, modulates tumor growth via a range of signaling pathways. The A(1)AR was found to play a role in preventing the development of glioblastomas. This antitumor effect of the A(1)AR is mediated via tumor-associated microglial cells. Activation of the A(2A)AR results in inhibition of the immune response to tumors via suppression of T regulatory cell function and inhibition of natural killer cell cytotoxicity and tumor-specific CD4+/CD8+ activity. Therefore, it is suggested that pharmacological inhibition of A(2A)AR activation by specific antagonists may enhance immunotherapeutics in cancer therapy. Activation of the A(2B)AR plays a role in the development of tumors via upregulation of the expression levels of angiogenic factors in microvascular endothelial cells. In contrast, it was evident that activation of A(2B)AR results in inhibition of ERK1/2 phosphorylation and MAP kinase activity, which are involved in tumor cell growth signals. Finally, A(3)AR was found to be highly expressed in tumor cells and tissues while low expression levels were noted in normal cells or adjacent tissue. Receptor expression in the tumor tissues was directly correlated to disease severity. The high receptor expression in the tumors was attributed to overexpression of NF-kappaB, known to act as an A(3)AR transcription factor. Interestingly, high A(3)AR expression levels were found in peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing animals and cancer patients, reflecting receptor status in the tumors. A(3)AR agonists were found to induce tumor growth inhibition, both in vitro and in vivo, via modulation of the Wnt and the NF-kappaB signaling pathways. Taken together, A(3)ARs that are abundantly expressed in tumor cells may be targeted by specific A(3)AR agonists, leading to tumor growth inhibition. The unique characteristics of these A(3)AR agonists make them attractive as drug candidates.


Subject(s)
Neoplasms/etiology , Receptors, Purinergic P1/physiology , Adenosine A2 Receptor Antagonists , Adenosine A3 Receptor Antagonists , Animals , Antineoplastic Agents/pharmacology , Humans , Immunotherapy , NF-kappa B/physiology , Neoplasms/immunology , Receptor, Adenosine A1/physiology , Receptor, Adenosine A2A/physiology , Receptor, Adenosine A2B/physiology , Receptor, Adenosine A3/physiology , Signal Transduction , Wnt Proteins/physiology
7.
Bone Marrow Transplant ; 43(6): 477-80, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18978825

ABSTRACT

Graft failure after allogeneic blood or marrow transplantation, although generally uncommon, can be a devastating complication. This report includes the outcome of nine patients who received a salvage transplant for failure to engraft after one (n=8) or 2 (n=1) prior transplants. Eight patients received allografts from the original donor. All received fludarabine 30 mg/m(2) i.v. and alemtuzumab 20 mg i.v. daily from days -6 to -2. Daily CYA was begun on day -2, and the allograft was infused on day 0. The therapy was well tolerated with low toxicity, and all nine patients engrafted, recovering neutrophils at a median of 12 days after transplant. Four patients died: two of relapse, one of a fungal infection in the setting of GVHD and one of multiple sclerosis. The combination of fludarabine and alemtuzumab is an effective and well-tolerated salvage conditioning regimen for patients who experience graft failure after blood or marrow transplants.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Neoplasm/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Bone Marrow Transplantation/methods , Transplantation Conditioning/methods , Vidarabine/analogs & derivatives , Adult , Alemtuzumab , Antibodies, Monoclonal, Humanized , Female , Graft Rejection , Humans , Immunosuppressive Agents/therapeutic use , Male , Middle Aged , Recurrence , Retrospective Studies , Salvage Therapy/methods , Treatment Outcome , Vidarabine/administration & dosage
8.
Biol Reprod ; 70(4): 1121-30, 2004 Apr.
Article in English | MEDLINE | ID: mdl-14668208

ABSTRACT

The epididymal epithelium contributes to formation of a luminal fluid that is essential for the protection of spermatozoa from a variety of insults including changes in oxygen tension. A key regulator of the response to oxygen debt in many cells is hypoxia-inducible factor-1 (HIF-1). A transcription factor composed of alpha and beta subunits, HIF-1 activates genes that mediate oxygen homeostasis and cell survival pathways or trigger cell death responses. Previously we have shown that HIF-1alpha mRNA is expressed in the adult rat epididymis. Goals of this study were to determine whether HIF-1alpha protein is activated by ischemia in the rat epididymis, to determine whether epididymal HIF-1alpha mRNA expression is androgen dependent, and to identify epididymal cell types expressing HIF-1alpha and beta. Immunoblot analysis revealed that HIF-1alpha protein is primarily present in corpus and cauda of the normoxic epididymis and unaffected by ischemia, whereas HIF-1beta was detected equally in all regions and also unaffected by ischemia. HIF-1alpha mRNA expression in all regions was not affected by 15 days bilateral orchiectomy. Principal cells stained positive for HIF-1alpha by immunocytochemistry, with the epithelium of initial segment and caput epididymidis staining less intensely than corpus and cauda. HIF-1beta immunoreactivity was equally present in principal cells in all regions. Clear, narrow, and basal cells were unreactive for HIF-1alpha and beta. The presence of HIF-1 in normoxic epididymis and the regional distribution of HIF-1alpha suggests fundamental differences in how proximal and distal regions of the epididymis maintain oxygen homeostasis to protect the epithelium and spermatozoa from hypoxia.


Subject(s)
DNA-Binding Proteins/metabolism , Epididymis/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Transcription Factors/metabolism , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator , Epididymis/blood supply , Hypoxia-Inducible Factor 1, alpha Subunit , Immunoblotting , Immunohistochemistry , Ischemia/metabolism , Male , Orchiectomy , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Spermatozoa/metabolism , Testosterone/blood , Testosterone/pharmacology , Tissue Distribution , Transcription Factors/genetics
9.
Vet Ther ; 4(2): 128-34, 2003.
Article in English | MEDLINE | ID: mdl-14506588

ABSTRACT

Xylazine hydrochloride was administered i.m. at 0.35 mg/kg to 13 steers and 10 lactating dairy cows at Time 0. Ten minutes later, tolazoline hydrochloride was given i.v. at 4 mg/kg. Tissue and milk samples were analyzed using gas chromatography with nitrogen and phosphorous detection to determine concentrations of xylazine, 2,6-dimethylaniline (a toxic metabolite of xylazine), and tolazoline (at various intervals). Concentrations of xylazine and 2,6- dimethylaniline were below the limit of quantitation (10 microg/kg) by 72 hours in tissues and 12 hours in milk. The concentration of tolazoline was below 10 microg/kg by 96 hours in tissues and 48 hours in milk. Based on the results of these residue studies submitted by the sponsoring agency to the Ministry of Agriculture and Forestry in New Zealand, withholding periods for both xylazine hydrochloride and tolazoline hydrochloride injection were established.


Subject(s)
Adrenergic alpha-Agonists/pharmacokinetics , Adrenergic alpha-Antagonists/pharmacokinetics , Aniline Compounds/pharmacokinetics , Cattle/metabolism , Milk/metabolism , Tolazoline/pharmacokinetics , Xylazine/pharmacokinetics , Adrenergic alpha-Agonists/administration & dosage , Adrenergic alpha-Agonists/blood , Adrenergic alpha-Antagonists/administration & dosage , Adrenergic alpha-Antagonists/blood , Aniline Compounds/blood , Animals , Dairying , Drug Residues/metabolism , Female , Hypnotics and Sedatives/administration & dosage , Hypnotics and Sedatives/antagonists & inhibitors , Infusions, Intravenous/veterinary , Injections, Intramuscular/veterinary , Kidney/metabolism , Liver/metabolism , Muscle, Skeletal/metabolism , Random Allocation , Tolazoline/administration & dosage , Tolazoline/blood , Xylazine/administration & dosage , Xylazine/blood
10.
Eur J Immunol ; 31(12): 3737-46, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11745394

ABSTRACT

We have proposed a model in which the prevention of anergy by costimulation is the result of IL-2-induced G1 to S phase cell cycle progression. Here we demonstrate that the reversal of anergy by exogenous IL-2 also occurs during this window of the cell cycle. Recently, it has been proposed that the cell cycle inhibitor p27(Kip1) is an anergic factor. In contrast, our data demonstrate that during the induction, maintenance and rechallenge phases of anergy, p27(Kip1) levels do not correlate with the anergic phenotype. Although p27(Kip1) levels were down-regulated by IL-2 during the G1 to S phase transition, the amount of IL-2 required to produce this effect was far lower than that required to prevent the induction of anergy. Furthermore, T cell lines from p27(Kip1) knockout mice were anergized as well as T cells from mice that were heterozygous for p27(Kip1). Interestingly, the forced overexpression of p27(Kip1) was able to decrease IL-2 promoter-induced transcription, suggesting that the cell cycle machinery may be involved in T cell activation; however, physiological levels of p27(Kip1) did not prevent IL-2 transcription. Overall, our data serve to disassociate the ability of IL-2 to down-regulate p27(Kip1) and its ability to prevent or reverse anergy.


Subject(s)
Cell Cycle Proteins/physiology , Immune Tolerance , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes/immunology , Tumor Suppressor Proteins/physiology , Animals , Cell Cycle , Cell Line , Cyclin-Dependent Kinase Inhibitor p27 , Down-Regulation , Gene Expression Regulation , Interleukin-2/genetics , Interleukin-2/pharmacology , Mice , Mice, Knockout , Protein Kinases/physiology , RNA, Messenger/analysis , TOR Serine-Threonine Kinases
11.
J Immunol ; 166(1): 506-16, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11123330

ABSTRACT

Despite high viral loads, T cells from sooty mangabey (SM) monkeys that are naturally infected with SIV but remain clinically asymptomatic, proliferate and demonstrate normal Ag-specific memory recall CD4(+) T cell responses. In contrast, CD4(+) T cells from rhesus macaques (RM) experimentally infected with SIV lose Ag-specific memory recall responses and develop immunological anergy. To elucidate the mechanisms for these distinct outcomes of lentiviral infection, highly enriched alloreactive CD4(+) T cells from humans, RM, and SM were anergized by TCR-only stimulation (signal 1 alone) and subsequently challenged with anti-CD3/anti-CD28 Abs (signals 1 + 2). Whereas alloreactive CD4(+)T cells from humans and RM became anergized, surprisingly, CD4(+) T cells from SM showed marked proliferation and IL-2 synthesis after restimulation. This resistance to undergo anergy was not secondary to a global deficiency in anergy induction of CD4(+) T cells from SM since incubation of CD4(+) T cells with anti-CD3 alone in the presence of rapamycin readily induced anergy in these cells. The resistance to undergo anergy was reasoned to be due to the ability of CD4(+) T cells from SM to synthesize IL-2 when incubated with anti-CD3 alone. Analysis of phosphorylated kinases involved in T cell activation showed that the activation of CD4(+) T cells by signal 1 in SM elicited a pattern of response that required both signals 1 + 2 in humans and RM. This function of CD4(+) T cells from SM may contribute to the resistance of this species to SIV-induced disease.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cercocebus atys/immunology , Clonal Anergy , Lymphocyte Activation , Simian Acquired Immunodeficiency Syndrome/immunology , Adult , Animals , Base Sequence , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , Clonal Anergy/drug effects , Clonal Anergy/genetics , Cyclosporine/pharmacology , Cytokines/biosynthesis , Epitopes, T-Lymphocyte/immunology , Humans , Hydroxyurea/pharmacology , Immunity, Innate , Kinetics , Lymphocyte Activation/drug effects , Lymphocyte Activation/genetics , MAP Kinase Signaling System/immunology , Macaca mulatta , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Muromonab-CD3/pharmacology , Simian Acquired Immunodeficiency Syndrome/genetics , Sirolimus/pharmacology
12.
Hypertension ; 36(4): 523-30, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11040230

ABSTRACT

Omapatrilat is a member of the new drug class of vasopeptidase inhibitors that may offer benefit in the treatment of heart failure (HF) through simultaneous inhibition of angiotensin-converting enzyme and neutral endopeptidase. We examined the effects of omapatrilat in a placebo-controlled crossover study using a pacing model of HF. Seven sheep were paced sequentially at 180 bpm (mild HF) and then 225 bpm (severe HF) for 7 days each. Omapatrilat (0.005 mg/kg) or vehicle was administered by intravenous bolus on days 4 to 7 of each paced period. Omapatrilat lowered mean arterial and left atrial pressure and increased cardiac output acutely and chronically in both mild and severe HF (P<0.01 for all). Plasma atrial and brain natriuretic peptide and cGMP levels were stable acutely (P=NS), while brain natriuretic peptide increased after repeated dosing in severe HF (P<0.05). Plasma renin activity rose, whereas angiotensin II and aldosterone levels fell after acute and repeated dosing in both states (P<0.01 for all). Omapatrilat increased urinary sodium excretion by day 7 in both mild and severe HF (P<0.05). Effective renal plasma flow and glomerular filtration rate increased or were stable after omapatrilat in mild and severe HF after both acute and repeated dosing. Omapatrilat exhibited pronounced acute and sustained beneficial hemodynamic and renal effects in both mild and severe heart failure.


Subject(s)
Cardiovascular Agents/administration & dosage , Heart Failure/drug therapy , Hemodynamics/drug effects , Kidney/drug effects , Pyridines/administration & dosage , Thiazepines/administration & dosage , Aldosterone/blood , Angiotensin II/blood , Animals , Atrial Natriuretic Factor/blood , Blood Pressure/drug effects , Cardiac Output/drug effects , Cross-Over Studies , Cyclic GMP/blood , Disease Models, Animal , Glomerular Filtration Rate/drug effects , Heart Failure/physiopathology , Injections, Intravenous , Natriuretic Peptide, Brain/blood , Renin/blood , Sheep , Sodium/urine
13.
J Immunol ; 164(8): 3996-4002, 2000 Apr 15.
Article in English | MEDLINE | ID: mdl-10754290

ABSTRACT

Ag presented by activated APCs promote immunogenic responses whereas Ag presented by resting APCs leads to tolerance. In such a model, the regulation of cytokine release by the presence or absence of costimulation might potentially play a critical role in dictating the ultimate outcome of Ag recognition. C-C chemokines are a structurally defined family of chemoattractants that have diverse effects on inflammation. We were interested in determining the activation requirements for chemokine production by CD4+ T cells. Our data demonstrate for T cell clones and previously activated T cells from TCR-transgenic mice that stimulation with anti-TCR alone results in the production of copious amounts of macrophage-inflammatory protein-1alpha (MIP-1alpha) and other C-C chemokines, and that addition of anti-CD28 gives very little augmentation. Furthermore, MIP-1alpha production is nearly equivalent from both anergic and nonanergic cells. For naive T cells, anti-CD3 stimulation alone led to as much MIP-1alpha production as Ag + APC stimulation. The addition of costimulation gave a 3-10-fold enhancement, but this was 70-fold less than the effect of costimulation on IL-2 production. Thus, although C-C chemokines play a broad role in influencing inflammation, their production by signal 1 alone makes them unlikely to play a critical role in the decision between a tolerogenic and an immunogenic response. Furthermore, the production of MIP-1alpha by anergic T cells, as well as following signal 1 alone, raises the possibility that in vivo this chemokine serves to recruit activated T cells to become tolerant.


Subject(s)
Chemokines, CC/biosynthesis , Clonal Anergy/immunology , Interleukin-2/biosynthesis , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , CD28 Antigens/physiology , CD4 Antigens/biosynthesis , Calcineurin/physiology , Calcium Signaling/immunology , Cells, Cultured , Chemokine CCL3 , Chemokine CCL4 , Clone Cells , Interphase/immunology , Macrophage Inflammatory Proteins/biosynthesis , Mice , Mice, Inbred A , Mice, Inbred C57BL , Mice, Transgenic , Mitogen-Activated Protein Kinases/physiology , NF-kappa B/physiology , Receptors, Antigen, T-Cell/genetics , Th1 Cells/immunology , Th1 Cells/metabolism
14.
J Immunol ; 163(12): 6631-9, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10586058

ABSTRACT

Anergic T cells display a marked decrease in their ability to produce IL-2 even in the presence of optimal TCR and costimulatory signals. Using IL-2 enhancer/promoter-driven reporter constructs, we have previously identified a region that appears to be a target for cis transcriptional repression in anergy. This region of the promoter, which shares partial homology with a consensus AP-1-binding sequence, is located about -180 bp from the transcriptional start site. In the present study, we demonstrate that cAMP response element-binding protein/cAMP response element modulator (CREB/CREM), activating transcription factor-2/c-Jun, and Jun-Jun/Oct complexes bind to this site. However, the induction of anergy by prolonged stimulation through the TCR led to an increase in binding of only the CREB/CREM complex. Furthermore, the level of binding of this complex appeared to be up-regulated in both resting and restimulated anergic T cells. Finally, an IL-2 promoter-driven reporter construct that contained a mutation that specifically reduced the binding of the CREB/CREM complex displayed a decreased ability to be affected by anergy, while a construct that contained a mutation that decreased the binding of the Jun-Jun/Oct complex was still susceptible to anergy. These findings suggest that the -180 region of the IL-2 promoter is the target of a CREB/CREM transcriptional inhibitor that contributes to the repression of IL-2 production in T cell anergy.


Subject(s)
Clonal Anergy/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins/metabolism , Interleukin-2/genetics , Promoter Regions, Genetic/immunology , Repressor Proteins , T-Lymphocytes/immunology , 5' Untranslated Regions/immunology , 5' Untranslated Regions/metabolism , Animals , Binding, Competitive/genetics , Binding, Competitive/immunology , Clonal Anergy/immunology , Clone Cells , Cyclic AMP Response Element Modulator , Cyclic AMP Response Element-Binding Protein/isolation & purification , Cyclic AMP Response Element-Binding Protein/physiology , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/physiology , Lymphoma, T-Cell , Macromolecular Substances , Mice , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-jun/metabolism , T-Lymphocytes/metabolism , Tumor Cells, Cultured
15.
J Immunol ; 162(5): 2775-84, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10072524

ABSTRACT

Costimulation (signal 2) has been proposed to inhibit the induction of T cell clonal anergy by either directly antagonizing negative signals arising from TCR engagement (signal 1) or by synergizing with signal 1 to produce IL-2, which in turn leads to proliferation and dilution of negative regulatory factors. To better define the cellular events that lead to the induction of anergy, we used the immunosuppressive agent rapamycin, which blocks T cell proliferation in late G1 phase but does not affect costimulation-dependent IL-2 production. Our data demonstrate that full T cell activation (signal 1 plus 2) in the presence of rapamycin results in profound T cell anergy, despite the fact that these cells produce copious amounts of IL-2. Similar to conventional anergy (induction by signal 1 alone), the rapamycin-induced anergic cells show a decrease in mitogen-activated protein kinase activation, and these cells can be rescued by culture in IL-2. Interestingly, the rapamycin-induced anergic cells display a more profound block in IL-3 and IFN-gamma production upon rechallenge. Finally, in contrast to rapamycin, full T cell activation in the presence of hydroxyurea (which inhibits the cell cycle in early S phase) did not result in anergy. These data suggest that it is neither the direct effect of costimulation nor the subsequent T cell proliferation that prevents anergy induction, but rather the biochemical events that occur upon progression through the cell cycle from G1 into S phase.


Subject(s)
Cell Cycle Proteins , Clonal Anergy/drug effects , Immunosuppressive Agents/pharmacology , Sirolimus/pharmacology , T-Lymphocytes/drug effects , Tumor Suppressor Proteins , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Cell Cycle/drug effects , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p27 , Cyclosporine/pharmacology , Humans , Interferon-gamma/biosynthesis , Interleukin-2/biosynthesis , Interleukin-2/pharmacology , Interleukin-3/biosynthesis , Lymphocyte Activation/drug effects , Microtubule-Associated Proteins/physiology , Receptors, Antigen, T-Cell/physiology , Ribosomal Protein S6 Kinases/physiology , T-Lymphocytes/immunology
16.
Immunol Rev ; 165: 287-300, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9850868

ABSTRACT

The consequences of T-cell receptor engagement (signal 1) are profoundly affected by the presence or absence of co-stimulation (signal 2). T-cell receptor (TCR) stimulation in the absence of CD28-mediated co-stimulation not only results in little interleukin (IL)-2 production, but induces a long lasting hyporesponsive state known as T-cell clonal anergy. The addition of CD28 ligation to signal 1, on the other hand, results in the production of copious amounts of IL-2. Our laboratory has utilized CD4+ Th 1 clones in an effort to understand the molecular events resulting in enhanced IL-2 production by co-stimulation and the inhibition of IL-2 production in anergy. Our current studies have focused on defining the post-transcriptional effects of CD28-enhanced IL-2 production. The data suggest that a major component of CD28's ability to regulate IL-2 production occurs at the level of message stability and involves the 3'-untranslated region of the message. In terms of anergy, our recent studies support the notion that it is not the result of TCR engagement in the absence of co-stimulation, but rather signal 1 in the absence of IL-2 receptor signaling and proliferation. Furthermore, T-cell anergy appears to be an active negative state in which IL-2 production is inhibited both at the level of signal transduction and by cis-dominant repression at the level of the IL-2 promoter.


Subject(s)
CD28 Antigens/immunology , Clonal Anergy/immunology , Interleukin-2/immunology , 3' Untranslated Regions , Animals , B7-1 Antigen/immunology , Humans , Interleukin-2/genetics , Ligands , Protein Biosynthesis , RNA, Messenger , Receptors, Antigen, T-Cell/immunology
17.
J Am Vet Med Assoc ; 212(1): 90-2, 1998 Jan 01.
Article in English | MEDLINE | ID: mdl-9426787

ABSTRACT

OBJECTIVE: To test effectiveness of IV administration of tolazoline hydrochloride in reversing xylazine hydrochloride-induced sedation in calves. DESIGN: Prospective study. ANIMALS: 12 female and 12 male Friesian-cross calves from 5 to 7 months old. PROCEDURE: Calves were assigned to 1 of 4 treatment groups. Calves were given xylazine (0.3 mg/kg [0.14 mg/lb] of body weight, IM). Twenty minutes later, calves were treated with saline (0.9% NaCl) solution (1 ml/50 kg [1 ml/110 lb], IV) or tolazoline (1, 2, or 4 mg/kg [0.45, 0.9, or 1.8 mg/lb], IV). Behavioral and physiologic measurements included elapsed time from xylazine administration to recumbency, arousal and standing times after reversal drug administration, heart rate, and respiratory rate. RESULTS: Mean (+/- SD) recumbency time for all calves was 5.4 +/- 1.8 minutes. Compared with administration of saline solution, all 3 doses of tolazoline significantly decreased arousal and standing times. Mean arousal time for calves receiving saline solution was 27.8 +/- 11.5 minutes. Administration of tolazoline at 1, 2, and 4 mg/kg resulted in mean arousal times of 4.7 +/- 3.8, 0.9 +/- 0.5, and 0.7 +/- 0.3 minutes, respectively. Mean standing time for calves receiving saline solution was 38.8 +/- 2.8 minutes. Administration of tolazoline at 1, 2, and 4 mg/kg resulted in mean standing times of 14.0 +/- 11.0, 3.0 +/- 1.2, and 2.4 +/- 1.1 minutes, respectively. CLINICAL IMPLICATIONS: For routine use, tolazoline doses of 1 to 2 mg/kg should suffice. In cattle, IV administration of tolazoline reverses pharmacologic effects of xylazine, thereby hastening recovery from xylazine-induced sedation.


Subject(s)
Adrenergic alpha-Antagonists/pharmacology , Cattle/physiology , Hypnotics and Sedatives/antagonists & inhibitors , Tolazoline/pharmacology , Xylazine/antagonists & inhibitors , Adrenergic alpha-Antagonists/administration & dosage , Animals , Arousal/drug effects , Female , Hypnotics and Sedatives/administration & dosage , Injections, Intramuscular/veterinary , Injections, Intravenous/veterinary , Male , Time Factors , Tolazoline/administration & dosage , Xylazine/administration & dosage
18.
Immunol Lett ; 51(1-2): 59-68, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8811346

ABSTRACT

Nonhuman primates naturally infected with simian immunodeficiency virus (SIV), while maintaining chronic viremia, do not develop any disease associated with lentiviral infection. Thus they provide a unique model to define the mechanism(s) by which they remain infected but disease-resistant. The purpose of this article is to summarize our current knowledge of the virological and immunological studies that have been performed in sooty mangabeys naturally infected with SIVsmm and in disease-susceptible rhesus macaques experimentally infected with SIVsmm. Data on virological studies demonstrate that the naturally infected sooty mangabeys are infected predominantly with SIV that have nef sequences distinct from those shown to cause disease in the inappropriate host, a factor which may contribute to disease resistance. Hyperimmunization with a variety of antigens or chronic infection contributes to accelerated disease and death in rhesus macaques if hyperimmunizations are initiated at the time of SIV infection, whereas similar hyperimmunization and chronic infection do not lead to disease in naturally infected seropositive sooty mangabeys. However, in both species infected with SIV, hyperimmunization leads to increased virus load, suggesting that virus load per se cannot account for disease, at least in naturally infected nonhuman primates. Immunological studies concerning changes in subsets of T cells, based on cytokine profile (TH0/TH1/TH2), showed that whereas rhesus macaques early post SIV infection show a dominant TH1 profile, this profile rapidly changes to TH0. On the other hand, mangabeys continuously demonstrate a TH2-like profile. Studies also showed a high frequency of in vivo-activated cells in the peripheral blood of SIV-infected rhesus macaques and mangabeys. Of interest, however, is the finding of a similar level of in vivo-activated cells from ELISA seronegative mangabeys. Although cells from SIV-infected mangabeys fail to show increased levels of apoptotic cells following incubation with immobilized anti-CD3, PBMC from rhesus macaques at varying time intervals do show increased levels of apoptotic cells, an increase which is predominantly seen in CD8+ T cells and is unrelated to levels of viremia. Sooty mangabeys maintain a high frequency of CD8+ T cells that regulate virus replication throughout their lifetime, a frequency that develops prior to ELISA-based seroconversion, whereas rhesus macaques only show a frequency of CD8+ T cells high enough to regulate virus replication shortly post infection, and this regulatory function is gradually lost prior to CD8+ cell loss and death. HIV and SIV infection do have profound effects on the expression of a number of costimulatory and adhesion molecules. There appear to be differences in the nature of the intracellular phosphorylated proteins in cells from activated rhesus macaques and mangabeys. We believe that careful studies of the detailed mechanisms of the issues described above may provide an understanding of the constellation of virological and immunological mechanisms responsible for the disease-resistant state of naturally infected sooty mangabeys. These findings can be employed for evaluating a nonvirus sterilizing form of SIV/HIV vaccines.


Subject(s)
Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Animals , Apoptosis/immunology , CD8-Positive T-Lymphocytes/immunology , Cercocebus atys , Gene Products, nef/immunology , Immunity, Innate/immunology , Lymphocyte Activation/immunology , Macaca mulatta , Signal Transduction/immunology , T-Lymphocytes, Helper-Inducer/immunology , Viral Load
19.
Mol Biol Evol ; 12(1): 103-12, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7877485

ABSTRACT

We introduce here a simple approach for rapidly determining restriction maps for a number of regions of a genome; this involves "anchoring" a map with a rare restriction site (in this case the seldom-cutting EagI) followed by partial digestion of a frequent-cutting enzyme (e.g., Sau 3A). We applied this technology to five species of the Anopheles gambiae complex. In a single Southern blot we obtained about a 15-kb restriction map each for the mtDNA, rRNA gene, and a scnDNA region for each of five species. Phylogenetic analyses of these regions yield trees at odds with the more traditional chromosome inversion-based trees. The value of the approach for systematic purposes is the ease with which several large, independent regions of the genome can be quickly assayed for molecular variation.


Subject(s)
Anopheles/genetics , Malaria/parasitology , NAD(P)H Dehydrogenase (Quinone)/genetics , Phylogeny , Animals , Anopheles/classification , Anopheles/enzymology , Blotting, Southern , DNA, Mitochondrial/genetics , DNA, Ribosomal/genetics , Deoxyribonucleases, Type II Site-Specific , Diptera/genetics , Genomic Library , Humans , Insect Vectors , Karyotyping , Restriction Mapping
20.
J Acquir Immune Defic Syndr (1988) ; 7(6): 539-50, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8176639

ABSTRACT

In vitro polyclonal activation of peripheral blood mononuclear cells (PBMCs) from 70% of the simian immunodeficiency virus (SIV) serum enzyme-linked, immunosorbent assay (ELISA)-negative sooty mangabeys leads to synthesis and release of low but significant and reproducible levels of SIV-reactive antibodies, as determined by ELISA and Western blot analysis. The predominant isotype of SIV-reactive antibodies in the pokeweed mitogen (PWM) supernatant fluids from serum ELISA-negative mangabeys is IgM, whereas the predominant isotype of SIV-reactive antibodies in seropositive mangabeys is IgG. Depletion of CD8+ cells led to a marked increase in the levels of SIV-reactive antibodies detected in supernatant fluids from PWM-induced cultures from the serum ELISA-negative mangabeys. No evidence for such SIV-reactive antibodies has been found, to date, in similar unfractionated or CD8+ T-cell-depleted PWM-induced PBMC cultures from uninfected macaques. Supernatant fluids from PWM cultures of PBMCs from a select group of serum ELISA-negative mangabeys, when concentrated five times, were shown to give a Western blot profile against SIV, similar to the profile seen with plasma from seropositive infected macaques and mangabeys. Evidence is presented to show that these serum ELISA-negative mangabeys are most likely latently infected with SIV. This evidence, which was obtained in samples from such ELISA-negative mangabeys, includes the detection of reverse transcriptase activity and the presence of SIV p27 in supernatant fluids of phytohemagglutinin-stimulated PBMCs in vitro. In addition, the data show the presence of CD8+ T cells that regulate SIV-specific Ig synthesis and show the detection of gag sequences by the polymerase chain reaction. Thus, the PWM assay described herein may provide a valuable additional tool for detection of lentivirus infection before or in the absence of seroconversion.


Subject(s)
Immunoglobulin G/biosynthesis , Immunoglobulin M/biosynthesis , Leukocytes, Mononuclear/microbiology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Animals , Antibodies, Viral/biosynthesis , Antibody Specificity , Blotting, Western , Cells, Cultured , Cercocebus atys , Enzyme-Linked Immunosorbent Assay , Leukocytes, Mononuclear/immunology , Macaca mulatta , Simian Acquired Immunodeficiency Syndrome/diagnosis , Simian Immunodeficiency Virus/physiology , T-Lymphocytes, Regulatory/immunology , Virus Latency
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