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1.  Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy 
Molecular Therapy  2012;21(1):217-227.
Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 1011 genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.
doi:10.1038/mt.2012.160
PMCID: PMC3538299  PMID: 23089734
2.  Induction of antigen-specific tolerance by oral administration of Lactococcus lactis delivered immunodominant DQ8-restricted Gliadin peptide in sensitized NOD AB° DQ8 transgenic mice 
Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (LL) provides a novel therapeutic approach for the induction of tolerance. Celiac disease is associated with either HLA-DQ2 or HLA-DQ8 restricted responses to specific antigenic epitopes of gliadin, and may be treated by induction of antigen-specific tolerance. We investigated whether oral administration of LL-delivered DQ8-specific gliadin epitope induces antigen-specific tolerance.
L. lactis
was engineered to secrete a deamidated DQ8 gliadin epitope (LL-eDQ8d) and the induction of antigen-specific tolerance was studied in NOD AB° DQ8 transgenic mice. Tolerance was assessed by delayed-type hypersensitivity reaction, cytokine measurements, eDQ8d-specific proliferation and regulatory T cell analysis. Oral administration of LL-eDQ8d induced suppression of local and systemic DQ8 restricted T-cell responses in NOD AB° DQ8 transgenic mice. Treatment resulted in an antigen-specific decrease of the proliferative capacity of inguinal lymph node cells and lamina propria cells. Production of IL-10 and TGF-β and a significant induction of Foxp3+ regulatory T-cells were associated with the eDQ8d-specific suppression induced by LL-eDQ8d.
These data provide support for the development of effective therapeutic approaches for gluten-sensitive disorders using orally administered antigen-secreting LL. Such treatments may be effective even in the setting of established hypersensitivity.
doi:10.4049/jimmunol.0802891
PMCID: PMC3480315  PMID: 19635921
3.  Adeno-associated virus mediated delivery of Tregitope 167 ameliorates experimental colitis 
AIM: To explore the anti-inflammatory potential of adeno-associated virus-mediated delivery of Tregitope 167 in an experimental colitis model.
METHODS: The trinitrobenzene sulfonate (TNBS) model of induced colitis was used in Balb/c mice. Subsequently after intravenous adeno-associated virus-mediated regulatory T-cell epitopes (Tregitope) delivery, acute colitis was initiated by intra-rectal administration of 1.5 mg TNBS in 40% ethanol followed by a second treatment with TNBS (0.75 mg in 20% ethanol) 8 d later. Control groups included mice not treated with TNBS (healthy control group) and mice treated by TNBS only (diseased group). At the time of sacrifice colon weight, the disease activity index and histology damage score were determined. Immunohistochemical staining of the colonic tissues was performed to asses the cellular infiltrate and the presence of transcription factor forkhead Box-P3 (Foxp3). Thymus, mesenteric lymph nodes, liver and spleen tissue were collected and the corresponding lymphocyte populations were further assessed by flow cytometry analysis for the expression of CD4+ T cell and regulatory T cell associated markers.
RESULTS: The Tregitope 167 treated mice gained an average of 4% over their initial body weight at the time of sacrifice. In contrast, the mice treated with TNBS alone (no Tregitope) developed colitis, and lost 4% of their initial body weight at the time of sacrifice (P < 0.01). The body weight increase that had been observed in the mice pre-treated with Tregitope 167 was substantiated by a lower disease activity index and a decreased colon weight as compared to the diseased control group (P < 0.01 and P < 0.001, respectively). Immunohistochemical staining of the colonic tissues for CD4+ showed that inflammatory cell infiltrates were present in TNBS treated mice with or without administration with tregitope 167 and that these cellular infiltrates consisted mainly of CD4+ cells. For both TNBS treated groups CD4+ T cell infiltrates were observed in the sub-epithelial layer and the lamina propria. CD4+ T cell infiltrates were also present in the muscularis mucosa layer of the diseased control mice, but were absent in the Tregitope 167 treated group. Numerous Foxp3 positive cells were detected in the lamina propria and sub-epithelium of the colon sections from mice treated with Tregitope 167. Furthermore, the Foxp3 and glycoprotein A repetitions predominant markers were significantly increased in the CD4+ T lymphocyte population in the thymus of the mice pre-treated with adeno-associated virus serotype 5 (cytomegalovirus promoter-Tregitope 167), as cytomegalovirus promoter compared to lymphocyte populations in the thymus of diseased and the healthy control mice (P < 0.05 and P < 0.001, respectively).
CONCLUSION: This study identifies adeno-associated virus-mediated delivery of regulatory T-cell epitope 167 as a novel anti-inflammatory approach with the capacity to decrease intestinal inflammation and induce long-term remission in inflammatory bowel disease.
doi:10.3748/wjg.v18.i32.4288
PMCID: PMC3436043  PMID: 22969191
Adeno-associated virus; Regulatory T cell epitope; Inflammatory bowel diseases; Adeno-associated virus
4.  Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100 
BMC Biotechnology  2012;12:42.
Background
Controlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds.
Results
Highly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA.
Conclusion
Our results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.
doi:10.1186/1472-6750-12-42
PMCID: PMC3424168  PMID: 22827812
5.  Gene and cell therapy based treatment strategies for inflammatory bowel diseases 
Inflammatory bowel diseases (IBD) are a group of chronic inflammatory disorders most commonly affecting young adults. Currently available therapies can result in induction and maintenance of remission, but are not curative and have sometimes important side effects. Advances in basic research in IBD have provided new therapeutic opportunities to target the inflammatory process involved. Gene and cell therapy approaches are suitable to prevent inflammation in the gastrointestinal tract and show therefore potential in the treatment of IBD. In this review, we present the current progress in the field of both gene and cell therapy and future prospects in the context of IBD. Regarding gene therapy, we focus on viral vectors and their applications in preclinical models. The focus for cell therapy is on regulatory T lymphocytes and mesenchymal stromal cells, their potential for the treatment of IBD and the progress made in both preclinical models and clinical trials.
doi:10.4291/wjgp.v2.i6.114
PMCID: PMC3240904  PMID: 22180846
Viral vector; Gene therapy; Cell therapy; Inflammatory bowel diseases; Immune tolerance; Regulatory T lymphocytes; Mesenchymal stromal cells
6.  Genetically Modified Lactococcus lactis for Delivery of Human Interleukin-10 to Dendritic Cells 
Interleukin-10 (IL-10) plays an indispensable role in mucosal tolerance by programming dendritic cells (DCs) to induce suppressor Th-cells. We have tested the modulating effect of L. lactis secreting human IL-10 (L.  lactisIL-10) on DC function in vitro. Monocyte-derived DC incubated with L.  lactisIL-10 induced effector Th-cells that markedly suppressed the proliferation of allogenic Th-cells as compared to L. lactis. This suppressive effect was only seen when DC showed increased CD83 and CD86 expression. Furthermore, enhanced production of IL-10 was measured in both L.  lactisIL-10-derived DC and Th-cells compared to L. lactis-derived DC and Th-cells. Neutralizing IL-10 during DC-Th-cell interaction and coculturing L.  lactisIL-10-derived suppressor Th-cells with allogenic Th-cells in a transwell system prevented the induction of suppressor Th-cells. Only 130 pg/mL of bacterial-derived IL-10 and 40 times more exogenously added recombinant human IL-10 were needed during DC priming for the generation of suppressor Th-cells. The spatially restricted delivery of IL-10 by food-grade bacteria is a promising strategy to induce suppressor Th-cells in vivo and to treat inflammatory diseases.
doi:10.1155/2012/639291
PMCID: PMC3146989  PMID: 21811497
7.  In vivo knock-down of multidrug resistance transporters ABCC1 and ABCC2 by AAV-delivered shRNAs and by artificial miRNAs 
ABC transporters export clinically-relevant drugs and their over-expression causes multidrug resistance. In order to knock-down ABC transporters, ABCC1 and ABCC2, 13 shRNAs were developed. Four shRNA candidates were tested in vivo using self-complementary adeno-associated virus serotype 8. A strong, specific knock-down of Abbc2 was observed in mice liver, but at the cost of toxicity caused by oversaturation of the RNAi machinery due to high shRNA expression. Subsequent generation of artificial miRNAs showed better efficacy profile. These results demonstrate the feasibility of knocking down Abbc2 via AAV-delivered shRNAs to the liver, and encourage the use of miRNA in further therapeutics development.
PMCID: PMC3131674  PMID: 21769296
shRNA; miRNA; AAV; Abbc1; Abbc2; multidrug resistance; hepatocellular carcinoma
8.  Prediction of antitumour necrosis factor clinical efficacy by real‐time visualisation of apoptosis in patients with Crohn's disease 
Gut  2006;56(4):509-517.
Background
The human anti‐tumour necrosis factor (TNF) antibody infliximab binds to the membrane TNF and subsequently induces apoptosis of activated lamina propria T lymphocytes in patients with Crohn's disease in vitro.
Aim
To test whether the ability of rapid anti‐TNF‐induced apoptosis in the gut predicts the efficacy of anti‐TNF treatment in inflammatory bowel disease.
Methods
99mTechnetium–annexin V single‐photon emission computer tomography (SPECT) was performed in 2 models of murine experimental colitis and in 14 patients with active Crohn's disease as assessed by the Crohńs Disease Activity Index (CDAI) to study the effect of anti‐TNF treatment on apoptosis in the intestine during active colitis. Disease activity was evaluated 2 weeks after infliximab infusion using the CDAI (definition response: drop of >100 points).
Results
Colonic uptake of 99mTc‐annexin V significantly increased in 2,4,6‐trinitrobenzene sulphonate‐induced colitis as well as in transfer colitis on administration of anti‐TNF antibodies compared with a control antibody as determined with dedicated animal pinhole SPECT. In addition, uptake of 99mTc–annexin V significantly increased in patients with active Crohn's disease responding to infliximab treatment. Colonic 99mTc–annexin V uptake ratio (mean (SEM)) increased from 0.24 (0.03) to 0.41(0.07) (p<0.01), 24 h after infliximab infusion (5 mg/kg). A mean increase of 98.7% in colonic uptake of 99mTc–annexin V could be detected in 10 of the 14 responding patients (CDAI >100 points at week 2) compared with 15.2% in non‐responding patients (p = 0.03). Analysis of the mucosal biopsy specimens identified lamina propria T cells as target cells undergoing apoptosis.
Conclusions
These in vivo observations support the notion that colonic uptake of 99mTc–annexin V correlates with clinical benefit of anti‐TNF treatment and might be predictive of therapeutic success.
doi:10.1136/gut.2006.105379
PMCID: PMC1856870  PMID: 17082252
9.  Prevention of experimental colitis by parenteral administration of a pathogen‐derived immunomodulatory molecule 
Gut  2006;56(3):351-357.
Background
Filamentous haemagglutinin (FHA) of Bordetella pertussis subverts host immune responses by inhibiting interleukin (IL)12 and enhancing IL10 production by macrophages and dendritic cells, and promoting the induction of regulatory T cells.
Hypothesis
Injection of FHA would ameliorate disease in a T cell‐dependent model of colitis via the induction of anti‐inflammatory cytokines and regulatory T cells.
Methods
Colitis was induced by injection of CD4CD45RBhigh naive T cells into severe combined immunodeficient (SCID) mice. Mice were treated with four subcutaneous injections of FHA or buffer alone.
Results
Parenteral injection of FHA stimulated IL10 and/or transforming growth factor β production in local and mesenteric lymph nodes and Peyer's patches of mice 2–6 h after administration. Compared with phosphate‐buffered saline‐treated mice, FHA‐treated SCID mice had significantly (p<0.01) less weight loss, lower colon weight, less colon shrinkage and reduced inflammatory lesions. The therapeutic effect of FHA was associated with enhanced IL10 and reduced type 1 and type 2 T helper cytokine production by spleen cells. Finally, FHA also attenuated the symptoms of colitis in SCID mice transferred with CD4CD45RBhigh T cells from IL10‐deficient mice.
Conclusions
Our finding shows that FHA suppresses type 1 T helper and pro‐inflammatory cytokines, and ameliorates disease activity in a chronic T cell‐dependent model of colitis, an effect that was not dependent on IL10 production by T cells, but was associated with induction of anti‐inflammatory cytokines in vivo. Having already been used as a pertussis vaccine component in children, FHA is a promising candidate for clinical testing in patients with Crohn's disease.
doi:10.1136/gut.2006.099861
PMCID: PMC1856816  PMID: 16952913
10.  Treatment and Prevention of Staphylococcus epidermidis Experimental Biomaterial-Associated Infection by Bactericidal Peptide 2▿  
Antimicrobial Agents and Chemotherapy  2006;50(12):3977-3983.
Biomaterial-associated infections (BAI) are the major cause of failure of indwelling medical devices and are predominantly caused by staphylococci, especially Staphylococcus epidermidis. We investigated the in vitro microbicidal activity of the synthetic antimicrobial peptide bactericidal peptide 2 (BP2) and its efficacy in a murine model of S. epidermidis BAI. BP2 showed potent microbicidal activity at micromolar concentrations against a broad spectrum of microorganisms, including antibiotic-resistant bacteria. The staphylocidal activity of BP2 was not affected by physiological salt concentrations and was only slightly affected by the presence of human plasma. In the BAI model, injection of BP2 (5 mg/kg of body weight) 1 h after challenge with S. epidermidis resulted in an 80% reduction in the number of culture-positive implants and a 100-fold reduction in survival of S. epidermidis in peri-implant tissue at 24 h postchallenge. When BP2 was injected along implants 3 h prior to bacterial challenge, the median numbers of CFU cultured from biomaterial implants and peri-implant tissue were reduced by 85% and 90%, respectively. In conclusion, BP2 has potent, broad-spectrum in vitro microbicidal activity and showed potent in vivo activity in a murine model of S. epidermidis biomaterial-associated infection.
doi:10.1128/AAC.00575-06
PMCID: PMC1693978  PMID: 17000746
11.  Distribution and Kinetics of Lipoprotein-Bound Lipoteichoic Acid  
Infection and Immunity  2003;71(6):3280-3284.
Lipoteichoic acid (LTA), a major cell wall component of gram-positive bacteria, is an amphipathic anionic glycolipid with structural similarities to lipopolysaccharide (LPS) from gram-negative bacteria. LTA has been implicated as one of the primary immunostimulatory components that may trigger the systemic inflammatory response syndrome. Plasma lipoproteins have been shown to sequester LPS, which results in attenuation of the host response to infection, but little is known about the LTA binding characteristics of plasma lipid particles. In this study, we have examined the LTA binding capacities and association kinetics of the major lipoprotein classes under simulated physiological conditions in human whole blood (ex vivo) by using biologically active, fluorescently labeled LTA and high-performance gel permeation chromatography. The average distribution of an LTA preparation from Staphylococcus aureus in whole blood from 10 human volunteers revealed that >95% of the LTA was associated with total plasma lipoproteins in the following proportions: high-density lipoprotein (HDL), 68% ± 10%; low-density lipoprotein (LDL), 28% ± 8%; and very low density lipoprotein (VLDL), 4% ± 5%. The saturation capacity of lipoproteins for LTA was in excess of 150 μg/ml. The LTA distribution was temperature dependent, with an optimal binding between 22 and 37°C. The binding of LTA by lipoproteins was essentially complete within 10 min and was followed by a subsequent redistribution from HDL and VLDL to LDL. We conclude that HDL has the highest binding capacity for LTA and propose that the loading and redistribution of LTA among plasma lipoproteins is a specific process that closely resembles that previously described for LPS (J. H. M. Levels, P. R. Abraham, A. van den Ende, and S. J. H. van Deventer, Infect. Immun. 68:2821-2828, 2001).
doi:10.1128/IAI.71.6.3280-3284.2003
PMCID: PMC155762  PMID: 12761109
12.  Arginine deficiency affects early B cell maturation and lymphoid organ development in transgenic mice 
The Journal of Clinical Investigation  2002;110(10):1539-1548.
Apart from its role in the synthesis of protein and nitric oxide (NO), and in ammonia detoxification, the amino acid arginine exerts an immunosupportive function. We have studied the role of arginine in immune defense mechanisms in the developing postnatal immune system. In suckling mice, arginine is produced in the small intestine. In F/A-2+/+ transgenic mice, which overexpress arginase in their enterocytes, circulating and tissue arginine concentrations are reduced to 30–35% of controls. In these mice, the development and composition of the T cell compartment did not reveal abnormalities. However, in peripheral lymphoid organs and the small intestine, B cell cellularity and the number and size of Peyer’s patches were drastically reduced, and serum IgM levels were significantly decreased. These phenotypes could be traced to an impaired transition from the pro– to pre–B cell stage in the bone marrow. Cytokine receptor levels in the bone marrow were normal. The development of the few peripheral B cells and their proliferative response after in vitro stimulation was normal. The disturbance in B cell maturation was dependent on decreased arginine levels, as this phenotype disappeared upon arginine supplementation and was not seen in NO synthase– or ornithine transcarbamoylase–deficient mice. We conclude that arginine deficiency impairs early B cell maturation.
doi:10.1172/JCI16143
PMCID: PMC151816  PMID: 12438451
13.  Feed a Cold, Starve a Fever? 
An English old wives’ tale advises us to “feed a cold and starve a fever.” Here we report that the nutritional status modulates the T helper 1 (Th1)-Th2 balance of activated T cells in human volunteers. Food intake resulted in increased levels of gamma interferon production, whereas food deprivation stimulated interleukin-4 release.
doi:10.1128/CDLI.9.1.182-183.2002
PMCID: PMC119893  PMID: 11777851
14.  CpG Oligodeoxynucleotides Enhance Host Defense during Murine Tuberculosis  
Infection and Immunity  2002;70(1):147-152.
Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate immune cells to produce cytokines. CpG ODNs protect mice against infections with intracellular bacteria by the induction of a T helper 1 (Th1) response. To determine the effect of CpG ODNs in pulmonary tuberculosis, mice were treated with CpG ODNs or control ODNs at the time of intranasal infection. CpG ODNs reduced mycobacterial outgrowth for up to 5 weeks after Mycobacterium tuberculosis infection and were associated with a decrease in inflammation in lung tissue. CpG treatment was also associated with elevated levels of gamma interferon (IFN-γ) and decreased levels of interleukin 4 in the lungs and an increased capacity of splenocytes to secrete Th1-type cytokines. CpG ODNs given 2 weeks after infection were still able to reduce mycobacterial outgrowth and to enhance a Th1 response 5 weeks postinfection. Administration of CpG ODNs to IFN-γ-gene-deficient mice failed to reduce mycobacterial outgrowth. These data suggest that CpG ODNs improve host defense during pulmonary tuberculosis by an IFN-γ-dependent mechanism.
doi:10.1128/IAI.70.1.147-152.2002
PMCID: PMC127605  PMID: 11748176
15.  Role of Endogenous Interleukin-12 in Immune Response to Staphylococcal Enterotoxin B in Mice 
Infection and Immunity  2001;69(9):5949-5952.
In the present study, the roles of interleukin 12 (IL-12) and IL-18 and their possible interaction during superantigen-induced responses were studied by injection of staphylococcal enterotoxin B (SEB) into mice. These data suggest that the role of IL-12 in SEB-induced responses is limited to sustaining gamma interferon release by an IL-18-independent mechanism.
doi:10.1128/IAI.69.9.5949-5952.2001
PMCID: PMC98720  PMID: 11500480
16.  Concurrent Upregulation of Urokinase Plasminogen Activator Receptor and CD11b during Tuberculosis and Experimental Endotoxemia 
Infection and Immunity  2001;69(8):5182-5185.
Patients with tuberculosis had higher expression of monocyte urokinase receptor (uPAR) and CD11b than controls. In vitro, lipoarabinomannan and lipopolysaccharide (LPS) from Escherichia coli shared the ability to enhance uPAR and CD11b expression on monocytes and granulocytes. In healthy volunteers, LPS induced increases in monocyte and granulocyte uPAR and CD11b.
doi:10.1128/IAI.69.8.5182-5185.2001
PMCID: PMC98617  PMID: 11447203
17.  Thalidomide Inhibits Granulocyte Responses in Healthy Humans after Ex Vivo Stimulation with Bacterial Antigens 
Ingestion of thalidomide was associated with a reduction in the upregulation of the granulocyte activation marker CD11b and a reduced capacity to release elastase and lactoferrin after stimulation with lipopolysaccharide or lipoteichoic acid. A single oral dose of thalidomide attenuates neutrophil activation upon ex vivo stimulation with bacterial antigens.
doi:10.1128/AAC.45.5.1547-1549.2001
PMCID: PMC90503  PMID: 11302825
18.  Induction of Cell-Associated Chemokines after Endotoxin Administration to Healthy Humans 
Infection and Immunity  2001;69(4):2736-2738.
Erythrocytes express the Duffy antigen receptor for chemokines. Endotoxin injection into humans induced high levels of interleukin-8 (IL-8), growth-related oncogene α, and monocyte chemoattractant protein 1 in circulating erythrocytes. IL-8 was also recovered from mononuclear and polymorphonuclear cells. Cell-associated chemokines may more accurately reflect their production than plasma concentrations.
doi:10.1128/IAI.69.4.2736-2738.2001
PMCID: PMC98216  PMID: 11254644
19.  A Single Oral Dose of Thalidomide Enhances the Capacity of Lymphocytes to Secrete Gamma Interferon in Healthy Humans 
Thalidomide is increasingly being used as adjuvant therapy for patients with mycobacterial and human immunodeficiency virus (HIV) infections. The T-helper (Th)1 cytokine–Th2 cytokine balance critically determines the outcomes of these diseases. To obtain insight into the effect of thalidomide on the capacity of lymphocytes to produce Th1 and Th2 cytokines, six healthy volunteers received an oral dose (400 mg) of thalidomide. Before and at 3, 6, and 24 h after ingestion of thalidomide, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated for 24 h with the T-cell stimulant staphylococcal enterotoxin B (SEB) or anti-CD3/CD28. In all six volunteers ingestion of thalidomide was associated with enhanced SEB- and anti-CD3/CD28-induced production of the Th1 cytokine gamma interferon (P < 0.05) and a decrease in the level of anti-CD3/CD28-induced interleukin-5 (IL-5) production (P < 0.05). The levels of IL-2 (Th1) and IL-4 (Th2) released remained unchanged. These changes were accompanied by an increase in the amount of IL-12p40 released by the PBMCs 6 h after ingestion of thalidomide (P < 0.05). Thus, a single oral dose of thalidomide causes a Th1-type response in healthy humans. This finding offers a potential explanation for the positive effect of thalidomide in patients with mycobacterial and HIV infections.
PMCID: PMC90059  PMID: 10952569
20.  The CXC Chemokines Gamma Interferon (IFN-γ)-Inducible Protein 10 and Monokine Induced by IFN-γ Are Released during Severe Melioidosis 
Infection and Immunity  2000;68(7):3888-3893.
Gamma interferon (IFN-γ)-inducible protein 10 (IP-10) and monokine induced by IFN-γ (Mig) are related CXC chemokines which bind to the CXCR3 receptor and specifically target activated T lymphocytes and natural killer (NK) cells. The production of IP-10 and Mig by various cell types in vitro is strongly dependent on IFN-γ. To determine whether IP-10 and Mig are released during bacterial infection in humans, we measured plasma levels of IP-10 and Mig in patients with melioidosis, a severe gram-negative infection caused by Burkholderia pseudomallei. IP-10 and Mig were markedly elevated in patients with melioidosis on admission, particularly in blood culture-positive patients, and remained elevated during the 72-h study period. Levels of IP-10 and Mig showed a positive correlation with IFN-γ concentrations and also correlated with clinical outcome. In whole blood stimulated with heat-killed B. pseudomallei, neutralization of IFN-γ and tumor necrosis factor alpha (TNF-α) partly attenuated IP-10 and Mig release, while anti-interleukin-12 (IL-12) and anti-IL-18 had a synergistic effect. Stimulation with other bacteria or endotoxin also induced strong secretion of IP-10 and Mig. These data suggest that IP-10 and Mig are part of the innate immune response to bacterial infection. IP-10 and Mig may contribute to host defense in Th1-mediated host defense during infections by attracting CXCR3+ Th1 cells to the site of inflammation.
PMCID: PMC101663  PMID: 10858199
21.  The Metalloproteinase Inhibitor GI5402 Inhibits Endotoxin-Induced Soluble CD27 and CD16 Release in Healthy Humans 
Infection and Immunity  2000;68(5):3036-3039.
Metalloproteinases have been implicated in the cleavage of a number of cell surface immune receptors. Oral administration of the metalloproteinase inhibitor GI5402 attenuated the release of soluble CD27 and CD16 into the circulation after intravenous endotoxin injection in healthy humans.
PMCID: PMC97526  PMID: 10769011
22.  Upregulation of Monocyte Urokinase Plasminogen Activator Receptor during Human Endotoxemia 
Infection and Immunity  2000;68(4):2156-2160.
The receptor for urokinase-type plasminogen activator (uPAR) (CD87) plays an important role in leukocyte adhesion and migration. To assess the effect of endotoxin on cellular uPAR, uPAR expression was determined on leukocytes by fluorescence-activated cell sorter analysis in seven healthy subjects following intravenous injection of endotoxin (lot G; 4 ng/kg). Endotoxin induced a transient increase in uPAR expression on monocytes, reaching a 92% ± 46% increase over baseline expression after 6 h (P < 0.05). Endotoxin did not influence uPAR expression on granulocytes, while uPAR remained undetectable on lymphocytes. Endotoxin also increased soluble uPAR levels in plasma (P < 0.05). Stimulation of human whole blood with endotoxin or gram-positive stimuli in vitro also resulted in an upregulation of monocyte uPAR expression. Although tumor necrosis factor alpha (TNF) upregulated monocyte uPAR expression, anti-TNF did not influence the endotoxin-induced increase in monocyte uPAR expression. These data suggest that infectious stimuli may influence monocyte function in vivo by enhancing the expression of uPAR.
PMCID: PMC97398  PMID: 10722614
23.  Reduced Th1, but Not Th2, Cytokine Production by Lymphocytes after In Vivo Exposure of Healthy Subjects to Endotoxin 
Infection and Immunity  2000;68(3):1014-1018.
Endotoxin (lipopolysaccharide [LPS]) tolerance is characterized by a reduced capacity of monocytes to produce proinflammatory cytokines upon restimulation in vitro. To determine whether LPS exposure induces a change in lymphocyte cytokine production and whether this results in a shift in the T-helper 1 (Th1)/Th2 balance, whole blood obtained from seven healthy subjects before and after an intravenous injection of LPS (4 ng/kg) was stimulated in vitro with the T-cell stimulus anti-CD3/CD28 or staphylococcal enterotoxin B. Whole-blood production of the Th1 cytokines gamma interferon (IFN-γ) and interleukin-2 (IL-2) was markedly reduced at 3 and 6 h, while the production of the Th2 cytokines IL-4 and IL-5 was not influenced or was slightly increased. The IFN-γ/IL-4 ratio was strongly decreased at 6 h. Serum obtained after LPS exposure could slightly inhibit the release of IFN-γ but increased IL-4 production during stimulation of blood drawn from subjects not previously exposed to LPS. Normal serum also inhibited IFN-γ production, albeit to a lesser extent. LPS exposure influences lymphocyte cytokine production, resulting in a shift toward a Th2 cytokine response, an effect that may be mediated in part by soluble factors present in serum after LPS administration in vivo.
PMCID: PMC97242  PMID: 10678901
24.  Divergent Roles of Tumor Necrosis Factor and Platelet-Activating Factor in Endotoxin-Induced Release of Monocyte Chemoattractant Protein 1 and Macrophage Inflammatory Protein 1β in Chimpanzees 
Infection and Immunity  1999;67(10):5480-5482.
A platelet-activating factor receptor antagonist reduced the release of macrophage inflammatory protein 1β (MIP-1β) during endotoxemia in chimpanzees but did not influence the secretion of monocyte chemoattractant protein 1 (MCP-1). Anti-tumor necrosis factor alpha monoclonal antibody completely prevented MCP-1 release and simultaneously enhanced the secretion of MIP-1β. Levels of MIP-1β and MCP-1 release were differentially regulated during endotoxemia.
PMCID: PMC96909  PMID: 10496934
25.  Elevated Chemokine Concentrations in Sera of Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Patients with Tuberculosis: a Possible Role for Mycobacterial Lipoarabinomannan 
Infection and Immunity  1999;67(8):4295-4297.
Levels of interleukin 8 (IL-8), gamma interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1β (MIP-1β) were elevated in patients with tuberculosis. IP-10 and MCP-1 levels were higher in human immunodeficiency virus (HIV)-seropositive patients than in HIV-seronegative patients with tuberculosis. Lipoarabinomannan induced IL-8, MCP-1, and MIP-1β in vitro, which was partly inhibited by anti-tumor necrosis factor antibody.
PMCID: PMC96742  PMID: 10417209

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