Dendritic Macrophages Research Articles

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Variable processing and presentation of HIV epitopes in dendritic cells and macrophages to CD8 T cells

Background Whether HIV-infectable subsets, such as CD4 T cells, monocytes, macrophages and dendritic cells (DCs), have equivalent capacity to produce and present MHC-I restricted epitopes to HIV-specific CD8 T cells is unknown. MHC-I epitopes are processed by an intracellular degradation pathway involving multiple proteases. In this study we analyzed the effect of toll-like receptor (TLR) agonist-mediated maturation on the processing and presentation of HIV antigens in monocyte-derived DCs and macrophages.

Abstract 221: Chronic Alterations of the Tissue Dendritic Cell and Macrophage Network Underlies the Th1 Response in the Failing Heart

Little is known about changes in the immune cell network that underlie pro-inflammatory cytokine elaboration in chronic heart failure (HF). Dendritic cells (DCs) and macrophages are antigen-presenting cells that play a central role in inflammation and immune tolerance. We investigated the pattern of infiltrating cardiac DCs and macrophages in chronic post-ischemic HF. Myocardial infarction (MI) was induced by left coronary ligation in male C57BL/6 mice. Pathological remodeling and HF were confirmed by hemodynamics, gravimetry, and echocardiography at 8 weeks post-MI. Sham-operated mice served as controls. At 8 weeks, cardiac infiltrating immune cells were isolated by collagenase digestion followed by density gradient centrifugation. Trypan blue-negative viable cells were quantified using flow cytometry. DCs were characterized using specific markers for classical (cDC) (CD11C+B220-), plasmacytoid (pDC) (CD11C+B220+), and immunogenic (CD11c+B220+CD86hi) and tolerogenic (CD11c+B220+CD86low) DCs. Macrophages were classified as activated (CD11b+F480+), pro- or anti-inflammatory (CD11b+F480+Gr1hi or low), and M1 (CD11b+F480+CD206-) or M2 (CD11b+F480+CD206+). Significant increases in cDCs (1.4 fold; p=0.016) and pDCs (4.3 fold; p=0.0016) were observed in failing myocardium. Surface expression of the maturation marker CD86 was increased 8.6 fold (p=0.009), whereas immature DCs exhibited a robust 24.4 fold (p=0.0005) increase. Activated macrophages were also significantly elevated in the failing heart as compared with sham-operated hearts (19.2 ± 1.9 vs 10.9 ± 2.9%; p=0.033), with marked infiltration of pro-inflammatory and M1 macrophages (6.2 ± 0.6 vs 3.8 ± 0.7%; p=0.019). In contrast, levels of anti-inflammatory and M2 macrophages were unchanged. We conclude that in chronic HF, there is marked upregulation of cDC and pDC subsets and pro-inflammatory/M1 macrophages that alter immune homeostasis, contribute to the recruitment and activation of immune cells, and underlie the chronic Th1 response in failing myocardium that promotes pathological remodeling. This suggests that targeting or altering immune cell subsets in the failing heart, rather than specific cytokines, may be a better approach to modulating inflammation in HF.

Host Indoleamine 2,3-Dioxygenase: Contribution to Systemic Acquired Tumor Tolerance

Indoleamine 2,3-dioxygenase (IDO) is a natural mechanism of creating acquired tolerance in a variety of physiological settings. This endogenous tolerogenic pathway has important functions in regulating the magnitude of immune responses in settings of infection, pregnancy, tissue transplantation, mucosal interfaces and others. Whether for angiogenesis, stromal formation or immunologic tolerance, tumors often rely on recruiting host mechanisms. IDO is one such potent endogenous mechanism that appears to be frequently hijacked by tumors to establish systemic immune tolerance to tumor antigens. IDO can be expressed by tumors themselves, but, in addition, its natural site of expression is the host immune cells recruited by the tumor (particularly dendritic cells and macrophages). Therapeutic strategies that target the IDO pathway have been shown to synergize with standard chemotherapy and experimental immunotherapies to break tumor-induced tolerance. When such strategies target IDO expressed in host cells, they may be able to disrupt tolerance without creating intrinsic tumor cell drug resistance.

Sequestrated Intervertebral Discs Are Mainly Infiltrated by Dendritic Cells and not Macrophages: Evidence from a Prospective Study on Patients undergoing Microdiscectomy

Introduction Disk degeneration and mechanical compression of the spinal nerve roots by herniated disk tissue have been identified as pathogenic factors of sciatic pain. Since mechanical compression of the spinal nerve roots by herniated disk tissue has been identified as a pathogenic factor of sciatic pain, microdiscectomy has become the standard treatment for this common health problem.3 Herniated disk tissue has been suggested to induce an autoimmune response as nucleus pulposus (NP) is thought to be excluded from the establishment of immunological tolerance and hence been treated as foreign, if exposed to the immune system in association with disk herniation. From an immunological point of view manifestation of an autoimmune response comprises different steps including antigen processing through antigen-presenting cells (APCs) and differentiation of naïve T helper (TH) cells into TH effector cells. In humans, different types of APCs have been characterized including plasmacytoid dendritic cells (PDCs) and macrophages. There is an evidence suggesting that herniated disk tissue is mainly infiltrated by macrophages and T cells.2,4 The aim of the present study was to characterize the immune cells infiltrating sequestrated discs and to correlate the type of cells with postoperative outcome. Materials and Methods Total 11 patients with sequestrated disk herniations underwent neurologic examinations before microdiscectomy. Their surgically removed discs were weighted and digested for 150 minutes. After digestion the tissue clumps were separated from supernatants by centrifugation. Cells in supernatants were counted, stained with specific surface markers of macrophages (CD11c, CD14), PDCs (CD123, CD4) and activated CD4 + TH cells (CD4, CD45RO), and analyzed by flow cytometry. Recovery from deficits was assessed 2 years postoperatively by neurologic examination and SF36. Results Discs of patients with severe loss of muscle strength (grade 2 and 3) were found to be heavier (10.64g) as compared with patients displaying mild muscle weakness (grade 4 and 5). Disk infiltrates of patients with severe neurologic deficits were found to contain a high proportion of PDCs (28.01%) ( p < 0.04) but only few CD11c + CD14 + macrophages (0.31%) as compared with patients having mild motor deficits. Patients with severe neurologic deficits did not differ from patients with mild motor deficits in the proportion of activated CD4 + CD45RO + T cells infiltrating their discs (both 5.98%). Stronger infiltration of sequestrated discs with PDCs was associated postoperatively with longer duration of complete recovery from motor deficits. Conclusion The findings of the present study do not confirm previous findings as sequestrated disk were found to be infiltrated mainly by PDCs instead of macrophages. Since disc infiltrates did comprise mainly PDCs, the current findings indicate that this APC type may initiate an autoimmune response against sequestrated discs by stimulating naïve TH cells to acquire a TH2-like effector function.1 An autoimmune response may also provide a link between disk degeneration and manifestation of sciatic pain as exposure of NP does occur during disk degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Geiss A., Larsson K., Junevik, K., Rydevik, B., Olmarker, K. Autologous nucleus pulposus primes T cells to develop into interleukin-4-producing effector cells: An experimental study on the autoimmune properties of nucleus pulposus. Journal of Orthopaedic Research 2009;27:97–103 Nerlich AG, Weiler C, Zipperer J, Narozny M, Boos N. Immunolocalization of phagocytic cells in normal and generated intervertebral discs. Spine 2002:27(22):2484–2490 Mixter WJ., Barr J. Rupture of the intervertebral disk with involvement of the spinal canal. New England Journal of Medicine 1934:211:210–215 Virri J, Grönblad M, Seitsalo S, Habtemariam A, Kääpä E, Karaharju E. Comparison of the prevalence of inflammatory cells in subtypes of disk herniations and associations with straight leg raising. Spine 2001:26(21):2311–2315

Abstract 5411: Rb1 controls the differentiation of myeloid-derived suppressor cells subsets

Abstract Myeloid derived suppressor cells (MDSC) consist of two distinct subsets, polymorphonuclear/granulocytic-MDSC (PMN-MDSC), and monocytic-MDSC (M-MDSC) that differ in morphology, phenotype and preferred suppression mechanisms. PMN-MDSC is the predominant subset of MDSC that accumulates in spleen, bone marrow, blood, and tumor tissues in various mouse tumor models. M-MDSC produced higher level of cytokines and chemokines including G-CSF, IL-1b, IL-6, TNF-a, MCP-1 and RANTES compared to PMN-MDSC. The current paradigm considers PMN-MDSC and M-MDSC to differentiate from different progenitors alongside different lineages (granulocytic and monocytic, respectively). M-MDSC differentiate into dendritic cells and macrophages, whereas PMN-MDSC are terminally differentiated cells. Our study demonstrated that in cancer M-MDSC represent highly proliferative population of cells that differentiate not only to DC and macrophages but also to PMN-MDSC. The retinoblastoma gene (Rb1), a central regulator of the cell cycle and cell differentiation, is known to be involved in monocytic and granulocytic lineage commitment. When examining Rb1 levels in freshly isolated cells, M-MDSC expressed high levels of Rb1 similar to those in dendritic cells, macrophages, and neutrophils isolated from naïve mice. In sharp contrast, PMN-MDSC had undetectable Rb1. Lack of Rb1 in mice with conditional deletion of the gene promoted an expansion of splenic CD11b+Ly6G+Ly6Clo cells, similar to the phenotype of PMN-MDSC in tumor-bearing mice. Lack of Rb1 in PMN-MDSC was caused by transcriptional repression of the gene. In culture with GM-CSF, Rb1 expression in M-MDSC remained the same while in PMN-MDSC it was increased over time to the level seen in control neutrophils. Inhibition of rb1 expression in PMN-MDSC correlated with the level of histone acetylation of rb1 promoter. Treatment of PMN-MDSC with inhibitors of histone deacetilases (HDAC) resulted in the increase in rb1 expression suggesting that it may be controlled by histone modification. HDAC inhibitors abrogated differentiation of M-MDSC to PMN-MDSC in the presence of tumor explants conditioned medium. These results suggest that down-regulation of Rb1 plays a major role in accumulation of MDSC in cancer by regulating PMN-MDSC differentiation from M-MDSC. HDAC may be considered as potential targets for therapeutic regulation of MDSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5411. doi:1538-7445.AM2012-5411

Abstract 1536: A novel immunocytokine generates a CD8+ T cell-mediated anti-tumor response in vivo

Abstract Interleukin-12 (IL-12) is a potent TH1 cytokine produced by dendritic cells and macrophages to activate NK cells and T cells. Data from preclinical studies in mice indicate that this cytokine may be useful for the treatment of cancer. However, cases of severe toxicity in human trials have impeded its successful clinical development. Targeting delivery of IL-12 to a tumor while reducing systemic IL-12 exposure could make this a safer, more effective cancer therapeutic agent. One strategy is to conjugate IL-12 to a tumor-binding antibody, as antibody conjugates have previously been used to deliver radioisotopes, other cytokines, and small molecule therapeutics to tumors in vivo. NHS-IL12 is a novel immunocytokine consisting of two molecules of the IL-12 heterodimer covalently fused to a fully humanized antibody (NHS76). This antibody targets necrotic portions of tumors because of its high affinity for single- and double-stranded DNA, which are often exposed as tumors outgrow their blood supply. NHS-IL12 may therefore be useful against a wide range of solid tumors, and mouse models can be used to study its mechanisms of action. Initial imaging studies clearly showed superior in vivo localization of IL-12 to mouse tumors following subcutaneous NHS-IL12 injection as compared to administration of recombinant IL-12 (rIL-12) or IL-12 fused to a control antibody (BC1). In the MC38/CEA+ colorectal carcinoma model in CEA.Tg mice, treatment with NHS-IL12 significantly delayed subcutaneous tumor growth more effectively than treatment with rIL-12. NHS-IL12 did not inhibit MC38/CEA+ tumor cell growth in vitro, suggesting that NHS-IL12 acts indirectly on tumors in vivo most probably via an immune-mediated mechanism. Subsequent findings revealed that tumor-bearing mice treated with NHS-IL12 developed a strong CD8+ T cell response directed against the endogenous p15E tumor antigen. In one study, a subset of mice bearing MC38/CEA+ tumors had complete tumor regression following NHS-IL12 treatment. Each of these mice still had a strong p15E-specific CTL response more than two months after tumor rejection, indicating that p15E may be the major tumor rejection antigen for this in vivo tumor model. Subsequent in vivo depletion studies are planned to investigate which immune cell subsets contribute to the anti-tumor efficacy of this agent. Taken together, these data show that targeted delivery of IL-12 to tumors can overcome the immunosuppressive tumor microenvironment and prime a robust CD8+ T cell response. These findings suggest that NHS-IL12 may be a promising cancer therapeutic as a single agent and/or as a potent vaccine adjuvant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1536. doi:1538-7445.AM2012-1536

Abstract LB-496: Low-dose aspirin targets tumor-associated inflammation and delays neuroblastoma tumor growth in vivo

Abstract ABSTRACT Tumor-associated inflammation is a driving force in several adult cancers, and low doses of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin have proven to reduce cancer incidence. Little is known about tumor-associated inflammation in pediatric neoplasms and no in vivo data exists on the effectiveness of low-dose aspirin on established tumors. Our study evaluates inflammatory patterns paralleling neuroblastoma (NB) tumor growth in vivo and low-dose aspirin as a therapeutic option for high-risk NB. Using the TH-MYCN mouse model of NB, this study demonstrates the presence and progression of tumor-associated inflammation during NB tumor growth. Ex vivo analysis of tumors revealed a transition from an adaptive immune response predominated by CD8+ T-cells in neoplastic lesions from 5 week old homozygous mice, towards an enrichment in immature cells of the innate immune system, including myeloid-derived suppressor cells (MDSCs), immature dendritic cells (DCs) and M2 tumor-associated macrophages (TAMs) during tumor progression. An ongoing M1 to M2 transition of TAMs was demonstrated, which was paralleled by a gradual deterioration of DC status. Ten days of consecutive anti-inflammatory treatment with low-dose aspirin significantly reduced tumor burden (p<0.01) and the presence of tumor-associated cells of the innate immune system (p<0.01) in homozygous mice. In conclusion, our study depicts how inflammatory pathways assist tumor progression through sculpturing of the tumor microenvironment, and for the first time demonstrates an in vivo effect of low-dose aspirin on established MYCN amplified NB tumors in a transgenic mouse model, suggesting a potential new treatment option for high-risk NB patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-496. doi:1538-7445.AM2012-LB-496

Cancer immunotherapy; integration of T cell biology with nanogel- and vector-technology in translational research

Molecular definition of cancer specific antigens recognized by T cells opened an approach to develop cancer specific immunotherapy. Through a series of key findings in cancer immunology, for development of effective therapy major effort has been directed to how to induce T cells with fine specificity, sufficient quantity and high quality in hosts. We intended to integrate immunobiological strategy of T cells with two technologies, nanogel technology and retroviral vector technology for translational research of cancer immunotherapy. Cholesterol-bearing hydrophobizedpullulan (CHP), physically cross-linked nanogels by self-assembly, form nanoparticle complex with protein in water.We found that antigen protein with multiple T cell epitopes, when complexed with CHP, was efficiently transported to lymph nodes and well captured by antigen presenting cells such as dendritic cells and macrophages leading to cross presentation. Hence, CHP-antigen protein complex may become excellent cancer vaccine to induce both CD8+ killer T cells and CD4+ helper T cells of high quality. Intrinsic weakness of insufficiency in number of cancer specific T cells in hosts, prompted us to develop adoptive T cell therapy with lymphocytes engineered to possess cancer specificity. For this purpose, we developed novel retroviral vectors to highly express exogenously transduced cancer specific T cell receptor (TCR), yet suppressing expression of endogenous polyclonal TCR. This approach allowed us to prepare T cells with finer specificity of expressed TCR. In addition, use of RetroNectin®, a recombinant fragment of fibronectin opened a way to ex vivo prepare T cells of sufficient quantity and good quality for clinical use. Translational clinical trials of these cancer vaccine and adoptive T cell therapy are now on-going. An open innovation to promote fusion of different fields of science and technology played an essential role in our development of cancer immunotherapy.

A Francisella tularensis Live Vaccine Strain That Improves Stimulation of Antigen-Presenting Cells Does Not Enhance Vaccine Efficacy

Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS), does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system.

Visualising innate immune responses in the skin in real time

Peripheral organs such as the skin harbour innate immune cells that act as sentinels for intruding pathogens. Our group has used intravital multi-photon imaging to study the behaviour of cutaneous immune cells in the steady-state and during microbial infections. This has led to the dissection of the migratory and interactive behaviour of dendritic cells, neutrophils and macro-phages in infections caused by Leishmania major and Staphylo-coccus aureus . In the course of our studies, we have identified a novel population of dermal gamma-delta T cells. These cells are the main producers of the pro-inflammatory cytokine IL-17 in response to bacterial challenge, regulate the influx of neutrophils into the dermis, and consequently, pathogen transport to the draining lymph node. We now describe a novel, rare population of dermal innate lymphoid-like cells that produce high amounts of Th2 cytokines, such as IL-13. Using multi-photon microscopy, we show that these cells screen the dermis in the steady-state. We further show that they increase in numbers in lymphocyte-deficient mice, indicating that they can fill the dermal niche in the absence of other immunocytes. Together, the skin serves as a paradigm for the understanding of the specialised functions of a diverse array of innate immune cells.

A tribute to Ralph M. Steinman

A tribute to Ralph M. Steinman

Transcriptional Modulation in a Leukocyte-Depleted Splenic Cell Population During Prion Disease

Prion replication in the periphery precedes neuroinvasion in many experimental rodent scrapie models, and in natural sheep scrapie and chronic wasting disease (CWD) in cervids. Prions propagate in the germinal centers of secondary lymphoid organs and are strongly associated with follicular dendritic cells (FDC) and possibly circulating dendritic cells and macrophages. Given the importance of lymphoid organs in prion disease transmission and pathogenesis, gene expression studies may reveal host factors or biological pathways related to prion replication and accumulation. A procedure was developed to enrich for FDC, dendritic cells, and macrophages prior to the investigation of transcriptional alterations in murine splenic cells during prion pathogenesis. In total, 1753 transcripts exhibited fold changes greater than three (false discovery rates less than 2%) in this population isolated from spleens of prion-infected versus uninfected mice. The gene for the small leucine-rich proteoglycan decorin (DCN) was one of the genes most overexpressed in infected mice, and the splenic protein levels mirrored this in mice infected with scrapie as well as bovine spongiform encephalopathy (BSE) and variant Creutzfeldt–Jakob disease (vCJD). A number of groups of functionally related genes were also significantly decreased in infected spleens. These included genes related to iron metabolism and homeostasis, pathways that have also been implicated in prion pathogenesis in the brain. These gene expression alterations provide insights into the molecular mechanisms underlying prion disease pathogenesis and may serve as a pool of potential surrogate markers for the early detection and diagnosis of some prion diseases.

Variant cell types and host glycoproteins influencing HIV-1 infection

A number of variant cell types are associated with HIV1 transmission and disease progression, including dendritic cells (DCs), macrophages and CD4 lymphocytes. The cell types and their phenotypes associated with infection are still poorly defined and will greatly influence HIV-1 transmission as well as disease progression. The types of immune responses mounted against infectious pathogens will also influence the pool of cells to be infected and thereby influence disease course. We have described CD4 lymphocyte cellular phenotypes induced within the same HIV-1 infected individuals against variant pathogens (namely CMV and TB antigens) and shown that these responses are highly variable. We further demonstrate that their distinctive cytokine and chemokine expression patterns correlate to their HIV-1 infectivity profile both in vitro and in vivo. We have additionally addressed whether viruses produced from variant cellular populations have similar or distinctive phenotypes. Here we show that viruses produced from macrophages opposed to Th1 or Th2 CD4 lymphocytes vary for their interaction with DC-SIGN expressed on DCs and have variant neutralization profiles for 2G12. Furthermore, we have identified a number of host glycopoteins (BSSL and MUC6) that can inhibit HIV-1 from interacting with DCs through binding DC-SIGN. We show that genetic polymorphisms within the BSSL gene can associate with strength of binding to DC-SIGN and that these variants can also be associated with markers of disease progression as well as time to coreceptor switch. In conclusion, the interactions between the adaptive and innate immune response can influence HIV-1 transmission and modulate disease course. Published: 3 October 2011

Cellular Infiltration and Cytokine Expression Correlate with Fistulizing State in Crohn's Disease

The purpose of this study was to determine the degree of infiltration of different cell subpopulations (tissue dendritic macrophages, T-helper cells, cytotoxic T lymphocytes, monocytes, neutrophils, and B cells) and the expression of the cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) in inflamed and noninflamed resected tissues from Crohn's disease (CD) and non-CD patients. Twenty-one resected full-thickness intestinal tissue specimens representing 13 subjects (8 CD and 5 non-CD patients) were included in this study. Sections of 20 μm in thickness were cut and then stained using immunohistochemistry. The sections were analyzed using confocal laser scanning microscopy (CLSM). Patterns of staining for inflamed CD and noninflamed CD tissues versus non-CD tissues demonstrated significant differences in the macrophage and T-helper subpopulations. Surprisingly, the T-helper subset was decreased significantly in the inflamed CD sections compared to the noninflamed CD and non-CD sections. The staining patterns also suggested differences in the expression of both IL-12 and TNF-α between the groups, with cytokine overexpression directly relating to the fistulizing state in CD patients. Cytokine expression is upregulated in chronic CD patients; therefore, the degree of inflammation and tissue damage in CD is dependent on the expression of specific cytokines within the tissue. Differentiation of cell subpopulations may be important for establishing a direct relationship with each state of CD (inflammatory, stricturing, and fistulizing states).

Abstract 413: Involvement of myeloid derived suppressor cells (MDSC) in the changes of plasma amino acid profiles in cancer disease

Abstract Although maintained in healthy human, it is well known that plasma amino acid profiles are changed in various pathologic conditions, including liver disorder and cancer. We have previously reported the potential use of plasma amino acid profiling as diagnosis for several cancer disease. However, some questions remained for the mechanisms. The changes in plasma amino acid profiles in tumor disease were regulated by a lot of factors, including metabolic changes of tumors and immune cells. In this study, we focused on the role of immune cells, especially myeloid derived suppressor cells (MDSC). MDSC are characterized as immature myeloid cells that are precursors of dendritic cells, macrophages, and/or granulocytes, they are considered to accumulate in the blood, lymph nods, and at tumor sites in most patients and experimental animals with cancer and inhibit anti-tumor inflammation. Therefore we hypothesized that MDSC were involved in the changes in plasma amino acid profiles in tumor disease. To test our hypothesis, we employed C57BL/6 mice tumor model in which Lewis lung carcinoma (3LL) was injected subcutaneously. Using flow cytometry analysis, a significant percentage of MDSC were detected in spleens of tumor-bearing mice as compared to control normal mice. To assess the role of MDSC in the changes in plasma amino acid profile, we eliminated MDSC from the tumor-bearing mice by inducing apoptosis cell death on MDSC selectively in vivo. Flow cytometry results showed a significant decrease in the percentage of CD11b+ Gr1+ cells in splenocytes derived from MDSC deleted tumor-bearing mice as compared to tumor-bearing mice. To study the immunogenic effects of the MDSC deletion on the tumor-bearing mice, we performed histological analysis (H&E staining) and extensive areas of inflammation within 3LL subcutaneous tumors were observed after the MDSC deletion. In addition, we investigated the effects of the MDSC deletion on the percentage of splenic T cell subpopulations by Flow cytometry. The percentage of CD4+ and CD8+ T cells were increased in spleens of MDSC deleted tumor-bearing mice compared to untreated tumor-bearing mice. To evaluate the effects of the MDSC depletion on the plasma amino acid profiles, plasma samples were collected from normal mice, tumor-bearing mice, and MDSC deleted tumor-bearing mice. Plasma amino acid concentration were measured by LC-MS. The MDSC deletion significantly changed the concentrations of some amino acids in plasma compared to the untreated tumor-bearing mice and normal mice. These changes in plasma amino acid concentrations were partially impeded by adoptive transfer of MDSC from tumor-bearing mice. Altogether, these results suggest that MDSC may be involved in the changes in plasma amino acid profiles in tumor disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 413. doi:10.1158/1538-7445.AM2011-413

Impact of streptozotocin-induced diabetes on functional responses of dendritic cells and macrophages towardsBurkholderia pseudomallei

Diabetes mellitus is a documented risk factor for melioidosis, a tropical infection caused by Burkholderia pseudomallei. The increased susceptibility of diabetic individuals to infections with other pathogens has been associated with immune dysregulation. However, the impact of diabetes on the functional responses of dendritic cells (DC) and macrophages during B. pseudomallei infection has not been investigated. This study compared the responses of macrophages and DC towards B. pseudomallei using bone marrow-derived DC (BMDC) and peritoneal elicited macrophages (PEM) isolated from streptozotocin-induced diabetic C57BL/6 mice exhibiting hyperglycaemia for 9 days (acute) or 70 days (chronic) and age-matched nondiabetic C57BL/6 mice. Following coincubation of BMDC and PEM with a highly virulent B. pseudomallei isolate, maturation, bacterial internalization plus intracellular survival and cytokine gene expression profiles were assessed. No significant differences in functional responses of BMDC or PEM isolated from acute diabetic and nondiabetic mice were observed. However, significant differences in BMDC and PEM function were observed when chronic diabetic and nondiabetic mice were compared. This study demonstrates that diabetic mice with extended periods of uncontrolled hyperglycaemia have impaired DC and macrophage function towards B. pseudomallei, which may contribute to the high susceptibility observed in clinical practice.

Progress of bone marrow stromal cells and hematopoietic regulation

Stromal cells are the important part of the bone marrow microenvironment. They provide a suitable environment for the self-renewal, proliferation and differentiation of the hematopoietic stem cells. Stromal cells include dendritic cells, macrophages, fibroblasts, endothelial cells and adipocytes. They secrete cytokines,extracellular matrix and provide direct cellular contaction to regulate hematopoiesis. Dysfunction of the stromal cells will lead to all kinds of hemopathy and immune system diseases directly. Key words: Bone marrow; Stromal cells; Hematopoiesis; Regulation

The Fourth Major Restriction Factor Against HIV/SIV

The Fourth Major Restriction Factor Against HIV/SIV

Transplantation Tolerance Induced in Humans at the Fetal or the Neonatal Stage

Patients transplanted with HLA-mismatched stem cells from fetal livers develop transplantation tolerance to donor antigens. Engraftment needs no conditioning regimen prior to transplantation in neonates with severe combined immunodeficiency disease or in human fetal patients having not yet developed any immune maturity, especially T-cell differentiation. The chimeric patients have donor-derived T lymphocytes which progressively demonstrate positive interactions with other host cells. They also can be shown to be tolerant toward both host and donor antigens. The latter tolerance relies upon clonal deletion from the T-cell repertoire, and it results from the contact between thymocytes of donor origin and dendritic cells or macrophages also deriving from donor stem cells. The former tolerance does not imply clonal deletion of T-cells with host reactivity. Numerous T-cells recognizing the allogeneic, host-type antigens are identified in these patients, but these cells are anergized, following interaction with epithelial cells of the host thymus. Induction of transplantation tolerance at the fetal stage requires minimal engraftment only; in the future it will be possible to further amplify the clinical benefit, using additional cell transplants after birth.