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Michael T Lotze

Publications and source records attributed to Michael T Lotze.

At least 37 records · Page 2Linked to original sources
PubMed2005

A basis for distinguishing cultured dendritic cells and macrophages in cytospins and fixed sections.

There is a burgeoning literature on the contrasting role of intratumoral dendritic cells (DCs) and tumor-associated macrophages, making reliable identification of both cell types in clinical and experimental tissue sections important. However, because these cell types are closely related and share several differentiation antigens, their absolute distinction in tissue sections is difficult. We differentiated DCs and macrophages from monocytes in vitro, prepared cytospins and paraffin-embedded sections of the various cell populations, and tested a variety of antibodies that purportedly recognize monocytes and DCs for their capacity to react and distinguish cells after conventional formalin fixation. Cultured DCs but not macrophages were detected by fascin, DC-LAMP, and CD83 with a predictable increase in staining that paralleled their maturation. Staining by CD1a was found on immature DCs but was weak and absent on mature DCs and macrophages, respectively. CD14 and CD163 were characteristic for macrophages and absent on DCs. CD68, HLA-DR, and S100 did not discriminate between DCs and macrophages. We conclude that antigens such as HLA-DR and S100 are not in themselves sufficient for identification of DCs in formalin-fixed tissue sections, but that additional macrophage-specific (CD14, CD163) and DC-specific (CD1a, CD83, fascin, DC-LAMP) antigens should be used to distinguish cell types from each other and to provide information on their state of maturation.

Biomarkers↗
PubMed2005

Monocytes promote natural killer cell interferon gamma production in response to the endogenous danger signal HMGB1.

Substantial attention has been paid to the role of the toll-like receptor (TLR) ligands of late and their role in regulating the innate immune response. They serve as exogenous danger signals important in informing and driving the distal adaptive immune response to pathogens. Less clear has been the role of the nominal endogenous danger signals released and recognized in stressed cells following genotoxic or metabolic stress as occurs in progressively growing tumors. HMGB1 (high-mobility group B1) is a nuclear protein well characterized for its ability to modify DNA access to transcriptional proteins that is released from necrotic cells as well as secreted through the endosomal route from hematopoietic cells, serving as a late mediator of sepsis. It interacts with high-affinity RAGE (receptor for advanced glycation end products) and TLR2 receptors. Here we show that HMGB1 enhances interferon gamma release from macrophage (but not dendritic cell)-stimulated NK cells. This is effective only when coupled with other pro-inflammatory cytokines particularly with IL-2 in combination with IL-1 or IL-12. We have used this information to suggest that HMGB1, which also promotes epithelial migration and proliferation, drives repair in the absence or inhibition of other factors but enhances inflammation in their presence. The implications for tumorigenesis and tumor progression are quite important as they may be for other states of chronic inflammation.

Cell Communication↗
PubMed2005

High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal.

High-mobility group box 1 protein (HMGB1), which previously was thought to function only as a nuclear factor that enhances transcription, was recently discovered to be a crucial cytokine that mediates the response to infection, injury and inflammation. These observations have led to the emergence of a new field in immunology that is focused on understanding the mechanisms of HMGB1 release, its biological activities and its pathological effects in sepsis, arthritis, cancer and other diseases. Here, we discuss these features of HMGB1 and summarize recent advances that have led to the preclinical development of therapeutics that modulate HMGB1 release and activity.

Amino Acid Sequence↗
PubMed2005

Regulatory balance between the immune response of tumor antigen-specific T-cell receptor gene-transduced CD8 T cells and the suppressive effects of tolerogenic dendritic cells.

Tumor immune responses, including some immunotherapy strategies, can fail because of a number of reasons, such as poor tumor cell immunogenicity or local suppressive cytokine release by dendritic cells (DC) at tumor sites. The retroviral transfer of T-cell receptor (TCR) genes encoding tumor-specific receptors into T cells is a fascinating approach to bypass antigen-presenting cells and allow T cells to directly recognize antigen. It also allows the generation and expansion of potent antitumor cytotoxic T lymphocytes with defined cancer antigen specificities more readily than naive T cells. However, interleukin-10 (IL-10)-exposed dendritic cells (IL-10-DC) have been labeled tolerogenic because of the suppressive effects they have on T cell responses. Whether TCR gene-transduced effector CD8(+) T cells can break through suppressive functions mediated by IL-10-DC is not known. In the current study, we demonstrate the role of IL-10 in modifying the function of DC that otherwise would activate potent TCR gene-transduced T cells against tumor antigens. TCR gene-transduced T cells maintained their cytolytic activity in the presence of DC exposed to low doses of IL-10 during maturation; however, they lost this activity in an antigen-specific manner when exposed to DC matured with high doses of IL-10.

CD8-Positive T-Lymphocytes↗
PubMed2005

Natural killer-dendritic cell cross-talk in cancer immunotherapy.

Natural killer (NK) cells and dendritic cells (DCs), two important components of the immune system, can exchange bidirectional activating signals in a positive feedback. Myeloid DCs, the cell type specialised in the presentation of antigen and initiation of antigen-specific immune responses, have recently been documented to be involved in supporting innate immunity, promoting the production of cytokines and cytotoxicity of NK cells, and enhancing their tumouricidal activity. Natural interferon-producing cells/plasmacytoid DCs (IPCs/PDCs) play an additional role in NK cell activation. Reciprocally, NK cells, traditionally considered to be major innate effector cells, have also recently been shown to play immunoregulatory 'helper' functions, being able to activate DCs and to enhance their ability to produce pro-inflammatory cytokines, and to stimulate T helper (Th) 1 and cytotoxic T lymphocyte (CTL) responses of tumour-specific CD4+ and CD8+ T cells. Activated NK cells induce the maturation of myeloid DCs into stable type-1 polarised DCs (DC1), characterised by up to a 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. In addition, the ability of NK cells to kill tumour cells may facilitate the generation of tumour-related antigenic material, further accelerating the induction of tumour-specific immunity. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumour-related suppressive factors, and demonstrate a strongly enhanced ability to induce Th1 and CTL responses in human in vitro and mouse in vivo models. Compared with the standard mature DCs that are used in clinical trials at present, human NK cell-induced DC1s act as superior inducers of anticancer CTL responses during in vitro sensitisation. This provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer and patients with chronic infections that are resistant to standard forms of treatment. Stage I/II clinical trials that are being implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK-DC therapy of melanoma and other cancers.

Animals↗
PubMed2005

Feature Selection for Classification of SELDI-TOF-MS Proteomic Profiles.

BACKGROUND: Proteomic peptide profiling is an emerging technology harbouring great expectations to enable early detection, enhance diagnosis and more clearly define prognosis of many diseases. Although previous research work has illustrated the ability of proteomic data to discriminate between cases and controls, significantly less attention has been paid to the analysis of feature selection strategies that enable learning of such predictive models. Feature selection, in addition to classification, plays an important role in successful identification of proteomic biomarker panels. METHODS: We present a new, efficient, multivariate feature selection strategy that extracts useful feature panels directly from the high-throughput spectra. The strategy takes advantage of the characteristics of surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) profiles and enhances widely used univariate feature selection strategies with a heuristic based on multivariate de-correlation filtering. We analyse and compare two versions of the method: one in which all feature pairs must adhere to a maximum allowed correlation (MAC) threshold, and another in which the feature panel is built greedily by deciding among best univariate features at different MAC levels. RESULTS: The analysis and comparison of feature selection strategies was carried out experimentally on the pancreatic cancer dataset with 57 cancers and 59 controls from the University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA. The analysis was conducted in both the whole-profile and peak-only modes. The results clearly show the benefit of the new strategy over univariate feature selection methods in terms of improved classification performance. CONCLUSION: Understanding the characteristics of the spectra allows us to better assess the relative importance of potential features in the diagnosis of cancer. Incorporation of these characteristics into feature selection strategies often leads to a more efficient data analysis as well as improved classification performance.

Journal Article↗
PubMed2004

Interleukin-17 enhances bFGF-, HGF- and VEGF-induced growth of vascular endothelial cells.

Interleukin-17 (IL-17) is a CD4 T cell-derived proinflammatry and proangiogenic cytokine. In this study, we investigated the effects of this cytokine on vascular endothelial cell growth induced by a well-known direct angiogenic factor bFGF, HGF, VEGF, CXCL5/ENA-78 or CXCL8/IL-8. While a wide range of doses of IL-17 alone did not show the ability to stimulate the growth of human dermal microvascular endothelial cells (HMVECs), bFGF, HGF, VEGF, CXCL5 or CXCL8 significantly induced the growth of HMVECs in vitro. When bFGF and IL-17 were used in combination, 10 or 100 ng/ml IL-17 enhanced 10 ng/ml bFGF-induced growth of HMVECs. Similarly, when HGF and IL-17 were combined together, 10 or 100 ng/ml IL-17 potentiated 10 ng/ml HGF-induced growth of HMVECs. When VEGF and IL-17 were used together, 10 ng/ml IL-17 did not significantly enhance 10 ng/ml VEGF-induced growth, whereas 100 ng/ml IL-17 clearly promoted 10ng/ml VEGF-mediated proliferation of HMVECs. On the contrary, IL-17 did not augment CXCL5- and CXCL8-mediated growth. These results indicate that IL-17 itself does not have the capability to stimulate the growth of vascular endothelial cells, whereas IL-17 is able to selectively enhance the mitogenic activity of bFGF, HGF, and VEGF for vascular endothelial cells. Our findings also suggest that IL-17 may promote bFGF-, HGF- and VEGF-mediated angiogenesis through enhancing bFGF-, HGF- and VEGF-induced growth of vascular endothelial cells.

Cell Proliferation↗
PubMed2004

Imaging analysis of STAT1 and NF-kappaB translocation in dendritic cells at the single cell level.

Rapid assessment of immune or stem cells, which are now widely applied in the clinical setting of cancer treatment, is necessary to speed their development and to determine their quality. We have evaluated immature dendritic cells (iDC) by semiautomated imaging cytometry which provides detailed assessment at a single cell level. Nuclear translocation of NF-kappaB was studied by imaging analysis as well as electrophoretic mobility shift assay with an excellent correlation (r=0.981) over a broad range of lipopolysaccharide (LPS) concentrations. Imaging analysis was time saving (5 h vs. 3 days), and required 30- to 100-fold less cells per analysis. Single cell information revealed remarkable heterogeneity between individual iDC and permitted detection of responses to 40 pg/ml of LPS. In IL-1beta/IFNgamma activated iDC, STAT1 responses preceded NF-kappaB responses, and the expression of both was strongly correlated in individual cells (p<0.001). IFNgamma amplified IL-1-induced NF-kappaB responses. NF-kappaB responses to IL-1beta, CD40L, and LPS were donor-dependent (n=7), correlated with the quality of iDC preparations (p=0.002), and IL-12 p70 production (p=0.010). NF-kappaB measurements in iDC within mixed cell cultures (iDC, NK, K562) demonstrated that these strategies are applicable for analyses of complex cell-cell interactions. Imaging analysis is a method that could be valuable for quality control of cell therapy preparations.

Antineoplastic Agents↗
PubMed2004

Interleukin-17 augments tumor necrosis factor-alpha-induced elaboration of proangiogenic factors from fibroblasts.

Interleukin-17 (IL-17) is a CD4 T cell cytokine. In this report, we investigated the effects of this cytokine on the elaboration of proangiogenic factors by lung fibroblasts. After stimulation with a wide range of doses of IL-17, fibroblasts produced more amount of various kinds of angiogenic factors including NO, HGF, MCP-1, KC, MIP-2, PGE1, PGE2 and VEGF in a dose-dependent manner. Treatment with a COX-1 and COX-2 inhibitor indomethacin did not impair IL-17-induced HGF and VEGF secretion in fibroblasts. In addition, TNF-alpha alone stimulated the elaboration of KC, MIP-2, PGE2 and VEGF in fibroblasts. IL-17 and TNF-alpha in combination up-regulated elaboration of these proangiogenic factors additively or synergistically. Moreover, conditioned media (CM) from IL-17-stimulated fibroblasts showed significantly higher activity on endothelial cell growth than those from non-treated control cells. These results indicate that IL-17 up-regulates elaboration of various proangiogenic factors, and modulates macrophage-derived TNF-alpha-induced production of KC, MIP-2, PGE2 and VEGF by fibroblasts. Our findings also demonstrate that IL-17 might be a potential contributor to the inflammatory angiogenesis via induction of proangiogenic factors by stromal fibroblasts.

Angiogenesis Inducing Agents↗
PubMed2004

A primer on cancer immunology and immunotherapy.

The role of immunity in cancer has been abundantly demonstrated in murine tumor models as well as in man. Induction of clinically effective antitumor immune responses, based on this information, in patients with cancer however, remains elusive. This is not because tumors lack recognizable antigens [in fact there is evidence that there are thousands of potential novel targets in each tumor cell] but rather due to the fact that the induction of responses is not adequate nor particularly well understood. Tumors seem to be rather effective at limiting immune responses. Many of the molecularly defined antigens that have been detected on tumor cells are derived from self-proteins and as such are subject to tolerizing mechanisms. Such tumors have also developed escape mechanisms capable of evading or suppressing immune responses. Understanding the role of dendritic cells during the effector phase of the immune response and the complex interactions of stromal, immune, and tumor cells in the tumor microenvironment represent the next challenges to be understood for tumor immunology.

Antigens, Neoplasm↗
PubMed2004

Identifying biomarkers and surrogates of tumors (cancer biometrics): correlation with immunotherapies and immune cells.

The presence of inflammatory cells within cancer has been described for quite some time by pathologists, with generally improved outcome associated with their presence in various epithelial neoplasms. Most remarkably, this has included dendritic cells and T cells but more recently NK cells as well. Coupled with the rapid evolution of molecular technology, microarray analyses of primary tumors, serum and tumor proteomics, tumor capture analyses in the peripheral blood (together with quantitative RT-PCR), and novel histochemical markers and tissue microarrays, this provides the opportunity to establish a more effective means to study and classify into subsets various forms of cancer. Much of the current controversy in cancer diagnosis and pathologic assessment of prognosis lies in the application of these techniques in concert with other molecular tools including DNA microarrays, expression of histochemically defined cytokines, proangiogenic factors, and oncogene products, and correlating this with clinical relevance. Molecular detection technologies such as reverse transcriptase polymerase chain reaction, proteomics, and microarray analyses will be validated based on their integration with conventional cancer pathology and cancer diagnostics. Further work is needed to establish which cancer biomarkers and surrogates should be routinely measured and in which settings, and determining the appropriate sample size for such assays that can be validated in retrospective and prospective clinical studies. The ability to integrate these rapidly evolving strategies will consume much of our coordinate effort in cancer and cancer therapeutics for the near future.

Biomarkers, Tumor↗
PubMed2004

Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness.

BACKGROUND: The tetracycline-regulatable system is one of the most valuable tools for controlling gene expression. In its current form, however, the system is less than ideal for in vivo or gene therapy uses due to difficulties in set-up procedures, high basal leakiness, and unpredictable delivery and efficiency. METHODS: To address these issues, we have devised a second generation of tetracycline-regulated promoters (TREs). The second-generation TRE (SG-TRE) contains a shortened cytomegalovirus (CMV) minimal promoter together with eight tet operator sequences positioned in an optimized manner upstream of the TATA box. This construct displays far greater reduction in basal leakiness than maximal transgene expression. Conversely, maximal transgene expression is increased to a greater degree than basal leakiness by post-translational stabilization with bovine growth hormone poly A. RESULTS: In transient studies, the SG-TRE displays over 100 000-fold regulation efficiency in HeLa cells at 1:1 ratio of transactivator to reporter plasmid in the Tet-Off system. This novel promoter achieves a regulation efficiency 500- to 1000-fold higher than that of the original TRE (P(hCMV*-1)) in HeLa cells by displaying undetectable levels of basal leakiness without compromised maximal expression. In other cell lines, the SG-TRE proves to be more efficient than the original P(hCMV*-1) in a cell-dependent manner. Furthermore, the SG-TRE preserves its enhanced regulation efficiency and its reduced basal leakiness in the context of a single positive feedback regulatory vector that presents ease of delivery of the system for use in vivo. Finally, in vivo, the biological function of granulocyte-macrophage colony stimulating factor is tightly regulated in the context of SG-TRE delivered via adeno-associated viruses.

Animals↗
PubMed2003

C16 ceramide accumulates following androgen ablation in LNCaP prostate cancer cells.

BACKGROUND: Adenocarcinoma of the prostate is the most frequently diagnosed non-cutaneous cancer and the second leading cause of cancer-related deaths among men in the United States. The most successful therapies to date for this tumor have involved some form of androgen ablation. However, these therapies become ineffective as the tumor evolves to an androgen-insensitive state. Ceramide is a lipid second messenger that has been shown to mediate growth arrest or cell death when added exogenously to prostate cancer cells. As a first step toward understanding the events that lead to the transition of prostate cancer cells to an androgen-independent state, we considered investigating the effect of androgen ablation on endogenous ceramide levels in androgen-sensitive and androgen-insensitive prostate cancer cells. METHODS: To investigate the mechanisms of growth arrest/apoptosis in androgen-sensitive (LNCaP) and insensitive (DU-145, PC-3) cells, we used various methods including nonyl acridine orange (NAO) staining, propidium iodide (PI) staining/cell-cycle analysis, lipid analysis, and Western blotting assays. RESULTS: In this study, we demonstrate that androgen ablation drives G(0)/G(1)-phase cell-cycle arrest followed by progressive apoptosis in vitro, in LNCaP cells. Lipid analysis indicated an increase in C16 ceramide, which was generated via the de novo pathway as revealed by blockade of ceramide synthase by fumonisin B1. The addition of 5alpha-dihydrotestosterone (DHT) or fumonisin B1 rescued LNCaP cells from apoptosis induced by androgen ablation, and decreased levels of intracellular C16 ceramide. Neither apoptosis nor an increase in C16 ceramide was observed in androgen-independent cell lines following androgen ablation.

Adenocarcinoma↗
PubMed2003

Natural killer, but not natural killer T, cells play a necessary role in the promotion of an innate antitumor response induced by IL-18.

IL-18 administration promotes innate immunity resulting in significant antitumor effects in multiple murine tumor models. Here, we examined the effector population mediating the innate immunity. Most NK cells and some NKT cells express IL-18Rs without prior stimulation (65% positive in NK cells, 18% positive in NKT cells), though few naive T cells do. In vivo depletion of NK cells, but not NKT cells, using AsGM1 antibody significantly reduces IL-18-induced cytotoxicity. However, NK-like activity of hepatic MNCs for the NK target YAC-1 was present in Valpha 14 NKT cell-deficient animals. Furthermore, administration of rIL-18 greatly reduced B16 pulmonary metastases in vivo in NKT cell-deficient animals. When sorted NK and NKT cells were exposed to IL-18 in vitro, NK cells showed more IFN-gamma production and cytolysis against YAC-1 than NKT cells in response to IL-18. These results are consistent with the notion that NK cells, but not NKT cells, are the major effectors in IL-18-induced innate immunity.

Animals↗
PubMed2003

Innate immunity mediated by the cytokine IL-1 homologue 4 (IL-1H4/IL-1F7) induces IL-12-dependent adaptive and profound antitumor immunity.

Recently, several novel members of the IL-1 family have been identified. The possible therapeutic utility and the underlying biologic role of these new members remain unclear. In the present study we analyzed the anti-tumor activity of human IL-1 homologue 4(IL-1H4; renamed IL-F7) by adenovirus-mediated gene transfer (AdIL-1H4) directly into murine tumors. In vitro expression analysis showed that IL-1H4 was a secretory protein. Treatment of an established MCA205 mouse fibrosarcoma by single intratumoral injection of AdIL-1H4 resulted in significant growth suppression. Furthermore, complete inhibition of tumor growth was observed following multiple injections of AdIL-1H4. The anti-tumor activity of IL-1H4 was abrogated in nude and SCID mice and in IL-12-, IFN-gamma-, or Fas ligand-deficient mice. In contrast, IL-1H4 was able to confer substantial anti-tumor effects in NKT-deficient mice. These results suggest that IL-1H4 could play an important role in the link between innate and adaptive immunity and may be useful for tumor immunotherapy.

Adenoviridae↗
PubMed2003

Advantages and limitations of particle-mediated transfection (gene gun) in cancer immuno-gene therapy using IL-10, IL-12 or B7-1 in murine tumor models.

BACKGROUND: Previous studies have shown that particle-mediated transfection (PMT; gene gun) is an efficient method of non-viral gene transfer. We have examined the advantages and limitations of PMT in cancer immuno-gene therapy using IL-10, IL-12 or B7-1, all of which have been shown to be effective in murine tumor models using retroviral vectors. METHODS: Murine cell lines (MCA205, MCA207, NIH-3T3) were treated with in vitro PMT, liposome-mediated transfection (LMT) or retroviral transfection. In vivo PMT was also examined for in vivo experiments using C57BL/6. RESULTS: Transfection efficiency and cytokine expression of PMT were similar to those of LMT, another non-viral approach, in culture when murine adherent cells were used. Tumor establishment of MCA205 was suppressed when they were transfected with the mIL-12 and/or mB7-1 gene using PMT in vitro. In a treatment of established tumor, vaccination with tumor cells transfected with an IL-12 gene suppressed tumor growth, whereas a B7-1 gene was not effective. When an IL-10 gene was used to supply a high level of expression for antitumor effects, neither in vitro nor in vivo PMT could suppress even tumor establishment. These failures appear to be caused by the characteristics of PMT which allows high, but transient, expression of transfected genes. CONCLUSIONS: Particle-mediated transfection is a useful non-viral transfection method in a system which does not require high-level gene expression for a prolonged time of period.

Analysis of Variance↗
PubMed2003

Synergistic interleukin-18 and low-dose interleukin-2 promote regression of established murine neuroblastoma in vivo.

BACKGROUND/PURPOSE: Severe systemic toxicities have limited the clinical applications of the potent cytokine, interleukin-2 (IL-2). Recent studies have shown that IL-18 synergizes with IL-2 to enhance cytolytic activity in vitro. Combination therapy allows for IL-2 dose reduction, thus, limiting its toxicity while augmenting natural killer cell activity. The authors hypothesize that IL-18 plus low-dose IL-2 may induce a potent and sustained antitumor response in vivo providing effective immunotherapy for neuroblastoma. METHODS: Four groups of A/J mice (n = 28) were inoculated subcutaneously in the right flank with 1 x 10(6) murine neuroblastoma cells (TBJ). On day 7, 5 consecutive daily peritumoral injections were performed with saline (control), human rIL-2 (30,000 IU), murine IL-18 (1 microg), or IL-2 plus IL-18. Tumor growth was monitored, and animals with tumor progression were killed on day 21. Seven weeks after the initial treatment, animals with rejected tumors were rechallenged with 5 x 10(6) cells in the opposite flank. Quantitative data were analyzed by Student's t test. RESULTS: Rapid tumor growth and death was noted in all control animals by 21 days. Complete tumor eradication was seen in 28% of mice treated with IL-2 (P =.03), 42% of mice treated with IL-18 (P <.05), and 57% of mice treated with of IL-2 plus IL-18 (P <.05). Despite the initial response, all animals failed rechallenge and developed new or recurrent tumors within 7 to 10 days. CONCLUSIONS: Coadministration of low-dose IL-2 plus IL-18 induced a potent primary response to murine neuroblastoma likely caused by activation of natural killer cells in the tumor microenvironment. This combined cytokine therapy strategy was unable to induce sustained immunity to rechallenge. However, dendritic cell vaccination combined with IL-2 plus IL-18 cytokine treatment did allow for the establishment of a complete and durable antitumor response.

Adjuvants, Immunologic↗