FLT3: receptor and ligand. Biology and potential clinical application.

Flt3 ligand (FL) is a recently identified cytokine having a central role in the proliferation, survival and differentiation of early murine and human hematopoietic precursor/stem cells. FL acts synergistically in vitro with a number of other hematopoietic growth factors such as IL-3, IL-6, IL-11, IL-12, KIT Ligand and GM-CSF. Recently, it has been shown the in vivo administration of FL results in a significant alteration of hematopoiesis in murine bone marrow (BM), spleen, peripheral blood, liver and lymph nodes. In addition, treatment with FL resulted in a significant accumulation of functionally active dendritic cells within murine lymphoid tissues. The possible applications of FL in dendritic cell-based immunotherapies are discussed.

FLT3 ligand administration inhibits tumor growth in murine melanoma and lymphoma.

Successful treatment of melanoma and lymphoma may result from the induction of specific antitumor immunity. Dendritic cells (DCs) are powerful antigen-presenting cells and show a remarkable capacity to stimulate antigen-specific T-cell responses. Administration of FLT3 ligand (FL) results in a reversible accumulation of functionally active DCs in both lymphoid and nonlymphoid tissues. Therefore, we evaluated the possible antitumor effect of FL in murine melanoma (B16 and CL8-1) and lymphoma (EL-4) models. In all experiments, tumor growth was significantly inhibited by FL administration. Analysis by immunohistochemistry revealed an increase in the DC accumulation within B16 and EL-4 tumors after treatment with FL. No change was observed for CL8-1 melanoma. These data suggest a potential role for FL in the immunotherapy of malignant skin tumors and possible DC involvement in this effect.

Tumor necrosis factor-alpha-promoted expression of Bcl-2 and inhibition of mitochondrial cytochrome c release mediate resistance of mature dendritic cells to melanoma-induced apoptosis.

Melanoma escapes host defenses through a variety of means, including the elimination of immune effector cells within the tumor microenvironment. We have reported recently that murine and human tumors including melanoma induce premature apoptosis of dendritic cells both in vitro and in vivo. In this study, we have demonstrated that overexpression of the Bcl-2 protein family member Bcl-xL rescued murine dendritic cells (DCs) from melanoma-induced death in vitro. Another successful protection approach was tumor necrosis factor (TNF)-alpha-promoted sustained expression of the antiapoptotic protein Bcl-2 within dendritic cells. This effect of TNF-alpha was mediated by inhibition of mitochondrial cytochrome c release. Thus, both Bcl-xL and Bcl-2 enhance survival of dendritic cells within the tumor microenvironment. In addition, mature DCs were more resistant to melanoma-induced apoptosis than immature dendritic cells. This finding suggests a stage-dependent sensitivity of DCs to tumor-induced cell death. We conclude that: (a) mature DCs might be more suitable for the use of cancer vaccination; and (b) Bcl-2 protein family members such as Bcl-xL and Bcl-2 rescue DCs from tumor-induced premature apoptosis.

Tumor's other immune targets: dendritic cells.

The induction of apoptosis in T cells is one of several mechanisms by which tumors escape immune recognition. We have investigated whether tumors induce apoptosis in dendritic cells (DC) by co-culture of murine or human DC with different tumor cell lines for 4-48 h. Analysis of DC morphological features, JAM assay, TUNEL, caspase-3-like and transglutaminase activity, Annexin V binding, and DNA fragmentation assays revealed a time- and dose-dependent induction of apoptosis in DC by tumor-derived factors. This finding is both effector and target specific. The mechanism of tumor-induced DC apoptosis involved regulation of Bcl-2 and Bax expression. Double staining of both murine and human tumor tissues confirmed that tumor-associated DC undergo apoptotic death in vivo. DC isolated from tumor tissue showed significantly higher levels of apoptosis as determined by TUNEL assay when compared with DC isolated from spleen. These findings demonstrate that tumors induce apoptosis in DC and suggest a new mechanism of tumor escape from immune recognition. DC protection from apoptosis will lead to improvement of DC-based immunotherapies for cancer and other immune diseases.

Differential regulation of epidermal and dermal dendritic cells by IL-12 and Flt3 ligand.

An abrogation of the decline in epidermal Langerhans cell numbers above melanoma might significantly improve the efficacy of immunotherapy for melanoma treatment. Systemic Flt3 ligand (FL) administration in mice induced a significant increase in mature dendritic cells (DC) within the skin, preferentially in the dermis, whereas IL-12 promoted a significant increase of immature DC preferentially in the epidermis. Both effects were abrogated in IL-12 knockout mice. Thus, IL-12 could promote FL-induced accumulation of skin DC. The involvement of FL and IL-12 in the regulation of DC accumulation within the skin may contribute, at least in part, to the stimulation of antimelanoma immunity by FL- and IL-12-based immunotherapies. Moreover, FL and IL-12 could be used for selective in vivo generation of DC in either epidermis or dermis for experimental and clinical purposes.

Identification of delta- and mu-type opioid receptors on human and murine dendritic cells.

The purpose of this study was to evaluate mu- and delta-opioid receptors (OR) on human and murine dendritic cells (DC). Expression of mu- and delta-OR mRNA on DC was demonstrated by RT-PCR. The immunocytochemical and Western blot analyses revealed the expression of OR protein in DC. Radioreceptor assay demonstrated the specific saturated temperature-dependent binding of [3H]-labeled opioid ligand on DC and B(max)=2.8+/-0.3 fmol/10(6) cells and K(D)=4.8+/-1.0 nM were calculated by a Scatchard analysis. Finally, OR ligands DADLE and DAGO dose-dependently modulated the capacity of DC to induce T cell proliferation in an MLR assay. Importantly, expression of functional OR on DC was significantly increased upon TNF-alpha-induced DC maturation. Thus, these data suggest a new mechanism of opioid-dependent neuroendocrine immunomodulation.

The use of dendritic cells for cancer vaccination.

Dendritic cells (DC) are the most potent antigen presenting cells (APC) and the only ones capable of presenting novel antigens to naïve T-cells. Large numbers of DC can be generated in vitro in the presence of appropriate cytokine cocktails using either adherent peripheral blood mononuclear cells (PBMC) or CD34+ precursors. More than 20 preclinical studies have demonstrated the effectiveness of antigen-loaded DC to mediate antitumor immune responses. Three clinical trials have been reported to date that show DC as a promising tool for the immunotherapy of cancer. However, completion and analysis of randomized trials to establish the appropriate antigen(s), adjuvant(s), dose, route and schedule will be crucial. Future DC-based therapies will include genetic modification of DC, the use of CD34+ precursors, direct delivery of DC to tumors, and application of tumor lysates or apoptotic cells as sources of additional, as yet undefined, antigens.

Cytokine-based therapy for melanoma: pre-clinical studies.

Incidence and mortality of human malignant melanoma has risen rapidly over recent decades. Systemic therapies for metastatic cutaneous melanoma, the most aggressive of all skin cancers, remain disappointing although immunological treatment has been more successful for melanoma than for most other tumours. With the availability of recombinant cytokines, immunotherapy for melanoma has entered a new era and a growing body of evidence suggests the efficacy of these approaches in pre-clinical models. Cytokine gene transfer to tumour cells has been demonstrated to induce tumour rejection in different murine melanoma models suggesting that vaccination with tumour cells genetically engineered to produce cytokines is an attractive strategy to enhance anti-tumour immune responses in patients with melanoma. Taken together, these data may hold significant promise for the development of effective ex vivo and in vivo systemic and gene therapy modalities to counter the highly metastatic nature of human melanoma.

TNF-alpha protects dendritic cells from prostate cancer-induced apoptosis.

We have recently shown that human prostate cancer (PCa) cells induced apoptotic death of the most potent antigen-presenting cells, dendritic cells (DC), which are responsible for the induction of specific antitumor immune responses. Here we have evaluated the effect of murine PCa cells RM-1 on the survival of immature and tumor necrosis factor-alpha (TNF-alpha)-stimulated mature DC. PCa cells and DC were co-incubated for 24-48 h and DC apoptosis was assessed by morphologic criteria, Annexin V assay, and TUNEL staining. We have shown that co-incubation of RM-1 cells with DC is accompanied by an increased level of DC apoptosis, which was mediated by decreased expression of anti-apoptotic protein Bcl-2. Stimulation of DC maturation by TNF-alpha resulted in increased resistance of DC to PCa-induced apoptosis. In TNF-alpha treated mature DC, but not in immature DC, the expression of Bcl-2 was not blocked after exposure to RM-1-derived factors. Thus, these data suggest that TNF-alpha-induced maturation of DC increases their resistance to PCa induced apoptosis. This is likely to be due to the stabilizing of the expression of anti-apoptotic protein Bcl-2. The difference in the sensitivity of mature and immature DC to PCa-induced cell death should be considered during the design of DC-based clinical trials for PCa patients.Prostate Cancer and Prostatic Diseases (2001) 4, 221-227.

Interlaboratory and intralaboratory variabilities in the Environmental Lead Proficiency Analytical Testing (ELPAT) Program.

The Environmental Lead Proficiency Analytical Testing (ELPAT) Program evaluates over 400 laboratories that perform lead measurements in paints, soils, and dusts. A previous National Institute for Occupational Safety and Health study, based on the ELPAT data over a 3-year period (1992-1995), found no large biases among common hotplate and microwave digestion techniques, but did detect small consistent bias between two common instrumental methods. This study expands on the earlier study by examining the total sample variability and its variation components (interlaboratory and intralaboratory). A correlation model was used to separate the variation components by estimating a variation ratio. The correlation model leads to a more general approach than a sample pairing technique developed by Youden. This study found no significant evidence that the relative contribution of intralaboratory and interlaboratory variability to total variability changes with lead loading levels. There were no significant differences in the relative contribution of variation components among three most commonly used analytical methods (combinations of sample preparation techniques and instrumental methods). The interlaboratory relative standard deviation is about 1.7 times the intralaboratory relative standard deviation. Both variation components are important parts of total variation although the laboratory-to-laboratory (including analyst-to-analyst) difference is greater than the within laboratory (including sample-to-sample) variation.

Muir-Torre syndrome: clinical features and molecular genetic analysis.

We report a 62-year-old man with rectal cancer, two keratoacanthomas and multiple sebaceous adenomas, epitheliomas and sebaceous hyperplasia. His brother and father died from colorectal cancer. A subgroup of patients with the Muir-Torre syndrome (MTS) is allelic to the cancer family syndrome. This genetic disorder is caused by an autosomal dominant inherited germline mutation in one of the DNA mismatch repair genes. It is thought that a somatic mutation of the other allele leads to a genomic instability responsible for tumorigenesis. In the patient presented here the instability was detected in two characteristic skin lesions; sebaceous adenoma and epithelioma. The search for a causal germline mutation revealed a frameshift mutation in the mismatch repair gene hMSH2 leading to a truncated protein. A presymptomatic molecular diagnosis can be offered to the children of the patient.

Interleukin-12 and Flt3 ligand differentially promote dendropoiesis in vivo.

Interleukin-12 (IL-12) and Flt3 ligand (FL) regulate hematopoiesis by stimulating proliferation, differentiation and migration of progenitor and/or stem cells. In this study, we evaluated the in vivo effect of IL-12 alone or in combination with FL on dendritic cell (DC) generation and accumulation in murine spleen, lymph node, thymus, liver, and tumor tissues. Both cytokines induced accumulation of functional DC in lymphoid and non-lymphoid tissues. IL-12 promoted predominantly myeloid dendropoiesis, while FL induced both myeloid and lymphoid dendropoiesis. Combination treatment resulted in a dramatic increase in CD86+, and particularly, NLDC-145+ cells within the liver, which was largely due to cell proliferation. Combination therapy also revealed the ability of FL to protect bone marrow cell populations from IL-12-induced depletion in vivo. In vitro, we found a significant FL-induced up-regulation of IL-12 production by DC at both mRNA and protein levels. Thus, our study suggests that (i) the antitumor activity of IL-12 may, at least in part, be mediated by the stimulation of dendropoiesis and (ii) IL-12 might contribute to the antitumor activity of FL. Furthermore, induction of DC generation in vivo by a combination of IL-12 and FL might become a new approach for immunotherapy of cancer.

FLT3-ligand administration inhibits liver metastases: role of NK cells.

FLT3-ligand (FL) is a recently described cytokine that stimulates the proliferation and differentiation of hematopoietic progenitors both in vivo and in vitro and, when administered to mice, induces an accumulation of dendritic cells (DC) in different lymphoid and nonlymphoid organs and tissues, including the liver. We have studied the antitumor effect of FL administered alone or in combination with IL-12 in a day 3 murine liver metastasis model. FL significantly reduced the number of hepatic metastases (36.00 +/- 11.00 vs 92.00 +/- 10.19 in control group, p < 0.05). Histologic evaluation of the livers revealed that FL induced a significant infiltration of the tumor border by lymphocytes and DC associated with increased number of apoptotic figures. Immunohistochemical analysis demonstrated that FL significantly enhanced the number of DC in the liver parenchyma and within the liver metastases, as well as the number of CD4+ and CD8+ T lymphocytes. These data support the suggestion that DC may be directly involved in the antitumor effect of FL. Interestingly, the antitumor effect of FL was greatly reduced by the NK depletion. Combination of FL and IL-12 resulted in greater antitumor efficacy than these cytokines alone. In summary, we have shown that FL has significant antitumor effect on preexisting murine C3 liver tumors that is mediated by NK cells. We have also demonstrated that the FL/IL-12 combination has an enhanced antitumor activity in the same murine tumor model.

Human prostate cancer regulates generation and maturation of monocyte-derived dendritic cells.

BACKGROUND: The progression of prostate cancer is accompanied by a marked suppression of the immune system, including the apoptotic death of dendritic cells (DC) responsible for the induction of antitumor immunity. In this study, we evaluated whether prostate cancer might inhibit DC generation and maturation in vitro. METHODS: DC were generated from peripheral blood monocytes in the presence of the human prostate cell line LNCaP or nonmalignant cells, and characterized by light microscopy, FACScan analysis, and ability to stimulate T-cell proliferation. RESULTS: Prostate cancer significantly inhibited the conversion of monocytes into DC, which was assessed by the expression of DC markers CD1a and CD83. These cells were weak stimulators of T-cell proliferation, suggesting that DC generated in the prostate cancer microenvironment are functionally inhibited. CONCLUSIONS: Prostate cancer not only kills mature DC, but also inhibits their generation and maturation, resulting in decreased production of antigen-presenting cells and inhibition of their functional activity.

Effect of vascular endothelial growth factor and FLT3 ligand on dendritic cell generation in vivo.

The cytokine FLT3 ligand (FL) enhances dendritic cell (DC) generation and has therefore been proposed as a means to boost antitumor immunity. Vascular endothelial growth factor (VEGF) is produced by a large percentage of tumors and is required for development of tumor neovasculature. We previously showed that VEGF decreases DC production and function in vivo. In this study, we tested the hypothesis that VEGF regulates FL effects on DC generation. In seven experiments, four groups of mice were treated with PBS, VEGF alone (100 ng/h), FL alone (10 microgram/day), or with the combination of FL and VEGF. VEGF and PBS were administered continuously for 14 days via s.c. pumps. FL was given s.c. daily for 9 days, beginning on day 4. Tissues were collected and the number, phenotype, and function of lymph node, splenic, and thymic DCs were analyzed on day 14. As expected, treatment with FL resulted in a marked increase in the number of lymph node and spleen DCs and a smaller increase in thymic DC. Pretreatment of mice with VEGF inhibited these FL effects in lymph nodes and thymus by about 50%, whereas spleen DC numbers were undiminished by VEGF. VEGF treatment in vivo also inhibited the ability of FL to increase the number of hemopoietic precursor cells and the level of maturity exhibited by DC derived from these hemopoietic precursor cells in vitro. VEGF inhibited FL-inducible activation of transcription factor NF-kappaB. These data suggest that VEGF interferes with the ability of FL to promote dendritic cell differentiation from bone marrow progenitor cells in mice and therefore may decrease the therapeutic efficacy of FL in settings where increased numbers of DCs might provide clinical benefits.

Ultrasonic extraction and portable anodic stripping voltammetric measurement of lead in paint, dust wipes, soil, and air: an interlaboratory evaluation.

Recent studies have demonstrated the utility of ultrasonic extraction (UE), followed by portable anodic stripping voltammetry (ASV), for the on-site determination of lead in environmental and industrial hygiene samples. The aim of this work was to conduct an interlaboratory evaluation of the UE-ASV procedure, with a goal of establishing estimates of method performance based on results from collaborative interlaboratory analysis. In this investigation, performance evaluation materials (PEMs) with characterized lead concentrations were used for interlaboratory testing of the UE-ASV procedure. The UE-ASV protocol examined has been promulgated in the form of two separate national voluntary consensus standards (one for UE and another for electroanalysis, which includes ASV). The PEMs consisted of characterized and homogenized paints, soils, and dusts (the last of which were spiked onto wipes meeting national voluntary consensus standard specifications), and air filter samples (mixed cellulose ester membrane) generated using characterized paints within an aerosol chamber. The lead concentrations within the PEMs were chosen so as to bracket pertinent action levels for lead in the various sample matrices. The interlaboratory evaluation was conducted so as to comply with an applicable national voluntary consensus standard that can be used to estimate the interlaboratory precision of a given analytical test method. Based on the analytical results reported by the participating laboratories, relative standard deviations (RSDs) for repeatability and reproducibility were computed for three different lead contents of the four PEMs. RSDs for repeatability were 0.019-0.100 for paints; 0.030-0.151 for soils; 0.085-0.134 for dust wipes; and 0.095-0.137 for air filters. RSDs for reproducibility were 0.127-0.213 for paints; 0.062-0.162 for soils; 0.085-0.134 for dust wipes; and 0.114-0.220 for air filters. With the exception of one of the air filter samples and one of the paint samples, the precision estimates were within the +/- 20% precision requirement specified in the US Environmental Protection Agency National Lead Laboratory Accreditation Program (NLLAP). The results of this investigation illustrate that the UE-ASV procedure is an effective method for the quantitative measurement of lead in the matrices evaluated in this study.

Interleukin-2: developing additional cytokine gene therapies using fibroblasts or dendritic cells to enhance tumor immunity.

PURPOSE: Recombinant interleukin (IL)-2 administration can mediate regression of solid tumors in patients with melanoma and renal cell carcinoma. A better understanding of the mechanisms of IL-2-mediated antitumor effects has led to the investigation of novel immunotherapeutic approaches. The rationale for these immunotherapeutic approaches and the results of preliminary clinical studies are presented. PATIENTS AND METHODS: The therapeutic potential of dendritic cells and the role of FLT3 ligand, a potent hematopoietic growth factor, was investigated in a variety of preclinical models. In addition, a clinical study with autologous dendritic cells pulsed with synthetic melanoma peptides derived from the MART1/ Melan A, gp100, and tyrosinase proteins was conducted. Twenty-eight human leukocyte antigen (HLA)-A2+ melanoma patients received an average of 106 dendritic cells a week for 4 weeks. RESULTS: In a murine liver metastases model, FLT3 ligand administration alone or in combination with IL-12 or IL-2 had significant antitumor effects and resulted in significant infiltration of the tumor border by lymphocytes and dendritic cells, which was associated with an increased number of apoptotic figures. Administration of melanoma peptide-pulsed dendritic cells to 28 patients with advanced metastatic melanoma produced a complete response in two patients and a partial response in one. Significant infiltration of T cells and dendritic cells into melanoma lesions was observed. CONCLUSION: These studies confirm the feasibility of immunotherapeutic approaches using dendritic cells and FLT3 ligand and demonstrate their potential antitumor activity. These approaches may be effective for patients with metastatic melanoma and other solid tumors and will likely be used to improve the efficacy of IL-2-based immunotherapy.