The lipophilic muramyltripeptide MTP-PE, a biological response modifier, is an activator of protein kinase C

The lipophilic immunomodulator MTP-PE is able to activate purified protein kinase C (PKC) by substituting phosphatidyl-serine (PS) or the synthetic diacylglycerol, DiC8, in the assay system. In addition, MTP-PE inhibited [3H]-phorbol-12, 13-dibutyrate ([3H]-PDBu) binding to PKC in a reconstituted receptor system as well as on intact cells (MCF-7). Furthermore, MTP-PE was also able to reduced the epidermal growth factor binding of MCF-7 cells to an extent similar to that found with DiC8 or PDBu. These data indicate that MTP-PE is able to compete for the phorbol ester binding site on PKC both in vivo and in vitro. The components of the MTP-PE molecule, MTP (muramyl-tripeptide) and PE (phosphatidylethanolamine) exerted only marginal effects on PKC activity, did not affect the phorbol ester binding of PKC and the EGF binding of intact MCF-7 cells. Our results suggest that only the complete molecule of the immunomodulator MTP-PE is able to interact with PKC.     

PEGylated liposomes of anastrozole for long-term treatment of breast cancer: in vitro and in vivo evaluation

The aim of present study was to develop conventional and PEGylated (long circulating), liposomes containing anastrozole (ANS) for effective treatment of breast cancer. ANS is a third-generation non-steroidal aromatase inhibitor of the triazole class used for the treatment of advanced and late-stage breast cancer in post-menopausal women. Under such disease conditions the median duration of therapy should be prolonged until tumor regression ends (>31 months). Liposomes were prepared by the thin film hydration method by using ANS and various lipids such as soyaphosphatidyl choline, cholesterol and methoxy polyethylene glycol distearoyl ethanolamine in different concentration ratios and evaluated for physical characteristics, in vitro drug release and stability. Optimized formulations of liposome were studied for in vitro cytotoxic activity against the BT-549 and MCF-7 cell lines and in vivo behavior in Wistar rats. Preformulation studies, both Fourier transform infrared study and differential scanning calorimetry analysis showed no interaction between the drug and the excipients used in the formulations. The optimized formulations AL-07 and AL-09 liposomes showed encapsulation efficiencies in the range 65.12?±?1.05% to 69.85?±?3.2% with desired mean particle size distribution of 101.1?±?5.9 and 120.2?±?2.8?nm and zeta potentials of ?43.7?±?4.7 and ?62.9?±?3.5 mV. All the optimized formulations followed Higuchi-matrix release kinetics and when plotted in accordance with the Korsemeyer–Peppas method, the n-value 0.5?n?in vitro cytotoxicity studies (p?(0–∞) values when compared to pure drug (p?相似文献   

Sterically stabilized liposomes as a potent carrier for shikonin

The ability of pegylated liposomes (sterically stabilized liposomes-SSL) to localize in solid tumors via the enhanced permeability and retention (EPR) effect, partly depends on their long circulating properties which can be achieved by grafting polyethylene glycol (PEG) to the liposomes’ surface. Alkannin and shikonin (A/S) are naturally occurring hydroxynaphthoquinones with a well-established spectrum of wound healing, antimicrobial, anti-inflammatory, antioxidant, and recently established antitumor activity. The purpose of this work was to prepare and characterize shikonin-loaded pegylated liposomes as a new drug carrier for shikonin, as a continuation of authors’ previous work on conventional shikonin-loaded liposomal formulations. Three new pegylated liposomal formulations of shikonin (DSPC-PEG₂₀₀₀, EPC-PEG₂₀₀₀, and DPPC-PEG₂₀₀₀) were prepared and characterized in terms of physicochemical characteristics, pharmacokinetics, and stability (at 4?°C, for 28?d) and compared with the corresponding conventional ones. Particle size distribution, ζ-potential, entrapment efficiency, and release profile of the entrapped drug were measured. Results indicated the successful incorporation of shikonin into liposomes alongside with their good physicochemical characteristics, high entrapment efficiency, satisfactory in vitro release profile, and good physical stability. The results are considered promising and could be used as a road map for designing further in vivo experiments.     

The ATP binding cassette transporter A1 contributes to the secretion of interleukin 1beta from macrophages but not from monocytes

Deficiency of ABCA1 causes high density lipoprotein deficiency and macrophage foam cell formation in Tangier disease. ABCA1 was also postulated to mediate the secretion of IL-1beta from monocytes and macrophages. We investigated the contribution of ABCA1 to IL-1beta secretion from human monocytes and macrophages of normal donors and Tangier disease patients. Neither an anti-ABCA1 antisense oligonucleotide nor ABCA1 deficiency interfered with LPS-induced secretion of IL-1beta from full blood or freshly isolated monocytes. By contrast, anti-ABCA1 antisense oligonucleotides decreased the LPS-induced secretion of IL-beta from macrophages by 30-50%. The secretion of the precursor pro-IL-1beta and TNFalpha was not inhibited. Compared to normal macrophages, LPS-stimulated Tangier disease macrophages secreted less IL-1beta relative to TNFalpha. Also the spontaneous secretion of IL-1beta by Tangier macrophages was lower than by control cells. We conclude that IL-1beta is secreted from monocytes by an ABCA1-independent pathway and from macrophages by ABCA1-dependent and -independent pathways.     

Temperature-sensitive liposomes for co-delivery of tamoxifen and imatinib for synergistic breast cancer treatment

Co-delivery of chemotherapeutic agents using nanocarriers is a promising strategy for enhancing therapeutic efficacy of anticancer agents. The aim of this work was to develop tamoxifen and imatinib dual drug loaded temperature-sensitive liposomes to treat breast cancer. Liposomes were prepared using 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), monopalmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC), and different surface active agents. The liposomes were characterized for the average particle size, zeta potential, transition temperature, and drug release below and above liposomal transition temperature. The temperature-sensitive liposomes co-encapsulated with tamoxifen and imatinib were investigated for their synergistic activity against MCF-7 and MDA-MB-231 breast cancer cells. The liposomal nanoparticles showed a transition temperature of 39.4?°C and >70% encapsulation efficiency for tamoxifen and imatinib. The temperature-responsive liposomes showed more than 80% drug released within 30?min above transition temperature. Dual drug loaded liposomes showed synergistic growth inhibition against MCF-7 and MDA-MB-231 breast cancer cells. Co-delivery of tamoxifen and imatinib using temperature-sensitive liposomes can be developed as a potential targeting strategy against breast cancer.     

Inducible expression of the alpha2-macroglobulin signaling receptor in response to antigenic stimulation: a study of second messenger generation.

Thioglycollate (TG)-elicited murine, peritoneal macrophages express two receptors for activated forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M*)--namely, the low density lipoprotein receptor-related protein (LRP) and the alpha2M signaling receptor (alpha2MSR). We now report that resident peritoneal macrophages express only 400+/-50 alpha2MSR receptors/cell compared to 5000+/-500 receptor/TG-elicited macrophage. By contrast, LRP expression is only 2-2.5-fold greater on elicited cells. The low level of alpha2MSR expression by resident cells is insufficient to trigger signal transduction in contrast to TG-elicited cells which when exposed to alpha2M* demonstrate a rapid rise in inositol 1,4,5-trisphosphate and a concomitant increase in cytosolic free Ca2+. We then studied a variety of preparations injected subcutaneously for their ability to upregulate alpha2MSR. Macroaggregated bovine serum albumin (macroBSA) injection upregulated alpha2MSR and triggered signaling responses by splenic macrophages. Nonaggregated BSA injection alone or in the presence of alum, by contrast, did not alter alpha2MSR expression. Recombivax (hepatitis B antigen adsorbed to alum) injection also upregulated alpha2MSR on splenic macrophages while the alum carrier had no effect. We conclude that macrophage alpha2M* receptors are inducible and their expression may be regulated, in part, by potential antigens.     

Large anti-HER2/neu liposomes for potential targeted intraperitoneal therapy of micrometastatic cancer

Effective targeting and killing of intraperitoneally disseminated micrometastases remains a challenge.

Objective/Methods:?In this work, we evaluated the potential of antibody-labeled PEGylated large liposomes as vehicles for direct intraperitoneal (i.p.) drug delivery with the aim to enhance the tumor-to-normal organ ratio and to improve the bioexposure of cancer cells to the delivered therapeutics while shifting the toxicities toward the spleen. These targeted liposomes are designed to combine: (1) specific targeting to and internalization by cancer cells mediated by liposome-conjugated tumor-specific antibodies, (2) slow clearance from the peritoneal cavity, and (3) shift of normal organ toxicities from the liver to the spleen due to their relatively large size.

Results:?Conjugation of anti-HER2/neu antibodies to the surface of large (approximately 600?nm in diameter) PEGylated liposomes results in fast, specific binding of targeted liposomes to cancer cells in vitro, followed by considerable cellular internalization. In vivo, after i.p. administration, these liposomes exhibit fast, specific binding to i.p. cancerous tumors. Large liposomes are slowly cleared from the peritoneal cavity, and they exhibit increased uptake by the spleen relative to the liver, while targeted large liposomes demonstrate specific tumor uptake at early times. Although tissue and tumor uptake are greater for cationic liposomes, the tumor-to-liver and spleen-to-liver ratios are similar for both membrane compositions, suggesting a primary role for the liposome’s size, compared to the liposome’s surface charge.

Conclusions:?The findings of this study suggest that large targeted liposomes administered i.p. could be a potent drug-delivery strategy for locoregional therapy of i.p. micrometastatic tumors.     

Increase of anti-inflammatory cytokines in patients with esophageal cancer after perioperative treatment with G-CSF

Granulocyte colony-stimulating factor (G-CSF) has been shown to effectively stimulate granulopoiesis, in both neutropenic and in non-neutropenic patients. Recently, other effects of G-CSF on the immune system have attracted interest in treating non-neutropenic patients with a high risk of severe infection. In this phase II trial, we measured the effects of G-CSF on the serum cytokine levels in patients with esophageal cancer undergoing esophagectomy. Twenty subsequent patients (study group, 19 evaluable) received G-CSF (rhG-CSF, Filgrastim) at standard doses (300 microg or 480 microg) subcutaneously 2 days before and up to 7 days after surgery. G-CSF was well tolerated. Leukocytes increased from 7600/microl at study entry (day -2) to a maximum of 45 100/microl (day 6). In the study patients, we found a highly significant (P<0.001) postoperative increase of G-CSF, IL-1ra, sTNFRp55 and sTNFRp75 as compared with the baseline level. In contrast, IL-8 levels were decreased by a factor of 6.8; there were no changes in the very low TNF-alpha levels. The comparison of the study group with a control group of 21 cancer patients undergoing major surgery who were not treated with G-CSF showed significant differences in the serum levels of G-CSF, sTNFRp55, sTNFRp75, and IL-1ra, respectively. There was no infection in the study group up to 10 days after surgery as compared with 29.9% in a historical control group (P=0.008). Thus, the induction of anti-inflammatory cytokines and the downregulation of pro-inflammatory cytokines by G-CSF might be a promising adjuvant treatment of infectious complications in patients undergoing esophagectomy.     

Use of liposomes as drug delivery vehicles for treatment of melanoma

Melanoma is a progressive disease that claims many lives each year due to lack of therapeutics effective for the long‐term treatment of patients. Currently, the best treatment option is early detection followed by surgical removal. Better melanoma therapies that are effectively delivered to tumors with minimal toxicity for patients are urgently needed. Nanotechnologies provide one approach to encapsulate therapeutic agents leading to improvements in circulation time, enhanced tumor uptake, avoidance of the reticulo‐endothelial system, and minimization of toxicity. Liposomes in particular are a promising nanotechnology that can be used for more effective delivery of therapeutic agents to treat melanoma. Liposomes delivering chemotherapies, siRNA, asODNs, DNA, and radioactive particles are just some of the promising new nanotechnology based therapies under development for the treatment of melanoma that are discussed in this review.     

Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

Abstract A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae, S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae. S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

The role of monocyte/macrophages as vehicles of dissemination of Simkania negevensis: an in vitro simulation model

Exposure to Simkania negevensis (Sn), an intracellular microorganism that has been associated with respiratory tract infections in infants and adults, is prevalent. Sn can multiply within free-living amoebae and has been detected in domestic water supplies, which may constitute a source of infection with the organism. Its path of transport from its portal of entry to the body to its target organs is unknown. In this study, the possibility that monocytes/macrophages may serve as vehicles of transmission was examined. In vitro cocultivation of Sn-infected Acanthamoeba polyphaga with the monocyte/macrophage cell line U937 resulted in the death of the amoebae and infection of the U937 cells. Sn entered and multiplied in U937 cells within short periods of time, and the microorganism could be transferred from U937 cells to cell cultures of various origins. Uninfected monocyte/macrophages could become infected when in contact with either actively or persistently Sn-infected cell cultures. Persistently infected cultures in contact with uninfected U937 cells became actively infected. The results of this study provide a basis for determination of the molecular mechanisms of monocyte/macrophage-cell interactions in transfer of infection and may contribute to a better understanding of the pathogenesis of Sn infections in vivo.     

Physical properties and biological interactions of liposomes developed as a drug carrier in the field of regenerative medicine

Liposomes are used for encapsulation of the active compounds in different therapies, with the increasing frequency. The important areas of clinical applications of liposomes are cancer targeted treatment, antibiotic delivery or regenerative medicine. The liposomes can transfer both hydrophilic and hydrophobic compounds and have the lipid bilayer which imitates the cell membrane. Liposomes additionally may extend half-live period of drugs and protect them against the elimination in different ways, such as phagocytosis, enzymatic cleavage or exclusion by detoxification. The size and charge of liposomes play an important role in drug distribution and absorption into the cell. Limited data is available on the effects of liposomes on stem cells and progenitor cells. In this article, we examined the effect of charged conventional liposomes on growth of mesenchymal and blood stem cells isolated from umbilical cord. The data suggest a likelihood, that positively charged liposomes could impair stem cell growth and metabolism. Different methodological approaches allowed for the selection of negatively charged liposomes for further experiments, as the only type of liposomes which has the lowest cytotoxicity and does not affect hematopoietic cell proliferation.     

Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages.

The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL-1, rIL-2, rIL-3, rIL-5, rIL-6, rTNFa, rGM-CSF, rM-CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL-2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rIFNa and rIL-2 were ineffective in purified monocytes or macrophages. rIL-4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose-dependently inhibited by rIL-4 and this inhibition is probably due to an IL-4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.     

Low-frequency electromagnetic fields do not alter responses of inflammatory genes and proteins in human monocytes and immune cell lines

The effects of low frequency electromagnetic fields (LF EMF) on human health are the subject of on-going research and serious public concern. These fields potentially elicit small effects that have been proposed to have consequences, either positive or negative, for biological systems. To reveal potentially weak but biologically relevant effects, we chose to extensively examine exposure of immune cells to two different signals, namely a complex multiple waveform field, and a 50 Hz sine wave. These immune cells are highly responsive and, in vivo, modulation of cytokine expression responses can result in systemic health effects. Using time course experiments, we determined kinetics of cytokine and other inflammation-related genes in a human monocytic leukemia cell line, THP-1, and primary monocytes and macrophages. Moreover, cytokine protein levels in THP-1 monocytes were determined. Exposure to either of the two signals did not result in a significant effect on gene and protein expression in the studied immune cells. Also, additional experiments using non-immune cells showed no effects of the signals on cytokine gene expression. We therefore conclude that these LF EMF exposure conditions are not expected to significantly modulate innate immune signaling.     

Influence of the galactosyl ligand structure on the interaction of galactosylated liposomes with mouse peritoneal macrophages

Liposomes bearing at their surface mono- and triantennary galactosyl ligands were prepared and their interaction with the galactose receptor of mouse peritoneal macrophages studied. Triantennary structures were synthesized by coupling derivatives of 1-thio--d-galactose to the amino groups of lysyl-lysine dipeptide. Galactosylated liposomes were obtained either by synthesis of neo-galactolipids followed by their incorporation into the vesicles or by neo-galactosylation of preformed liposomes by reaction between thiol-functionalized galactosyl ligands and vesicles bearing maleimido groups. The interaction of the galactosylated liposomes with the macrophage lectin was remarkably sensitive to the topology of the ligands, i.e., a spacer-arm length about 3 nm was necessary and, in contrast to results obtained with the galactose receptor of other cells, the triantennary structure did not provide additional binding. Related to the strategy of drug delivery with targeted liposomes, these results indicate that lectins from different cells might possibly be distinguished by using multiantennary ligands having optimal geometries.Abbreviations Gal d-galactose - GalNAc 2-acetamido-2-deoxy-d-galactose - PC l--phosphatidylcholine - PE l--phosphatidylethanolamine - DPPE dipalmitoyl-l--phosphatidylethanolamine - PG l--phosphatidylglycerol - SPDP N-succinimidyl-3-(2-pyridyldithio)propionate - SMPB succinimidyl-4-(p-maleimidophenyl)butyrate - MPB-PE 4-(p-maleimidophenyl)butyryl-PE - Succ-DPPE N-succinyl-DPPE - NHS N-hydroxysuccinimide - DCC N,N-dicyclohexylcarbodiimide - EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - NHS-Succ-DPPE NHS ester ofN-succinyl-DPPE - REV vesicles obtained by reversed phase evaporation - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - DMEM Dulbecco's modified Eagle's medium.