Regulation of the In Vitro Secondary Cell-Mediated Cytotoxic Response against Syngeneic FBL-3 Leukemia by Macrophages

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.     

In Vitro Evaluation of Mucoadhesive Films for Gingival Administration of Lidocaine

The aim of this study was the optimization of a lidocaine-based film formulation for the prevention of pain from needle prick during the injection of local anesthetic in dentistry. Film performances were evaluated in vitro by studying lidocaine permeation across pig esophageal epithelium as model for nonkeratinized buccal mucosa. The results obtained showed that the molecular weight of the film-forming polymer had no effect on lidocaine transport. The introduction of the adhesive Plastoid^® into the film determined a significant increase of drug permeation rate, which was further improved by the addition of Azone^®. On the contrary, the effect of sodium taurocholate was negligible.     

植物离体组织染色体加倍诱导同源四倍体

随着生物技术的迅速发展,通过植物离体组织人工诱导多倍体已经成为获得多倍体植株的有效途径。本文就植物离体组织染色体加倍诱导同源四倍体的研究进展做一介绍,详细评述了植物离体组织细胞加倍的途径、影响植物离体组织加倍的因素、利用不同诱导剂进行处理效果比较及离体组织材料的早期倍性鉴定技术等,并展望了植物离体诱导同源四倍体的前景。     

Interleukin-1 Hyperproduction by In Vitro Activated Peripheral Macrophages from Cerebellar Mutant Mice

Several mutations in mice produce complex patterns of neuronal degeneration of the cerebellum and of its afferent pathways. In the staggerer (sg/sg) mutant, atrophy of the lymphoid organs and immunological abnormalities have been described. To search for a possible link between the neurological and the immune disorders in this mutant, we studied the production by its peripheral macrophages of interleukin-1 (IL-1), which roles in both immune and nervous systems are well established. Suspensions of peritoneal and/or spleen macrophages from mutants and their appropriate controls were stimulated in vitro by lipopolysaccharide. Northern and dot blots, performed with murine IL-1 cDNA probes, revealed a clear-cut hyperexpression of IL-1 mRNA in staggerer macrophages. An IL-1 bioassay using the IL-1-responsive D10.G4 cell line also revealed a sixfold increase of IL-1 activity in the macrophage supernatants of staggerer mutant mice. The hyperproduction was found in 3-week to 1-year-old staggerer and also in heterozygous (+/sg) mice. A similar phenomenon existed in cerebellar mutants lurcher, Purkinje cell degeneration (pcd), and to a lesser extent reeler and wobbler, but was absent in the neurological mutants weaver, jimpy, and motor end plate disease (medH). These observations establish that in several point mutations in mice, central nervous degeneration is associated with dysregulation of IL-1 production by peripheral macrophages.     

In Vitro Development of Exoerythrocytic Forms of Plasmodium Gallinaceum Sporozoites In Avian Macrophages

ABSTRACT Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. the host cells were macrophage precursors from chicken bone marrow. At various times after introduction of Sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). the time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until Ac end of the primary exoerythrocytic schizogony.     

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response¹. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)².The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.     

Leishmania: Chemotaxic Responses of Promastigotes and Macrophages In Vitro1

Promastigotes of Leishmania move progressively up a concentration gradient of: various sugars, specific sugars attracting individual species of Leishmania; serum albumin and another unidentified constituent of serum; hemoglobin; and a factor generated by promastigotes in NNN medium. The movement of promastigotes up a concentration gradient of serum is optimal at a pH of 6.4 to 6.8 and a temperature of 28°C and above. Cholinergic and adrenergic agents did not affect the attraction of serum for promastigotes. and cyclic nucleotides, inflammatory mediators, and macrophage products were not chemotaxic. It is postulated that the sugar chem-otaxins influence the movement of promastigotes from the sand fly midgut to the esophagus, and serum chemotaxins may play a part in the entry of promastigotes into the skin of a mammal from the proboscis. Macrophages, the host cell of the obligate intracellular Leishmania species, were not attracted to any product of promastigotes. When, however, promastigotes interact with serum, complement is activated to form C5a which is chemotaxic for macrophages. Activation of complement by promastigotes is, at least partially, by the alternate pathway. Other chemotaxins resulting from promastigote interaction with serum may also be present. Promastigotes may also produce inhibitors of C5a activity.     

Plasmodium Sporozoite Interactions with Macrophages In Vitro: a Videomicroscopic Analysis

ABSTRACT. Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.     

Establishment of Self-Renewable GM-CSF-Dependent Immature Macrophages In Vitro from Murine Bone Marrow

Macrophages play a key role in the innate immune system. Macrophages are thought to originate from hematopoietic precursors or the yolk sac. Here, we describe the in vitro establishment of self-renewable GM-CSF-dependent immature macrophages (GM-IMs) from murine bone marrow (BM). GM-IMs grow continuously in vitro in conditioned medium containing GM-CSF. The immunophenotype of GM-IMs is F4/80^high CD11b^high CD11c^low Ly6C^low. By comparing gene expression in GM-IMs and BM dendritic cells, we found that GM-IMs expressed lower levels of chemokines, cytokines and their receptors. GM-IMs are round in shape, attach loosely to non-coated culture dishes and have a marked phagocytic capacity. These results indicate that GM-IMs are macrophage precursor cells. Following stimulation with LPS, monocyte-like GM-IMs converted to flat macrophage-like cells that tightly adhered to non-coated culture dishes and produced pro-inflammatory cytokines TNFα, IL-6 and IL-1β. These results indicated that GM-IMs differentiated to M1 pro-inflammatory macrophages. This was confirmed by stimulation of GM-IMs with IFNγ, an inducer of M1 markers. GM-IMs showed enhanced expression of M2 macrophage markers such as Arg1 and Retnla following stimulation by Th2 cytokines IL-4 and IL-13. When GM-IMs were injected into mice at sites of wounding, wound repair was enhanced. These results indicate that GM-IMs can differentiate to M2 macrophages. When GM-IMs were injected into clodronate-treated mice, they induced resident macrophage proliferation by producing M-CSF. In conclusion we have established self-renewable GM-CSF-dependent immature macrophages in vitro from murine BM, which differentiate to M1 or M2 macrophages.     

In Vitro Effects of Calcium Fructoborate upon Production of Inflammatory Mediators by LPS-stimulated RAW 264.7 Macrophages

The present study is supported by our previous findings suggesting that calcium fructoborate (CF) has anti-inflammatory and antioxidant properties. Thus, we investigated the effects of CF on a model for studying inflammatory disorders in vitro represented by lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. This investigation was performed by analyzing the levels of some mediators released during the inflammatory process: cytokines such as tumor necrosis factor-α (TNF-α), interleukins IL-1β and IL-6 as well as cyclooxygenase-2 (COX-2), the main enzyme responsible for endotoxin/LPS-induced prostaglandin synthesis by macrophages. We also measured production of nitric oxide (NO) that plays an important role in the cytotoxicity activity of macrophages towards microbial pathogens. After CF treatment of LPS-stimulated macrophages we found an up-regulation of TNF-α protein level in culture medium, no significant changes in the level of COX-2 protein expression and a decrease in NO production as well as in IL-1β and IL-6 release. Collectively, this series of experiments indicate that CF affect macrophage production of inflammatory mediators. However, further research is required in order to establish whether CF treatment can be beneficial in suppression of pro-inflammatory cytokine production and against progression of endotoxin-related diseases.     

未授粉子房和胚珠离体培养诱导植物雌核发育研究进展

未授粉子房和胚珠培养离体诱导雌核发育的研究已近半个世纪,目前共有20科40余种种子植物的未授粉子房或胚珠离体培养诱导了雌核发育,但成功获得雌性单倍体的植物只有10科25种2变种。本文重点对离体雌核发育的胚胎学的研究资料进行综述,包括离体条件下胚珠中胚囊的发育状况、雌核发育的模式、雌性单倍体形成的途径以及自发胚乳的离体诱导并指出该领域研究中存在的问题,进一步在研究方法上提出了设想和建议。     

Leishmania mexicana mexicana: Attachment and Uptake of Promastigotes to and by Macrophages In Vitro1

Promastigotes of Leishmania mexicana mexicana attach to mouse macrophages in vitro in the absence of serum by a wheat germ agglutinin-like ligand on the surface of the promastigote that binds to the N-acetyl glucosamine moiety of a receptor on the surface of the macrophage. The binding is temperature dependent, and the macrophage receptor is trypsin, cytochalasin B, and glutaraldehyde sensitive. The promastigote ligand is proteolytic enzyme and glutaraldehyde insensitive. Uptake follows attachment and is assisted or inhibited as for attachment. Treatment of promastigotes with proteolytic enzymes uncovers a receptor for a serum component that binds strongly to a mouse macrophage receptor in vitro. The strain of mice donating the macrophages had little effect upon attachment and uptake except that A strain mouse macrophages attached fewer promastigotes in 10 min than those of outbred mice, but took up as many promastigotes over 90 min as those of outbred mice. Low responder Biozzi mouse macrophages took up more promastigotes than high responder Biozzi mouse macrophages. Normal unheated human, rabbit, and guinea pig sera lysed promastigotes and so inhibited their attachment to macrophages in vitro. Unheated immune serum showed an enhanced inhibition of attachment. Heated normal serum allowed attachment and uptake, while promastigotes treated with heated immune serum showed enhanced attachment to and uptake by macrophages. Treatment of macrophages in vitro with immune serum enhanced their ability to attach promastigotes and to engulf them. Repeated 90-min exposures of a population of promastigotes to uptake by mouse macrophages in vitro did not deplete the population of any sub-population more likely to be taken by macrophages. The first sub-population to be taken up survived better in macrophages over 24 h than subsequently engulfed sub-populations.     

Processing of Doxorubicin-Containing Liposomes by Liver Macrophages in Vitro

Abstract

Previous results suggested that drug formation in macrophages is an important aspect of the mode of action of doxorubicin (DXR)-containing liposomes. Intracellular degradation of DXR-liposomes may result in the liberation of DXR molecules that subsequently are released from the macrophages. We investigated whether the rate of intracellular degradation of DXR-liposomes phagocytosed by rat liver macrophages (Kupffer's cells) in monolayer culture is dependent on the type of DXR-liposomes internalized and whether differences in degradation rate of DXR-liposomes are reflected in different DXR release profiles. Two DXR-liposome types that were previously shown to differ markedly both in antitumor activity and degradation rate in vivo were selected for this investigation: a liposome composed of egg-phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol (chol), and a liposome composed of distearoylphosphatidylcholine (DSPC), dipalmitoyl-phosphatidylglycerol (DPPG), and chol. To monitor the rate of intracellular degradation of DXR-liposomes, cholesterol-1-[¹⁴Cjoleate was used as marker of the liposomal lipid phase. DXR was monitored with the use of a high-performance liquid chromatography (HPLC) method capable of detecting not only intact DXR but also major metabolites.

Comparable amounts of both types of DXR-liposomes were taken up by in vitro cultured Kupffer's cells. Liposome-associated cholesteryloleate was metabolized by the cells in a liposome-type-dependent pattern. During the first 30 min after start of the incubation, degradation of cholesteryloleate occurred at a similar rate for both types of DXR-liposomes. During continued incubation, however, PC/PS/chol DXR-liposomes were degraded at a considerably higher rate than DSPC/DPPG/ chol DXR-liposomes. the difference in susceptibility to lysosomal degradation of the two liposome preparations was also demonstrated by incubating the DXR-liposomes with lysosomal fractions isolated from rat liver homogenates: PC/PS/chol DXR-liposomes were much more sensitive to lysosomal esterase than DSPC/DPPG/chol DXR-liposomes. DXR either free or in liposomal form was chemically stable for up to 26 hr during incubation with the lysosomal fractions. Following uptake of DXR-liposomes by the cells, DXR was released from the cells into the medium. the release of DXR from cells that internalized DSPC/DPPG/chol DXR-liposomes was significantly delayed compared to the release of DXR from cells that internalized PC/PS/chol DXR-liposomes. Correlation of the relatively slow intracellular degradation of the DSPC/DPPG/chol DXR-liposomes with the delayed release of DXR from the cells suggests that by varying the type of DXR-liposomes, the rate of intracellular degradation can be manipulated, which, in turn, determines the rate of extracellular DXR release and thereby the therapeutic availability of the drug.     

Cell Surface Interactions between Trypanosoma congolense and Macrophages during Phagocytosis In Vitro

ABSTRACT. Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.     

Homotypic Lysosome Fusion in Macrophages: Analysis Using an In Vitro Assay

Lysosomes are dynamic structures capable of fusing with endosomes as well as other lysosomes. We examined the biochemical requirements for homotypic lysosome fusion in vitro using lysosomes obtained from rabbit alveolar macrophages or the cultured macrophage-like cell line, J774E. The in vitro assay measures the formation of a biotinylated HRP–avidin conjugate, in which biotinylated HRP and avidin were accumulated in lysosomes by receptor-mediated endocytosis. We determined that lysosome fusion in vitro was time- and temperature-dependent and required ATP and an N-ethylmaleimide (NEM)-sensitive factor from cytosol. The NEM-sensitive factor was NSF as purified recombinant NSF could completely replace cytosol in the fusion assay whereas a dominant-negative mutant NSF inhibited fusion. Fusion in vitro was extensive; up to 30% of purified macrophage lysosomes were capable of self-fusion. Addition of GTPγs to the in vitro assay inhibited fusion in a concentration-dependent manner. Purified GDP-dissociation inhibitor inhibited homotypic lysosome fusion suggesting the involvement of rabs. Fusion was also inhibited by the heterotrimeric G protein activator mastoparan, but not by its inactive analogue Mas-17. Pertussis toxin, a Gα_i activator, inhibited in vitro lysosome fusion whereas cholera toxin, a Gα_s activator did not inhibit the fusion reaction. Addition of agents that either promoted or disrupted microtubule function had little effect on either the extent or rate of lysosome fusion. The high value of homotypic fusion was supported by in vivo experiments examining lysosome fusion in heterokaryons formed between cells containing fluorescently labeled lysosomes. In both macrophages and J774E cells, almost complete mixing of the lysosome labels was observed within 1–3 h of UV sendai-mediated cell fusion. These studies provide a model system for identifying the components required for lysosome fusion.     

Pepsinogen Induction in Chick Stomach Epithelia by Reaggregated Proventricular Mesenchymal Cells In Vitro

In vitro organ culture system which permits embryonic chick proventriculus (glandular stomach) to synthesize pepsinogen de novo was developed. Explants of the proventricular rudiment were cultured on Millipore filters in Medium 199 with Earle's salts supplemented with 50% 12-day embryo extract at 38°C in 95% air and 5% CO₂.
In these culture conditions, pepsinogen, a functional marker protein of proventriculus, was first detected after 3 days of cultivation of 6-day chick proventricular rudiment. When recombined and cultured with 6-day proventricular mesenchyme, 6-day oesophageal, proventricular or gizzard (muscular stomach) epithelium expressed pepsinogen while small intestinal epithelium did not. These results were consistent with the previous results obtained by chorioallantoic membrane (CAM) grafting, and showed that the culture conditions are permissive for pepsinogen expression.
When recombined and cultured with reaggregated mesenchymal cells isolated from 6-day proventricular mesenchymal fragments, both 6-day proventricular and gizzard epithelia formed glandular structure and expressed pepsinogen. This indicates that the proventricular mesenchymal cells retain the ability to induce morphogenesis and cytodifferentiation of the proventricular epithelium even if the normal organization of proventricular mesenchyme is once destroyed.     

大薯类原球茎的离体诱导及再生体系的建立

采用正交试验设计方法,以大薯带节茎段为外植体,离体诱导类原球茎并建立大薯类原球茎的再生体系,以解决愈伤组织分化成苗和试管苗移栽成活率低的难题。结果表明：以带节茎段为外植体诱导类原球茎的最适培养基为MS（含3×Ca2＋）＋1.0 mg·L-1 6-BA＋0.2 mg·L-1 NAA＋0.1%PVP＋3%蔗糖,诱导率高达93.33%;类原球茎增殖的最适培养基为MS＋4mg·L-1 6-BA＋80 mg·L-1 Ad＋0.1%PVP＋3%蔗糖;类原球茎生根的最适培养基：1/2MS＋0.10 mg·L-1 NAA＋0.1%PVP＋3%蔗糖。将诱导得到的生根类原球茎植株进行炼苗,移栽基质珍珠岩：蛭石=2：1,移栽成活率可达到95%。     

In Vitro Generation of Monocyte-Derived Macrophages under Serum-Free Conditions Improves Their Tumor Promoting Functions

The tumor promoting role of M2 macrophages has been described in in vivo models and the presence of macrophages in certain tumor types has been linked to a poor clinical outcome. In light of burgeoning activities to clinically develop new therapies targeting tumor-associated macrophages (TAMs), reliable in vitro models faithfully mimicking the tumor promoting functions of TAMs are required. Generation and activation of human monocyte-derived macrophages (MDM) in vitro, described as M1 or M2 macrophages attributed with tumoricidal or tumor-promoting functions, respectively, has been widely reported using mainly serum containing culture methods. In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype. We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions. More importantly, MDM differentiated under serum-free conditions displayed enhanced tumoricidal activity for M1 and tumor promoting property for M2 macrophages in contrast to MDM differentiated in the presence of serum. Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1. Our data therefore confirm the tumor promoting properties of M2 macrophages in vitro and encourage the targeting of TAMs for cancer therapy.