Migration studies and histology of injectable microspheres of different sizes in mice

Injectable dermal filler materials consist of either fluids, biological fragments, or suspensions of particles or microspheres. Particles and microspheres are said to "migrate," but migration can occur only when they are injected into blood vessels. To evaluate biocompatibility and transport, five nonresorbable polymethylmethacrylate microspheres of various sizes, suspended in different carriers, as well as resorbable polylactic acid and dextran microspheres were injected subcutaneously into mice. The five implantation sites were the right cheek, right axilla, right groin, urethra, and the right quadriceps muscle of the thigh. These sites were excised along with the local lymph nodes, lungs, liver, and spleen at 1, 3, 6, and 9 months after injection. Polymethylmethacrylate microspheres of 4 microm and 8 microm were phagocytosed but not transported to lymph nodes or distant organs. Larger microspheres of 20, 40, and 100 microm were encapsulated by connective tissue, macrophages, and giant cells. Polylactic acid microspheres caused a mild inflammatory response and had disappeared at 6 months. Dextran microspheres caused a pronounced foreign-body reaction and were phagocytosed at 9 months. The extremely large carbon-coated spheres of 200 to 500 microm in diameter "migrated" up to 1 cm from the implantation site. With the exception of an erroneous intravenous injection, no migration or transportation of any of the injected microspheres to lymph nodes or filter organs was seen. Obviously, the collagen glue released no microspheres. After subdermal injection, the collagen carrier substance kept the microspheres apart as a scaffold for tissue ingrowth, whereas all other carrier substances, such as gelatin, hyaluronic acid, or alginate, separated soon after injection, thereby causing agglomeration of the microspheres.     

Nonspecific entry of thoracic duct immunoblasts into intradermal foci of antigens

Lymph borne immunoblasts were obtained by collecting thoracic duct lymph from inbred rats 3–5 days after either killed C. parvum, B. abortus or B.C.G. organisms had been injected subcutaneously into the hindquarter regions to stimulate the caudal lymph nodes. By incubating the lymph cells with a radioactive precursor of DNA, 5-iodo-2-deoxyuridine-¹²⁵I, the immunoblasts became labelled but the small lymphocytes did not. The labelled cells were washed and injected intravenously into syngeneic recipients which had had intradermal injections of various antigens at various previous times. The entry of labelled cells into these injection sites was monitored by counting the radioactivity that they contained up to 24 hr later.It was found that the accumulation of radioactivity in the skin lesions was maximal 12 hr after the donor cells had been injected, but the immunological specificity of the donor immunoblasts did not affect significantly the extent to which they entered lesions which contained the same or unrelated antigens. It was found also that the sites of intradermal injections of B.C.G. or C. parvum always attracted more immunoblasts than sites containing other antigens; this was a non-specific effect, thought to be related to the adjuvant properties of these organisms.     

Observations on the effect of radiotherapy,followed by injection of pig lymph node cells,in reducing the number of pulmonary tumors induced by intravenous injection of tumor cells into isogenic mice

Summary Pulmonary tumors were produced in A. strain mice by intravenous injection of A. strain mammary carcinoma cells. The mesenteric lymph nodes of pigs were immunized by implantation of fragments from the same tumors into the pig mesentery.Tumor-immune pig lymph node cells when injected IV 7 days after tumor cells did not reduce the number of tumors, counted on day 14. However, when preceded by 200 rad thoracic irradiation on day 3 (which increased the number of pig cells settling in the lungs) tumor-immune cells given IV reduced the number of tumors compared with the effect of irradiation alone, or in combination with nonimmune pig cells.When tumor-immune pig cells were injected IP on day 7 (following thoracic irradiation on day 3), no antitumor effect was observed. Thus immediate pig cell/tumor cell contact is important in order to obtain an antitumor effect.Pig cells immunized against a human bladder carcinoma did not reduce pulmonary tumor formation by one of the mouse tumors.     

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e. intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 μl of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.     

Morphological observations on the fate of liposomes in the regional lymph nodes after footpad injection into rats

Multilamellar liposomes composed of equimolar egg phosphatidylcholine and cholesterol and containing carboxyfluorescein or colloidal gold were injected subcutaneously into the footpad of the hind-leg of rats. The draining popliteal lymph nodes of animals killed at time intervals after injection were then dissected and sections examined by fluorescence microscopy (carboxyfluorescein), light microscopy using an immunogold silver kit to enhance gold particles or by transmission electron microscopy. Morphological observations confirmed that subcutaneously injected liposomes accumulate in large numbers in the draining lymph node. The majority of liposomes arrived at the subcapsular sinuses, probably via afferent lymphatic vessels, as such, i.e., in a non-cell bound form. Subsequently, liposomes were dispersed throughout the lymph node either by permeation as free vesicles along the sinuses or by cells involved in vesicle uptake. The majority of such cells were free macrophages, littoral cells and reticular cells (fixed macrophages). Once within cells, liposomes were seen digested by the lysosomal apparatus with varying loss of their lamellar structure, leaving free gold particles within the lysosomes.     

Granulocyte-macrophage colony-stimulating factor-based melanoma cell vaccines immunize syngeneic and allogeneic recipients via host dendritic cells

Subcutaneous injection of GM-CSF-expressing cancer cells into experimental animals results in protective cancer immunity. To delineate the mode of action of such vaccines, we used trinitrophenyl, the antigenic moiety of the contact allergen trinitrochlorobenzene, as surrogate Ag. Trinitrophenyl-derivatized bone marrow-derived dendritic cells were found to elicit a contact hypersensitivity response in syngeneic, but not in allogeneic recipients, compatible with their expected mode of direct Ag presentation. When expressing GM-CSF, haptenized M3 melanoma cells were also able to induce a contact hypersensitivity response but, in contrast to bone marrow-derived dendritic cells, not only in syngeneic but also in allogeneic recipients. This argues for a critical role of host APC. To identify their nature, we introduced the beta-galactosidase (betagal) gene into M3-GM cells. Their administration activated betagal-specific, L(d)-restricted CTL in syngeneic BALB/c mice. Evaluation of lymph nodes draining M3-GM-betagal injection sites revealed the presence of cells presenting the respective L(d)-binding betagal peptide epitope. Based on their capacity to activate betagal-specific CTL, they were identified as being CD11c(+) dendritic cells. These experiments provide a rational basis for the use of GM-CSF-based melanoma cell vaccines in an allogeneic setting.     

The in vivo fate of APCs displaying minor H antigen and/or MHC differences is regulated by CTLs specific for immunodominant class I-associated epitopes

The goal of this work was to evaluate the fate of APCs following interactions with T cells in unprimed mice with a normal T cell repertoire. We elaborated a model in which male adherent peritoneal mononuclear cells were injected into the foreleg footpads of naive female recipients mismatched for either minor or major histocompatibility Ags. At various times after injection, APC numbers in the draining (axillary and brachial) lymph nodes were assessed using a Ube1y gene-specific PCR assay. Our experimental model was designed so that the number of APCs expressing the priming epitope was similar to what is observed under real life conditions. Thus, early after injection, the frequency of afferent lymph-derived APCs expressing the priming epitope was in the range of 101-102/106 lymph node cells. We found that APCs presenting some, but not all, nonself epitopes were killed rapidly after entrance into the lymph nodes. Rapid elimination of APCs occurred following interactions with MHC class I-restricted, but not class II-restricted, T cells and was observed when APCs presented an immunodominant (B6dom1/H7a), but not a nondominant (HY), epitope. Killing of APCs was mediated partly, but not exclusively, by perforin-dependent process. We propose that killing of APCs by CTLs specific for immunodominant MHC class I-restricted epitopes may be instrumental in regulating the intensity, duration, and diversity of T cell responses.     

抗原物质引起滤泡的形成:一次投入与多次投入的比较

实验以不同方式投入抗原物质,即一次投入与分次投入,并观察了新产生滤泡数,维持的滤泡数,实验用小鼠２４ 只, 于足底注入铝和钥孔　血蓝素附合物（ＡＫＬＨ）, 分一次注入与三次注入组; 三次注入组又分间隔５ 日及间隔二周注入。注入后第３ 周与第１２ 周分别取出腘淋巴结,应用免疫组化法,第三周末观察可见一次投入产生的滤泡多, 而第十二周发现分次投入维持的滤泡数多。可见反应性滤泡的形成, 不仅与刺激物的性状和投入量有关, 而且与投入的方法有关     

Production of hapten-specific T cell hybridomas and their use to study the effect of ultraviolet B irradiation on the development of contact hypersensitivity

We produced a series of T cell hybridomas that produce IL-2 when cultured with syngeneic APC coupled to FITC or TNP. These hybridomas are hapten specific and Ia restricted. The hybridomas were used to detect hapten-bearing APC in draining lymph nodes of mice sensitized with trinitrochlorobenzene or FITC in vivo. Hapten-bearing APC capable of stimulating the hybridomas were detectable in draining lymph nodes of hapten-painted mice within 3 h after sensitization. The ability of lymph node APC to stimulate the hybridomas peaked at 24 h and declined by 48 h. The dendritic cell subpopulation was the subpopulation of cells that were found in the regional lymph nodes of hapten-painted animals that were capable of stimulating the hybridomas to produce IL-2. Prior treatment of the skin with low dose UVB irradiation before epicutaneous application of contact sensitizers significantly reduced the capacity of hapten-bearing APC to stimulate the hybridomas. This observation was corroborated by results obtained from flow microfluorometry analysis of lymph node cells from FITC-sensitized mice. Lymph node dendritic cells obtained from FITC-painted mice contain a brightly staining group of cells by flow microfluorometry analysis. Lymph node dendritic cells from FITC-painted, UVB-irradiated mice did not contain this brightly staining population. These results indicate that low dose, local UVB irradiation may affect APC migration and/or function. We believe that these hybridomas will prove to be useful tools in the study of the development and regulation of contact hypersensitivity.     

Effect of thymus cell injections on germinal center formation in lymphoid tissues of nude (thymusless) mice

Nude mice, partially backcrossed to Balb/c or DBA/2, were injected iv with 5 × 10⁷ thymus cells from the respective inbred strain. The response of these mice to immunization with Brucella abortus antigen was studied, with respect to both antibody production and the formation of germinal centers in their lymphoid tissues. The results were compared to those obtained with nude mice to which no thymus cells were given, as well as to Balb/c, DBA/2, or +/? litter mate controls.Nude mice formed less 19S as well as 7S antibody than did litter mate controls and completely lacked germinal centers in lymph nodes and gut-associated lymphoid tissue. Those nude mice which had been injected with thymus cells made a much better secondary response, both for 19S and for 7S antibody, and had active germinal centers in their lymph nodes as early as 3 wk after thymus cell injection. Intestinal lymphoid tissue in nude mice showed only slight reconstitution of germinal center activity several months after thymus cell injection and none at earlier times. Irradiated (3000 R) thymus cells appeared as effective as normal cells in facilitating germinal center appearance and 7S antibody production in the nude mice.     

Lymph node localization of non-specific antibody-coated liposomes

Subcutaneously injected small unilamellar liposomes are drained into the lymphatics and localized in the regional lymph nodes, and thus they can be used for the detection of metastatic spread in breast cancer patients and for delivery of drugs to diseased lymph nodes (1-8). An aqueous phase marker, [125I]-polyvinylpyrrolidone, and a lipid phase marker, [3H]-cholesterol, were used to study the lymph node localization of IgG-coated liposomes injected subcutaneously into mouse and rat footpads. The results show that human immunoglobulin G (IgG) coated liposomes are rapidly removed from the site of injection and are localized in the regional lymph nodes to a greater extent than control liposomes (i.e. liposomes without IgG). Free IgG was found to inhibit the uptake of IgG-coated liposomes by the lymph nodes. The localization of IgG-coated liposomes in the regional lymph nodes is influenced by charge of the liposomes. The results presented here suggest that antibody-coated liposomes may provide a more efficient way of delivering therapeutic agents to the lymph nodes in the treatment of diseases such as breast cancer with lymph node involvement. Similarly, monoclonal antibody-coated liposomes containing lymphoscintigraphic material may improve the detection of lymph node metastases.     

Migration of specific antibody-forming cells during the secondary immune response against horseradish peroxidase

Mice were primed subcutaneously in the hind footpads with horseradish peroxidase (HRP) and boosted intravenously 10 weeks later. Removal of the popliteal lymph nodes (draining the site of primary immunization) before the booster injection markedly depressed the secondary immune response in the spleen and bone marrow. This was taken as evidence that the secondary humoral immune response against HRP in the spleen and bone marrow is largely dependent upon immigration of cells from the popliteal lymph nodes after the booster injection. In rats primed subcutaneously in the hind footpads with HRP, antibody-forming cells were demonstrated in the blood, but not in the thoracic duct lymph 3 days after an intravenous booster injection with HRP.     

The migration of lymphocytes into rat popliteal lymph nodes during antigenic promotion or competition between heterologous erythrocytes

The injection of chicken and sheep red blood cells (CRBC and SRBC) into rat popliteal lymph nodes either together or sequentially 2, 4, 6, or 8 days apart resulted in an enhanced immune response when the second antigen was injected 2 or 4 days after the injection of the first antigen (antigenic promotion) or a suppressed immune response when the second antigen was injected 6 days after the injection of the first antigen (antigenic competition). The immune response to either antigen was dependent upon the time of administration of the second antigen with respect to the first antigen. Lymphocyte migration into antigenically stimulated lymph nodes was greater when the two antigens were injected sequentially rather than together. Further, the migration of lymphocytes into the lymph node was enhanced when the second antigen was injected during the inductive or suppressive phase of the immune response to the first antigen (CRBC) regardless of whether the same (CRBC) or an antigenically unrelated antigen (SRBC) was used as the second antigen. While antigenic promotion may in part be explained by the increased rate at which lymphocytes migrate into lymph nodes, lymphocyte migration is also enhanced during antigenic competition. This suggests that while suppressor cells/factors may regulate the effector phase of an immune response they do not directly modulate the migration of blood-borne lymphocytes into the lymph node.     

THE DISTRIBUTION AND DIFFERENTIATION OF LYMPH-BORNE IMMUNOBLASTS AFTER INTRAVENOUS INJECTION INTO SYNGENEIC RECIPIENTS

Thoracic duct lymph from inbred, hooded rats was collected 3–5 days after antigenic stimulation of the caudal lymph nodes. During this period the lymph contained 10–15% of large, basophilic lymphoid blast cells (immunoblasts). By incubating the lymph cells at 38.C with radioactive DNA precursors, either ³H-thymidine or ¹²⁵I-deoxyuridine, the immunoblasts became labelled but the small lymphocytes did not. The lymph cells were then washed and injected intravenously into syngeneic recipients which were killed after various intervals up to 24 hr so that the radioactivity of their organs could be assayed by scintillation counting and autoradiography.
The main finding was that in animals killed after 4 or more hours the small gut always contained most of the recoverable activity and autoradiographs showed that this was because the injected cells had infiltrated the lamina propria in large numbers. Earlier, many of the injected cells were retained temporarily in the lungs, liver and spleen but many of them soon left those organs and entered the lamina propria of the small gut.
An electron microscope study of autoradiographs showed that 24 hr after injection the cells which entered the lamina propria of the gut had differentiated into plasma cells so that they displayed abundant, lamellar endoplasmic reticulum.     

Peritoneal macrophages introduced into mouse foot pads enter the germinal center of regional lymph nodes nonspecifically.

Male mice were injected into their foot pads with sheep erythrocytes (SRBC) to form lymph follicles in the germinal centers in the popliteal lymph nodes. 4 weeks later, peritoneal macrophages labeled with carbon from syngeneic donors sensitized with SRBC or typhoid-paratyphoid bacilli (TAB) were separately injected into the foot pads as well. The popliteal lymph nodes were histologically examined at 6 h to 5 days after injection. Labeled macrophages appeared in the marginal sinus, migrated straight across the cortex from the marginal sinus to the lymph follicles and then entered the germinal centers. There was no difference in the mode of appearance, migration and localization of labeled macrophages in the regional lymph nodes between the mice given labeled macrophages from SRBC-sensitized donors and those given macrophages from TAB-sensitized donors. The entrance of lymph macrophages into the germinal centers of the regional lymph nodes would be immunologically nonspecific. After the injection of Pelikan ink into the foot pads, the macrophages which have taken up carbon in the peripheral tissue reached the regional lymph nodes via the afferent lymphatics and then entered the germinal centers, mainly through the medullary pole of the lymph follicles, after migrating along their immediate exterior from their marginal sinus to their medullary pole.     

Hepal-6 RNA脉冲BMDCs瘤苗抗小鼠HCC免疫效应的研究

目的观察Hepal-6RNA脉冲BMDCs瘤苗能否激发有效的机体抗肿瘤免疫反应。方法用Hepal-6RNA脉冲骨髓分离培养5d的BMDCs,瘤苗的抗肿瘤效应通过C57BL/6J小鼠肝细胞癌(HCC)模型验证,即C57BL/6J小鼠皮下接种Hepal-6细胞8d后,成瘤小鼠分为2组:对照组(注射无血清RPMI-1640)和实验组(足垫部注射Hepal-6RNA脉冲BMDCs瘤苗),30d后处死小鼠并取淋巴结、脾和瘤体冰冻切片,进行CD4、CD8、CD25和Foxp3免疫组织化学染色。结果免疫组织化学检测显示,淋巴结、脾、肿瘤内有大量CD4+T细胞、CD8+T细胞、CD25+T细胞和Foxp3+Tregs细胞浸润。与对照组小鼠比较,足垫部注射Hepal-6RNA脉冲BMDCs瘤苗的实验组小鼠淋巴结、脾、瘤内CD4+T细胞和CD8+T细胞浸润显著增加,CD25+T细胞和Foxp3+Tregs浸润明显减少。结论 Hepal-6RNA脉冲BMDCs瘤苗能下调小鼠HCC瘤内CD25+T细胞和Foxp3+Tregs浸润,促进CD4+T细胞和CD8+T细胞的增殖和活化,增强D4+T细胞、CD8+T细胞归巢至淋巴结和脾,具有抗瘤免疫效应。     

Immunity status of guinea pigs infected with Brucella L forms

B. abortus L-forms injected subcutaneously into guinea pigs adapt in the lymph nodes of the animals in the absence of reversion to normal cells. Complete and incomplete antibodies belonging to macro- and microglobulins (IgM and IgG) were synthetized. The allergic transformation of the organism is faintly pronounced. After this form of infection guinea pigs become resistant to B. melitensis infection for 6 months (the term of observation).     

Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity

Epicutaneous application of haptens to UV-exposed skin induces hapten-specific tolerance. This is mediated via regulatory T cells (Tr), as i.v. injection of T cells from UV-tolerized mice into naive animals renders the recipients unresponsive to the respective hapten. However, when UV-induced Tr are injected i.v. into sensitized mice, contact hypersensitivity (CHS) is not suppressed, suggesting that Tr inhibit the induction, but not the elicitation, of CHS and are inferior to T effector cells. As sensitization takes place in the lymph nodes, but elicitation occurs in the area of challenge, we postulated that Tr injected i.v. locate to the lymph nodes and not to the periphery and therefore only suppress the induction, not the elicitation, of CHS. Indeed, i.v. injection of Tr into sensitized mice did not inhibit CHS, although injection of Tr into the ears of sensitized mice suppressed the challenge. Inhibition was hapten specific, as injection of dinitrofluorobenzene (DNFB)-specific Tr into the ears of oxazolone (OXA)-sensitized mice did not affect challenge with OXA. However, when ears of OXA-sensitized mice were injected with DNFB-specific Tr and painted with DNFB before OXA challenge, CHS was suppressed. Inhibition correlated with the local expression of IL-10. Depletion studies and FACS analysis revealed that Tr express the lymph node-homing receptor L-selectin, but not the ligands for the skin-homing receptors E- and P-selectin, suggesting that UV-induced Tr, although able to inhibit T effector cells, do not suppress the elicitation of CHS upon i.v. injection, because they obviously do not migrate into the skin.     

Comparative studies of the effects of recombinant GM-CSF and GM-CSF administered via a poxvirus to enhance the concentration of antigen- presenting cells in regional lymph nodes

Repeated subcutaneous (s.c.) injections of recombinant granulocyte-macrophage colony-stimulating factor (recGM-CSF) for 4-5 days can enrich an immunization site with antigen-presenting cells (APC), which has been correlated with improved immune responses in experimental and clinical studies. A recombinant vaccinia virus encoding the GM-CSF gene (rV-GM-CSF) has been developed and can generate specific antitumour immunity in a whole tumour cell vaccine. In the present study, we examined whether rV-GM-CSF could produce and release GM-CSF locally which, in turn, might enrich a site of immunization for APC as previously shown for recGM-CSF. S.c. injection of rV-GM-CSF significantly (P<0.05) enhanced the percentage and overall number of APC, measured by class II expression levels, in the regional lymph nodes that drain the injection site. Dose- and temporal-dependent studies showed class II expression levels in the draining lymph nodes were maximally enhanced 5-7 days after a single injection of 10(7)plaque-forming units (pfu) of rV-GM-CSF. Flow cytometry revealed that the increase in class II expression resulted from (i) a higher class II expression level on CD19(+)B cells and (ii) an increase in the number of CD11c(+)/class II(+)professional APC within the draining lymph nodes. Moreover, isolation of lymph nodes from rV-GM-CSF-treated mice revealed their capacity to support higher levels of antigen-specific T cell proliferation and allospecific cytotoxic responses. A comparison between a single injection of rV-GM-CSF and a 4-day course of recGM-CSF revealed comparable changes in class II expression and functional T cell assays. GM-CSF can be delivered in a recombinant poxvirus, and the local production of the cytokine results in cellular and phenotypic changes that are similar to those of recGM-CSF. The ability to utilize rV-GM-CSF as a single inoculum may be more compatible with traditional immunization strategies.     

Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates including immature dendritic cells into injected muscles

Plasmid-encoded GM-CSF (pGM-CSF) is an adjuvant for genetic vaccines; however, little is known about how pGM-CSF enhances immunogenicity. We now report that pGM-CSF injected into mouse muscle leads to a local infiltration of potential APCs. Infiltrates reached maximal size on days 3 to 5 after injection and appeared in several large discrete clusters within the muscle. Immunohistological studies in muscle sections from mice injected with pGM-CSF showed staining of cells with the macrophage markers CD11b, Mac-3, IA(d)/E(d) and to the granulocyte marker GR-1 from day 1 through day 14. Cells staining with the dendritic cell marker CD11c were detected only on days 3 to 5. Muscles injected with control plasmids did not stain for CD11c but did stain for CD11b, Mac-3, IA(d)/E(d), and GR-1. No staining was observed with the APC activation markers, B7.1 or CD40, or with markers for T or B cells. These findings are consistent with the infiltrating cells in the pGM-CSF-injected muscles being a mixture of neutrophils, macrophages, and immature dendritic cells and suggest that the i.m. APCs may be enhancing immune responses to coinjected plasmid Ags. This hypothesis is supported by data showing that 1) separation of injections with pGM-CSF and Ag-expressing plasmid into different sites did not enhance immune responses and 2) immune enhancement was associated with the presence of CD11c+ cells in the infiltrates. Thus, pGM-CSF enhancement may depend on APC recruitment to the i.m. site of injection.