Injection by various routes of melanoma antigen-associated macrophages: biodistribution and clinical effects

Patients' autologous macrophages (AM) were used as antigen-presenting cells (APC) in a vaccination protocol against malignant melanoma. AM were administered by various routes, including intralymphatic, since these cells did not express CCR7, a molecule required for APC migration to lymph nodes. Seven HLA-A2 patients with metastatic melanoma-two classified as M1 and five as M3-were included in the study. AM were produced from leukapheresis-separated mononuclear cells by 7-day culture with granulocyte-macrophage colony-stimulating factor. After separation by elutriation, AM were frozen in aliquots and subsequently thawed at monthly intervals, exposed to MAGE-3(271-279) peptide and injected subcutaneously into lymph nodes or into one peripheral lymph vessel. Intradermal tests were performed before and after treatment to determine peptide reactivity. No acute toxicity was observed following injection. One M1 patient had a 7-mm induration intradermal reaction response and was stabilized for 64 weeks. The M3 patients did not show any immunological or clinical response. In 11 patients, the biodistribution of 111In-labeled AM was investigated. There was no clear evidence that AM injected intradermally or subcutaneously left the site of injection. After injection into a lymph vessel of the foot region, scintigraphs showed five to ten popliteal and inguinocrural lymph nodes. This appeared to be the most efficient way to administer rapidly and safely large amounts of peptide-loaded APC into lymph nodes.     

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.     

Localization of retinal dehydrogenase type 1 in the stomach and intestine

Rats were injected with liposomes containing iodixanol (CTP10 Injection; 100 mg iodine per kg body weight) followed by a second injection of 125I-tyramine-cellobiose-albumin microspheres. The amounts of phagocytosed and degraded labelled albumin in liver were measured. A reduced uptake and degradation of albumin microspheres was observed when the labelled microspheres were injected 2 h or 24 h after the liposomes compared with that obtained in control animals receiving saline. No effect on the uptake and degradation of labelled microspheres was observed when the time lag between the injection of liposomes and labelled microspheres was 1 week. The data show that the uptake and degradation of 125I-tyramine-cellobiose-albumin microspheres can be used as indicators of Kupffer cell phagocytotic function following drug uptake by these cells.     

Cell-mediated DNA transport between distant inflammatory sites following intradermal DNA immunization in the presence of adjuvant

After intradermal genetic immunization, naked DNA is transported from the site of injection to regional lymph nodes. Little is known on how inflammation influences this process and whether DNA is transported beyond local lymph nodes. In the experiments herein reported, we injected naked DNA in the presence of adjuvant to address questions related to 1) the fate of naked DNA in the presence of inflammation; 2) the generation of immune responses to the encoded protein during inflammation; and, more in general, 3) the fate of ingested molecules beyond regional lymph nodes during inflammation. Two sites of inflammation were induced in vivo in mice. Naked DNA was injected in the nape together with adjuvant, and adjuvant only was injected at a distant peritoneal site. Injected DNA, uptaken at the primary dermal site of inflammation, was transported beyond regional lymph nodes to distant organs such as the spleen and to the distant peritoneal site of inflammation. This transport, mediated by CD11b+ cells, was cumulative during chronic inflammation. These results indicate a novel route of transport of DNA beyond regional lymph nodes and may have specific implications for DNA-based immune modulation.     

Lymphocyte migration in internally irradiated animals. Effect of internal gamma radiation on the migration properties of spleen lymphocytes labeled with 51Cr

In experiments on CBA mice it was shown that migration of 51Cr-labeled spleen lymphocytes, injected intravenously, to lymph nodes of intact recipients was suppressed 6-24 months after the administration of a radiopharmaceutic preparation of selenium-75-selenomethionine in a quantity forming the doses of 1 Gy and 1.5 Gy absorbed within the whole body and lymphoid organs, respectively. Migration of labeled lymphocytes to the liver, kidneys and lungs, as well as their retention in the circulating blood, were increased. As the result of the migration disorders the specific affinity of lymphocytes for peripheral lymphoid tissue decreased.     

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.     

Experimental allergic encephalomyelitis: changes in growth and proliferation within the draining lymph node following injection of basic encephalitogenic protein in Freund's complete adjuvant

Changes in wet weight, dry mass, and DNA synthesis of draining lymph nodes from rats injected with encephalitogenic basic protein in Freund's complete adjuvant (FCA) were studied. Lymph nodes of rats injected with encephalitogenic basic protein in FCA show accelerated enlargement from the second up to the fourth day after injection, as compared to lymph nodes of rats injected with FCA alone, or with nonencephalitogenic basic protein in FCA. The greatest difference in lymph node weight was found on the fourth day. At this time cell division is higher in the group injected with encephalitogenic protein in FCA than in the group injected with FCA alone. However, the increased division of cells in situ cannot account, in and by itself, for the enlargement of the lymph node which was observed. It is concluded that migration of lymphocytes into the lymph node makes a substantial contribution to the hyperplasia of the lymph node.The results suggest that accelerated lymph node enlargement may be specific, at least in part, for induction of experimental allergic encephalomyelitis.     

Anatomy and topography of external iliac lymph nodes in adults]

The investigation of the external iliac lymph nodes has been performed in 152 preparations of corpses of mature persons of both sex, who died from causes not connected with any disease of the lymphatic system, lower extremities and pelvic organs. The external iliac lymph nodes and their afferent and efferent lymphatic vessels have been revealed by means of interstitial injection of the lower extremities and pelvic organs, as well as by means of direct injection of Gerota mass into the lymphatic vessels. Form, amount, dimensions and topography of common iliac lymph nodes have been studied. Lymphatic vessels, running from certain parts and organs of the body to various subgroups of the external iliac lymph nodes have been described, as well as efferent lymph vessels of these nodes. The external iliac lymph nodes are constant formations; the largest of them--lymph nodes of the lacuna--are nodes of the I step for the lower extremity lymph vessels. In 54% of cases in persons of both sex positive (right-sided) asymmetry has been revealed. Total amount of the iliac lymph nodes prevails in men, while their size is greater in women. The size of these nodes in persons of both sex is greater to the left than to the right. There are connections (in 3% of cases) between the external iliac lymph nodes and aortal and lumbar nodes of the opposite side.     

Anatomy and topography of the common iliac lymph nodes in human beings in the 1st period of maturity

The investigation of common iliac lymph nodes has been performed in 20 corpses of the first mature age of both sex (5 male and 5 female corpses) of persons died from causes not connected with the lymphatic system diseases, the lower extremities and the pelvic organs. The common iliac lymph nodes with their afferent and efferent lymphatic vessels are revealed by means of interstitial injection into the lower extremities and the pelvic organs and with direct injection into the lymphatic vessels. The form, amount, size and topography of the common iliac lymphatic vessels have been studied. The lymphatic vessels, that go from certain body parts and organs to various subgroups of the common iliac lymph nodes, as well as the lymphatic vessels that connect the nodes both within the subgroup and also between the subgroups. The amount and size of the lymphatic nodes of the lateral subgroup predominate over the nodes of other subgroups of the common iliac lymph nodes; the amount of the common iliac lymph nodes predominates in men, and their size--in women. Amount of these nodes in the right and their size in the left predominate in both sex. Among the common iliac lymph nodes there are no teniform nodes, and efferent lymphatic vessels of the lateral and medial subgroup of the common iliac lymph nodes in 15% of cases run towards the lumbar nodes in the opposite side.     

Tissue localization and retention of antigen in relation to the immune response

Antigen persists for months or even years in lymphoid tissues of immune animals and this antigen is believed to participate in the induction and maintenance of B-cell memory as well as in the maintenance of serum antibody levels. In the present report we describe the phenomenon of antigen localization and long-term retention on mouse follicular dendritic cells (FDCs). The antigens used were injected in the hind footpads of immune mice and the popliteal lymph nodes were the lymphoid organs generally studied. In addition to presenting the morphological features of mouse FDCs, we report the results of a study of the mechanism of antigen migration from the site of initial localization in the lymph node subcapsular sinus to the regions of follicular retention in the cortex. The migration was followed by light and electron microscopy. The results support the concepts that immune complexes are trapped in the subcapsular sinus and are transported by a group of nonphagocytic cells to follicular regions. The mechanism of transport may involve either migration of pre-FDCs with a concomitant maturation into FDCs, or cell-to-cell transport utilizing dendritic cell processes and membrane fluidity; or a combination of the two mechanisms may be in operation.     

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides ''"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.     

Individual and sexual characteristics of the common iliac lymph nodes in elderly persons

The common iliac lymph nodes (CILN) have been investigated on 24 preparations from corpses of elderly persons (5 male and 7 female corpses), died from the causes not connected with the lymphatic system diseases, lower extremities and pelvic organs. The CILN with their afferent and deferent lymphatic vessels are revealed by means of interstitial injection into the lower extremities and pelvic organs, as well as by means of direct injection into lymphatic vessels. The form, amount, size and topography of CILN are studied. Lymphatic vessels, running from certain parts of the body and organs to various subgroups of CILN are described, as well as lymphatic vessels, connecting the nodes both within each subgroup and between the subgroups. There is a tendency in prevalence of amount and size of the lateral subgroup of the lymph nodes over the nodes of other subgroups of CILN; tendency in prevalence of amount of the lymph nodes in men, and their size--in women; prevalence of amount of right CILN and their size in the left--in persons of both sex; in 70% of the cases the amount of afferent lymphatic vessels to CILN prevails over that of the deferent lymph nodes.     

Comparative migration of B- and T-Lymphocytes in the rat spleen and lymph nodes.

B-lymphocytes were obtained either by thoracic duct cannulation of thymectomized, irradiated rats or by isolation of complement-receptor-bearing lymphocytes from normal rats. They were labeled in vitro with [³H]-leucine and injected iv into syngeneic recipients from which samples of spleen and lymph node were taken at intervals from 15 min to 48 hr after injection. The sites of initial localisation of B- and T-lymphocytes were identical suggesting that the cells migrated into both organs by a common entrance. The two cell types remained closely associated for several hours in the paracortex of lymph nodes and at the periphery of the periarteriolar lymphoid sheath of the spleen. After 1–6 hr, B-cells segregated from T-cells by moving on into the adjacent part of the lymphocyte corona in the follicular area. By 24 hr, B-cells were evenly distributed throughout the corona. A definite minority of B-cells but no T-cells were seen within the germinal centres. In the spleen, T-cells moved into the central area of the periarteriolar sheath before returning to the blood. The immunological significance of the routes of B- and T-cell migration is discussed.     

Schistosoma mansoni: the effect of regional lymphadenectomy on the level of protection induced in mice by radiation-attenuated cercariae

The lymph nodes which drain the sites of percutaneous vaccination with optimally irradiated cercariae of Schistosoma mansoni were surgically excised in studies to determine their role in the induction of protective immunity. Lymphadenectomy of the axillary and inguinal nodes which drain the abdominal exposure site, or of the cervical node which drains the aural site of exposure, five days prior to vaccination reduced the levels of resistance by two-thirds. Excision of these nodes on Days 5, 10, 15, or 20 postvaccination also significantly reduced the levels of immunity induced, though ablation was less effective at later times. Removal of lymph nodes not draining the site of vaccination had no effect on the induction of resistance. We interpret the results as indicating that successful vaccination of mice against S. mansoni requires the presentation of antigen to lymphocytes in local lymph nodes draining the vaccination site, rather than distant lymphoid organs such as the spleen.     

Characteristics of the cellular reactions of the T- and B-dependent areas of the regional lymph nodes in dogs to the administration of immunodepressants

Mesenteric, bifurcational, axillary and popliteal lymph nodes have been studied in 22 healthy mature male dogs. Amount of blast cells, small lymphocytes, plasma cells and macrophages has been taken into account in the paracortical zone, in the germinative centers and in the medullary cords. For two weeks to one group of the animals every day imuran in turn with aurantin (10 mg/kg and 25 mg/kg) are injected, or antilymphocytic serum (ALS) intraperitoneally every other day (0.1 ml/kg). The combined injection of imuran and aurantin produces a more pronounced toxic effect to the hemopoietic organs than ALS. ALS is more specific for T-dependent zones of the lymph nodes. In the dose and interval mentioned ALS is an immunostimulating preparation for the immunocompetent cells of the germinative centers of the lymph nodes. The reaction of the lymph nodes depends on their regional belonging.     

微球包裹pcDNA3-GRF（1-32）在小鼠肌肉组织的表达及对生长的影响

以乳酸-乙醇酸共聚物为载体,采用复乳液中蒸发法制备载生长激素释放因子（Growth hormone releasing factor,GRF）真核表达质粒pcDNA3-GRF（1-32）微球,其中包封率达69%,平均粒径为2.20μm,载药量80μg/mg,收率70%;体内转染小鼠肌肉组织, 提取注射部位肌肉组织DNA 和总RNA,经PCR和RT-PCR,发现注射pcDNA3-GRF（1-32）微球组的GRF表达水平最高（UVIBAND Version 99分析）,释放的GRF表达质粒在小鼠肌肉内存在并表达的时间至少30d。体重统计结果表明,30d后微球包裹质粒组累积增重与其它组相比差异极显著（P<0.01）,比裸质粒组、质粒和空白微球混合物组、生理盐水组分别高12.87%,19.72%,58.58%;结论认为,载pcDNA3-GRF（1-32）微球具有缓释作用,并可实现GRF基因体内局部基因转染、表达,发挥其相应的生物学效应,有希望成为提高质粒在动物肌肉组织表达效率的新方法。     

Radio-opaque and surface-functionalized polymer microparticles: potentially safer biomaterials for different injection therapies

Injectable polymer particles with a diameter in the range of 30-300 microm find applications as a biomaterial in different clinical fields, such as cosmetic surgery, reconstructive surgery, and urology. However, clinical effects tend to disappear after several months, either due to migration of the particles away from the injection site (caused by weak adherence with the surrounding soft tissues) or due to fibrosis (caused by excessive encapsulation of the particles by fibrous tissue). Little is known about the fate of injected microparticles, due to the fact that they are extremely difficult to trace in a noninvasive manner. Design, synthesis, and characterization of new polymeric microspheres with two additional features that can enhance safety and can help to overcome drawbacks of existing products are reported. First, the new microparticles feature clear radio-opacity (X-ray visibility) as they are prepared on the basis of a reactive methacrylic monomer that contains covalently bound iodine. Model experiments reveal that the level of X-ray contrast is sufficient for clinical monitoring; they can be visualized both during the injection and afterward. The particles feature excellent cytocompatibility in vitro and in vivo. Second, a method is explored to functionalize the surface of the particles, for example, through immobilization of collagen. Other extracellular matrix proteins can also be immobilized, and this provides a mechanism to control anchoring of the particles in soft tissue. The results are briefly discussed in the context of improved biomaterials, contemporary X-ray imaging, and control over biomaterial-soft tissue interactions in vivo.     

Reduction in adhesiveness to extracellular matrix components, modulation of adhesion molecules and in vivo migration of murine macrophages infected with Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite, able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. In order to investigate whether the parasite uses leukocyte trafficking to disseminate throughout the host, the adhesive potential to extracellular matrix components, the expression of adhesion molecules and the in vivo migration of murine macrophages infected with RH strain of T. gondii were investigated. Cellular adhesion to fibronectin, laminin and collagen IV decreased after 24 h of T. gondii infection. However, the decrease in adhesion of infected macrophages observed at early infection was reversed after 48 h. Moreover, decreased adhesion was dependent on active penetration, since heat-killed parasites were unable to reproduce it. Expression of integrins alphaL, alpha4 and alpha5 chains was downmodulated early postinfection, but a progressive regain of expression was observed after 12 h of infection. Expression of beta2, alphav and alpha4 integrins by peritoneal macrophages at late infection was also gradually reestablished. The assessment of in vivo migration of infected macrophages labeled with the fluorescent dye 5-chloromethylfluorescein diacetate showed a 48-h delay in migration to cervical lymph nodes when compared to LPS pre-stimulated macrophages. Furthermore, cells that migrate to distal lymph nodes were loaded with live parasites. Taken together, these results provide insights about T. gondii escape from the host immune response, placing the macrophage as a "Trojan horse", contributing to parasite dissemination and access to immunoprivileged sites.     

Metallic tin-induced lymphadenopathy in rat strains and hybrids

Metallic tin powder, injected into Lewis rats obtained from three different sources, caused enlargement of the regional draining lymph nodes. The histopathology featured epithelioid cell granulomas around phagocytosed particles of tin and an intense hyperplasia of plasma cells. The same material injected into August rats enlarged the lymph nodes but the enlargement was caused by granulomas without a major concomitant plasma cell response. In most other strains, tin produced less lymph node enlargement and the plasma cell response was minimal. However, F1 hybrids of Lewis rats with either the August, Brown-Norway (BN), or Dark Agouti (DA) strains developed plasma cell hyperplasia similar to that seen in the parental Lewis strain. The response to tin was the same whether the tin was injected into the feet or into the peritoneal cavity. Thus, the lymph node response to metallic tin varied from a slight, banal response to insoluble foreign particles, to an exuberant granulomatous hyperplasia, to an intense plasmacellular hyperplasia, depending on the genetic characteristics of the subjects.     

The morphological characteristics of globulin production during the vaccinal process induced by tularemia and brucellosis vaccines

Significant data on the dynamics of globulin production in guinea pigs in the process of immunogenesis after the injection of Francisella tularensis vaccine strain or conjugated brucellosis vaccine have been obtained by means of immunofluorescence and the enzyme immunoassay. The number of globulin-producing cells in lymphoid organs (the spleen, regional and remote lymph nodes) differs, depending on the injected antigen. The relationship between the character of immunomorphological changes in lymphoid organs and the dynamics of the increase of antibody titers in the peripheral blood of the animals after their immunization with conjugated brucellosis vaccine and the injection of avirulent F. tularensis has been established.