Enhanced lymph node retention of subcutaneously injected IgG1-PEG2000-liposomes through pentameric IgM antibody-mediated vesicular aggregation

An efficient strategy for enhancing the lymph node deposition of rapidly drained liposomes from the interstitial injection site is described. Subcutaneously injected small-sized immuno-poly(ethyleneglycol)-liposomes (immuno-PEG-liposomes), containing 10 mol% mPEG350-phospholipid and 1 mol% PEG2000-phospholipid in their bilayer and where IgG1 is coupled to the distal end of PEG2000, not only drain rapidly from the interstitial spaces into the initial lymphatic system, but also accumulate efficiently among the lymph nodes draining the region when compared with non-PEG-bearing immunoliposomes where IgG is directly coupled to the phospholipid. Liposome deposition among the draining lymph nodes, however, was further enhanced dramatically following an adjacent subcutaneous injection of a pentameric IgM against the surface attached IgG molecules (IgM:IgG, 10:1) without compromising vesicle drainage from the interstitium. This is suggested to arise either as a result of formation of large immuno-aggregates within the lymphatic vessels with subsequent transport to and trapping among the regional lymph nodes and/or following IgM binding to Fc receptors of the lymph node sinus macrophages forming a platform for subsequent trapping of drained IgG-coupled liposomes. This lymph node targeting approach may be amenable for the design and surface engineering of any rapidly drained nanoparticulate system bearing peptides and proteins that can be aggregated with a desired monoclonal pentameric IgM.     

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.     

Lack of age-associated immune dysfunction in mucosal-associated lymph nodes

The magnitude of the immune response in old and young mice to trinitrophenylated bovine gamma-globulin was measured in various lymphatic sites at a cellular level using the plaque-forming cell assay. As we have previously shown, the number of splenic IgM, IgG, and IgA anti-TNP PFC progressively declined in aging C57BL/6J male mice. In addition, mice receiving antigen in the 4 footpads and the base of the tail exhibited similar decline in the number of PFC in the draining peripheral lymph nodes with increasing age. In contrast, mesenteric and mediastinal lymph node IgM, IgG, and IgA anti-TNP PFC response to TNP-BGG in complete Freund's adjuvant, i.p. or via gastric intubation, in old mice remained unimpaired compared with the number in younger mice. The data support the view that the mucosal-associated lymphoid system differs from the systemic system with regard to immune competence with age. Furthermore, the findings imply a site preference for a decline in immune function with aging.     

Subcutaneous Administration of Liposomes for Lymphatic Targeting

Abstract

Liposomes have received considerable interest for targeting to regional lymph nodes after s.c. administration. Detailed information on factors influencing lymphatic uptake and lymph node localization of s.c. administered liposomes is, however, not readily available. The present paper provides a short overview of the outcome of recently performed studies on factors potentially affecting lymphatic disposition of liposomes after s.c. injection into rats. An important factor influencing lymphatic disposition was found to be the anatomical site of injection. S.c. injection into the dorsal side of the foot or in the footpad resulted in relatively high uptake (about 40% of the injected dose (%ID)) of small liposomes (mean size about 0.10 μm) from the site of injection compared to uptake from the s.c. injection site at the flank from which uptake was low (< 5 %ID). Liposome size was found to be the most important liposome characteristic influencing lymphatic disposition of s.c. administered liposomes. Small, liposomes (mean size about 0.04 μm) were taken up by the lymphatic system to a relatively high extent (about 74 %ID) compared to large, non-sized liposomes which remained present almost completely at the site of injection. Small liposomes were less efficiently retained by regional lymph nodes than larger liposomes. Liposomal lipid composition did not influence lymphatic disposition significantly with one exception: lymph node localization of liposomes was substantially enhanced by inclusion of phosphatidylserine into the liposomal bilayers. Remarkably, lymphatic uptake and lymph node localization was only slightly affected by distearoylphosphatidylethanolamine-poly(ethyleneglycol) (DSPE-PEG¹) mediated steric stabilization of the liposome surface. Studies designed to elucidate the intranodal fate of liposomes confirmed that liposomes are mainly taken up by lymph node macrophages. Small liposomes may also be taken up by other cells such as endothelial cells. In addition, it was found that PEG-liposomes retained by lymph nodes are also taken up by lymph node macrophages.     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery. Failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, 99mTc, and an aqueous marker, 125I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped 125I-labelled poly(vinyl pyrrolidone) were labelled with 99mTc by the SnCl2 method. 99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than 125I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, 99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than 125I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60-70% of 125I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of 99mTc-radioactivity was co-eluted with the liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all 99mTc-radioactivity does not represent the 60-70% of intact liposomes present in lymph nodes. Using the aqueous marker 125I-labelled poly(vinyl pyrrolidone), the lymph node localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that 125I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier, where the authors used 99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became 'leaky' to low-molecular-weight drugs, for example, methotrexate (Mr 454) to a much greater extent than with a large-molecular-weight substance, 125I-labelled poly(vinyl pyrrolidone) (Mr 30 000-40 000), when incubated with rat lymph at 37 degrees C. Using the two markers 99mTc and 125I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining lambda-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with 99mTc rather than using the SnCl2 technique, or using other radionuclides as markers for gamma-scan imaging.     

Distinctions between rabbit lymph node cell populations responding to primary and secondary injections of keyhole limpet hemocyanin

Rabbits were injected once or twice into the hind foot pads with alum-precipitated keyhole limpet hemocyanin. At the height of the primary or secondary responses individual rabbits were sacrificed for the preparation of lymph node cell suspensions from the regional lymph nodes. These cells were employed for the in vitro study of antibody synthesis by the incorporation of ¹⁴C-leucine and the secretion of antibody by the time of appearance of radioactive antibody in the medium. The primary response cells rapidly synthesized and secreted IgM and IgG antibodies. The secondary response cells rapidly synthesized IgM and IgG antibodies, secreted IgG antibody promptly, but secreted the IgM antibody with a lag of six to ten hours. Microsomal fractions could not be prepared from the primary response cells, but were readily produced from the cells of the secondary response. The primary response cells contained mainly free ribosomes, those of the secondary response predominantly membrane-bound ribosomes. It was postulated that IgM antibody was not secreted until it was glyco-sylated in the Golgi apparatus and that the lag in secretion entailed the time for this rate-limiting step to occur.     

Interaction between phosphatidylserine and the phosphatidylserine receptor inhibits immune responses in vivo

Phosphatidylserine (PS) on apoptotic cells promotes their uptake and induces anti-inflammatory responses in phagocytes, including TGF-beta release. Little is known regarding the effects of PS on adaptive immune responses. We therefore investigated the effects of PS-containing liposomes on immune responses in mice in vivo. PS liposomes specifically inhibited responses to Ags as determined by decreased draining lymph node tissue mass, with reduced numbers of total leukocytes and Ag-specific CD4(+) T cells. There was also a decrease in formation and size of germinal centers in spleen and lymph nodes, accompanied by decreased levels of Ag-specific IgG in blood. Many of these effects were mimicked by an agonistic Ab-specific for the PS receptor. TGF-beta appears to play a critical role in this inhibition, as the inhibitory effects of PS were reversed by in vivo administration of anti-TGF-beta Ab. PS-containing liposomes did not appear to directly inhibit dendritic cell maturation in vitro in response to a variety of stimuli, nor did it prevent their migration to regional lymph nodes in vivo, suggesting that the inhibitory effects may have resulted from complicated interactions between tissue cells and dendritic cells, subsequently inhibiting their ability to productively activate T lymphocytes.     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, ^99mTc, and an aqueous marker, ¹²⁵I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped ¹²⁵I-labelled poly(vinyl pyrrolidone) were labelled with ^99mTc by the SnCl₂ method [2]. ^99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, ^99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than ¹²⁵I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60–70% of ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of ^99mTc-radioactivity was co-eluted with liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all ^99mTc-radioactivity does not represent the 60–70% of intact liposomes present in lymph nodes. Using the aqueous marker ¹²⁵I-labelled poly(vinyl pyrrolidone), the lymphnode localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that ¹²⁵I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier [2], where the authors used ^99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became ‘leaky’ to low-molecular-weight drugs, for example, methotrexate (M_r 454) to a much greater extent than with a large-molecular-weight substance, ¹²⁵I-labelled poly(vinyl pyrrolidone) (M_r 30 000–40 000), when incubated with rat lymph at 37°C. Using the two markers ^99mTc and ¹²⁵I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining λ-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with ^99mTc rather than using the SnCl₂ technique [2], or using other radionuclides as markers for γ-scan imaging.     

Neutrophil Recruitment to Lymph Nodes Limits Local Humoral Response to Staphylococcus aureus

Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF- β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.     

Sustained Liver Targeting and Improved Antiproliferative Effect of Doxorubicin Liposomes Modified with Galactosylated Lipid and PEG-Lipid

In this study, a cleavable PEG-lipid (methoxypolyethyleneglycol 2000-cholesteryl hemisuccinate, PEG₂₀₀₀-CHEMS) linked via ester bond and galactosylated lipid ((5-cholesten-3β-yl) 4-oxo-4-[2-(lactobionyl amido) ethylamido] butanoate, CHS-ED-LA) were used to modify doxorubicin (DOX) liposome. DOX was encapsulated into conventional liposomes (CL), galactosylated liposomes (modified with CHS-ED-LA, GalL), pegylated liposomes (modified with PEG₂₀₀₀-CHEMS, PEG-CL), and pegylated galactosylated liposomes (modified with CHS-ED-LA and PEG₂₀₀₀-CHEMS, PEG-GalL) using an ammonium sulfate gradient loading method and then intravenously injected to normal mice. Both PEG-GalL DOX and GalL DOX gave relatively high overall drug targeting efficiencies to liver ((T _e)_liver) and were mainly taken up by hepatocyte. However, PEG-GalL DOX showed unique “sustained targeting” characterized by slowed transfer of DOX to liver and reduced peak concentrations in the liver. The biodistribution and antitumor efficacy of various DOX preparations were studied in hepatocarcinoma 22 (H22) tumor-bearing mice. The inhibitory rate of PEG-GalL DOX to H22 tumors was up to 94%, significantly higher than that of PEG-CL DOX, GalL DOX, CL DOX, and free DOX, although the tumor distribution of DOX revealed no difference between PEG-GalL DOX and PEG-CL DOX. Meanwhile, the gradual increase in the liver DOX concentration due to the sustained uptake of PEG-GalL DOX formulations resulted in lower damage to liver. In conclusion, the present investigation indicated that double modification of liposomes with PEG₂₀₀₀-CHEMS, and CHS-ED-LA represents a potentially advantageous strategy in the therapy of liver cancers or other liver diseases.     

Regeneration of autotransplanted lymph node fragments

Summary In normal young minipigs thin slices of autologous mesenteric or superficial inguinal lymph nodes were implanted either in the greater omentum or subcutaneously in the groin region. The regeneration was studied histologically and connections between the afferent lymphatics and the regenerated tissue were checked. In the greater omentum, no regenerated lymph node tissue was found. In the inguinal region, lymphoid tissue with all the typical lymph node compartments was identified following antigenic stimulation in the draining area. Sinuses, germinal centres with a lymphatic corona, and a paracortex with typical high endothelial venules were seen. There was evidence of afferent lymphatics, e.g., macroscopically visible lymphatics, the occurrence of a subcutaneously injected dye, the effect of antigenic stimulation and a normal lymph node structure. Avascular transplants of autologous lymph node fragments regenerate subcutaneously, possibly providing a future technique for treating lymphoedema after radical excision or irradiation of lymph nodes.     

Distribution pattern of drained antigens and antibodies in the subcapsular sinus of the lymph node of the rat

Summary A recent study of lymph nodes of the rat showed that they are morphologically and physiologically compartmentalized. A compartment of a node includes a portion of subcapsular sinus into which the lymph, entering via the related afferent lymphatic opening, is filtered. The study also showed that colloidal carbon injected locally in a small dose becomes predominantly associated with the areas of the inner wall of the subcapsular sinus that cover the extrafollicular zone of the peripheral cortex. Little carbon is seen over the folliculo-nodules (follicles with a nodule or germinal center). The question arose as to whether drained natural substances, as antigens and antibodies, follow the same pattern of distribution in the subcapsular sinus as the carbon. Therefore, small doses of fluorescein isothyocyanate (FITC)-conjugated antigens were injected locally into normal rats whose own antibodies in the nodes were stained by immunofluorescence. The pattern of distribution of the antigens in the draining nodes was found to be the same as that of the carbon. Furthermore, the lymph-carried antibodies of the rats were found to follow the same pattern. The morphological basis for such a pattern is explained. The results are further discussed with regard to the probable normal entry route of lymph-carried antigens in the parenchyma of nodes.Supported by a grant from the Université de Montréal     

Sentinel lymph node biopsy in the management of cutaneous head and neck melanoma

Sentinel lymph node biopsy has revolutionized the surgical management of primary malignant melanoma. Most series on sentinel lymph node mapping have concentrated on extremity and truncal melanomas. The head and neck region has a rich and unpredictable lymphatic system. The use of sentinel lymph node mapping in the management of head and neck melanoma is evaluated. The authors conducted a retrospective review of patients treated for clinical stage I and stage II malignant melanoma of the head and neck with dynamic lymphoscintigraphy and gamma probe-guided sentinel lymph node biopsy. One hundred thirty-two patients (99 male patients and 33 female patients) were identified. The primary melanoma sites were the scalp (n = 54), ear (n = 14), face (n = 37), and neck (n = 27). Primary tumor staging was as follows: T1, 11; T2, 38; T3, 39; and T4, 44. Dynamic lymphoscintigraphy visualized sentinel lymph nodes in 128 patients (97 percent). In 71 cases (55 percent), a single draining nodal basin was identified, and in 57 cases there were multiple draining nodal basins (two basins, 55; three basins, two). Sentinel lymph nodes were successfully identified in 176 of 186 nodal basins (95 percent). Positive sentinel lymph nodes were identified in 22 patients (17.6 percent). Sentinel lymph node positivity by tumor staging was as follows: T2, 10.8 percent; T3, 19.4 percent; and T4, 26.8 percent. Completion lymphadenectomy revealed residual disease in seven patients (33.3 percent). Sentinel lymph node mapping for head and neck melanoma can be performed with results comparable to those of other anatomical sites.     

Splenic regulation of lymphocyte trapping in lymph nodes draining tumor grafts.

We measured the trapping of 51chromium-labeled splenic lymphocytes in the lymph nodes and spleens of tumor-inoculated mice at various intervals after inoculation. Both histocompatible and incompatible tumors were studied. Significant trapping occurred in the draining lymph nodes over the entire course of the experiments. The splenic trapping response commenced very early after transplantation and abated after a few days. The magnitude of the lymph node trapping response correlated with the magnitude of antigenic difference between tumor and host. Splenectomy before tumor inoculation had bidirectional effects; it caused a marked augmentation of the trapping response in the early period after tumor inoculation, which declined with time so that late after tumor inoculation, splenectomized animals trapped less well than intact controls. Tumor resection always led to a rapid fall in the trapping response in normal mice. However, tumor resection at the time of the peak trapping response of splenectomized mice produced no change. These results support the notion that lymphocyte trapping is a valid and useful monitor of the immune response to tumors. They also serve to reemphasize the important regulatory role of the spleen plays in the immune response to tumor-associated antigens by demonstrating a new parameter of splenic control; regulation of lymphocyte traffic in response to antigenic stimuli.     

Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1.

We have examined the generation of CTL immunity immediately after localized footpad infection with herpes simplex virus 1 (HSV-1) using three coordinated in vivo T cell tracking methodologies. Tetrameric MHC class I containing the immunodominant peptide from HSV-1 glycoprotein B (gB) showed that after infection the proportion of Ag-specific T cells peaked at day 5 within draining popliteal lymph nodes and 2 days later in the spleen. Preferential expression of the activation marker CD25 by tetramer-positive cells in draining popliteal nodes but not spleen suggested that gB-specific T cells were initially activated within the lymph node. In vivo cytotoxicity assays showed that Ag-specific effector cells were present within the draining lymph nodes as early as day 2 after infection, with a further 2-day lag before detection in the spleen. Consistent with the very early arming of effector CTL in the draining lymph node, adoptive transfer of CFSE-labeled gB-specific transgenic T cells showed that they had undergone one to four rounds of cell division by day 2 after infection. In contrast, proliferating T cells were first detected in appreciable numbers in the spleen on day 4, at which time they had undergone extensive cell division. These data demonstrate that HSV-1-specific T cells are rapidly activated and armed within draining lymph nodes shortly after localized HSV-1 infection. This is followed by their dissemination to other compartments such as the spleen, where they further proliferate in an Ag-independent fashion.     

MAdCAM-1 expressing sacral lymph node in the lymphotoxin beta-deficient mouse provides a site for immune generation following vaginal herpes simplex virus-2 infection

The members of the lymphotoxin (LT) family of molecules play a critical role in lymphoid organogenesis. Whereas LT alpha-deficient mice lack all lymph nodes and Peyer's patches, mice deficient in LT beta retain mesenteric lymph nodes and cervical lymph nodes, suggesting that an LT beta-independent pathway exists for the generation of mucosal lymph nodes. In this study, we describe the presence of a lymph node in LT beta-deficient mice responsible for draining the genital mucosa. In the majority of LT beta-deficient mice, a lymph node was found near the iliac artery, slightly misplaced from the site of the sacral lymph node in wild-type mice. The sacral lymph node of the LT beta-deficient mice, as well as that of the wild-type mice, expressed the mucosal addressin cell adhesion molecule-1 similar to the mesenteric lymph node. Following intravaginal infection with HSV type 2, activated dendritic cells capable of stimulating a Th1 response were found in this sacral lymph node. Furthermore, normal HSV-2-specific IgG responses were generated in the LT beta-deficient mice following intravaginal HSV-2 infection even in the absence of the spleen. Therefore, an LT beta-independent pathway exists for the development of a lymph node associated with the genital mucosa, and such a lymph node serves to generate potent immune responses against viral challenge.     

Dynamic imaging of lymphatic vessels and lymph nodes using a bimodal nanoparticulate contrast agent

BACKGROUND: Evaluation of lymphedema and lymph node metastasis in humans has relied primarily on invasive or radioactive modalities. While noninvasive technologies such as magnetic resonance imaging (MRI) offer the potential for true three-dimensional imaging of lymphatic structures, invasive modalities, such as optical fluorescence microscopy, provide higher resolution and clearer delineation of both lymph nodes and lymphatic vessels. Thus, contrast agents that image lymphatic vessels and lymph nodes by both fluorescence and MRI may further enhance our understanding of the structure and function of the lymphatic system. Recent applications of bimodal (fluorescence and MR) contrast agents in mice have not achieved clear visualization of lymphatic vessels and nodes. Here the authors describe the development of a nanoparticulate contrast agent that is taken up by lymphatic vessels to draining lymph nodes and detected by both modalities. METHODS: A unique nanoparticulate contrast agent composed of a polyamidoamine dendrimer core conjugated to paramagnetic contrast agents and fluorescent probes was synthesized. Anesthetized mice were injected with the nanoparticulates in the hind footpads and imaged by MR and fluorescence microscopy. High resolution MR and fluorescence images were obtained and compared to traditional techniques for lymphatic visualization using Evans blue dye. RESULTS: Lymph nodes and lymphatic vessels were clearly observed by both MRI and fluorescence microscopy using the bimodal nanoparticulate contrast agent. Characteristic tail-lymphatics were also visualized by both modalities. Contrast imaging yielded a higher resolution than the traditional method employing Evans blue dye. MR data correlated with fluorescence and Evans blue dye imaging. CONCLUSION: A bimodal nanoparticulate contrast agent facilitates the visualization of lymphatic vessels and lymph nodes by both fluorescence microscopy and MRI with strong correlation between the two modalities. This agent may translate to applications such as the assessment of malignancy and lymphedema in humans and the evaluation of lymphatic vessel function and morphology in animal models.     

Leishmania major: recruitment of Gr-1+ cells into draining lymph nodes during infection is important for early IL-12 and IFN gamma production

The production of interleukin-12 and interferon-γ is a key event for controlling leishmaniasis. Here, we tested the hypothesis that after murine infection with Leishmania major, cell migration into draining lymph nodes is crucial for early production of those cytokines. We showed that inflammatory cells carrying the marker of recently migrated cells, the Gr-1 antigen, including polymorphonuclear and mononuclear cells, migrate rapidly into the site of promastigote infection and, subsequently, into draining lymph nodes. Treatment with RB6-8C5 monoclonal antibody reduced local inflammation and migration of Gr-1+ cells into the draining lymph nodes. This reduction was associated with a decrease of interleukin-12 production by draining lymph node cells from BALB/c mice but not C57BL/6 mice. Additionally, interferon-γ was also reduced in both mouse strains after depletion of Gr-1+ cells, suggesting that these cells are important for early interleukin-12 and interferon-γ production. Our findings suggest that recently migrated myeloid cells, more than resident cells, are the major source of the early IL-12 production after L. major infection.     

Chitosan-DTPA-Gd舌间质磁共振淋巴造影显示兔颈淋巴的研究

目的：探讨磁共振间质淋巴造影剂Chitosan-DTPA-Gd经舌黏膜下注射后,显示颈淋巴的应用价值。方法：选取健康成年纯种新西兰大白兔12只,麻醉并仰卧位固定磁共振平扫,再于每只兔双侧舌缘中后交界处粘膜下各注射0.1 mL造影剂,注射部位按摩30秒,按摩后5、10、15、20、25、30、35、40 min行三维增强磁共振淋巴造影成像,测量增强前后不同时间颈部淋巴结的信号强度（SI）,计算相对应的信号强化率（E%）,采用SPSS软件进行数据的统计学分析。结果：Chitosan-DTPA-Gd于兔双侧舌缘粘膜下注射后很快吸收进入颈部淋巴引流区,淋巴结及淋巴管明显、均匀的强化,血管无显影。颈部淋巴结于注射后15 min信号强度达到峰值,并可保持一段时间,25 min后信号强化率开始明显降低,40分钟后淋巴系统显影与周围组织无差别。结论：间质磁共振淋巴造影剂Chitosan-DTPA-Gd用量小,强化效应明显,能够有效的显示颈部淋巴结形态及淋巴管走形。     

A specific vaccine effective against stage I and stage II malignant disease in guinea pigs effect of variations in preparations and storage

Summary Guinea pigs, each with an established, syngeneic dermal line 10 tumor and lymph node metastases, were immunized by intradermal injection of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG. Immunization eradicated 7- or 10-day-old dermal tumors (about 10 or 12 mm in diameter, respectively) and prevented growth of microscopic lymph node metastases. Fourteen-day-old dermal tumors (about 15 mm in diameter) were not rejected by immunization.Guinea pigs with stage II disease (21-day-old dermal tumors and palpable metastases in the first draining lymph node) were treated by excision of the dermal tumor and the first draining lymph node, and by specific immunization. This treatment eliminated tumor cells remaining in the second draining lymph nodes. The surgical treatment alone was not curative, palpable metastases in the second draining lymph nodes progressed and the animals died (some with visible lung metastases).Emulsions containing killed BCG were good adjuvants even after prolonged storage at 4° C, but lost most of their adjuvant activity after autoclaving or freezing.