Lymph Nodes Of Rats Research Articles

Optimizing retention of multimodal imaging nanostructures in sentinel lymph nodes by nanoscale size tailoring

This study investigates the retention of different sized ultra-small superparamagnetic iron oxide nanoparticles (USPIOs) in lymph nodes of healthy rats, after subcutaneous injection. Three distinct sizes (15, 27 and 58 nm) of USPIOs were synthesized by only varying the thickness of the polymer coating surrounding the 10 nm cores. Particles were injected on the dorsal side of the hind paw of rats and the uptake in the popliteal, inguinal and iliac lymph nodes was monitored. The data reveal that the 15 nm particle accumulates more rapidly and to a higher amount in the first lymph node than the two larger particles. A clear contrast between the first and second lymph nodes could be detected indicating that even the rather small difference in particle size (15-58 nm) tested has significant effects on the retention of USPIOs in the lymph nodes. From the Clinical EditorFrom the Clinical Editor: In this study, the size-dependence of USPIO particles is studied from the standpoint of their accumulation characteristics in lymph nodes. The authors conclude that the smaller particles accumulated faster and at a higher concentration than the two larger sizes studied.

Diubiquitin (Ubd) is a susceptibility gene for virus-triggered autoimmune diabetes in rats

Genetic studies of type 1 diabetes (T1D) have been advanced by comparative analysis of multiple susceptible and resistant rat strains with a permissive class II MHC haplotype, RT1u. LEW.1WR1 (but not resistant LEW.1W or WF) rats are susceptible to T1D induced by a TLR3 agonist (poly I:C) followed by infection with parvovirus. We have mapped genetic loci for virus-induced T1D susceptibility, identifying a major susceptibility locus (Iddm37) near the MHC. Iddm37 homologues on mouse and human chromosomes are also diabetes-linked. We report that a major effect gene within Iddm37 is diubiquitin (Ubd). Gene expression profiling of pancreatic lymph nodes in susceptible and resistant rats during disease induction showed differences in Ubd transcript abundance. The LEW.1WR1 Ubd promoter allele leads to higher inducible levels of UBD than that of LEW.1W or WF. Using zinc finger nucleases (ZFN), we deleted a segment of the LEW.1WR1 Ubd gene and eliminated its expression. UBD-deficient rats show substantially reduced diabetes after viral infection. Complementation studies show that there may be another diabetes gene in addition to Ubd in the Iddm37 interval. These data prove that Ubd is a diabetes susceptibility gene, providing insight into the interplay of multiple genes and environmental factors in T1D susceptibility.

Prevention of Late PJI

The definition of late PJI is variable in literature. Majority of the members of the consensus felt that any infection occurring after one year should be considered as late. Coventry defined stages of PJI, where Stage I is an acute infection that occurred within 3 months of the index procedure, Stage II is a delayed infection that occurred between 3 months and 2 years after the index procedure where there was no pain-free interval, and Stage III is a hematogenous infection where there is a pain-free stage [1]. Garvin and Hanssen defined a late chronic PJI as one that occurred 4 weeks after the index procedure with an insidious clinical onset [2]. McPherson et al defined a chronic infection as one that had symptoms for 4 weeks or longer [3]. In Sweden, a late PJI is defined as one that occurs 2 years after the index procedure. Due to the huge variation in time frames, we did not find consensus in defining a timeframe for a late PJI. However, we classified late PJI as late hematogenous PJI, where there was an asymptomatic period followed by clinical and/or radiographical signs of infection. The workgroup feels that late PJI arises as a result of bacteremia at a later stage [4] and should be distinguished with infections arising as a result of intraoperative contamination. Risk factors for late PJI are similar to those described for PJI in Workgroup 1 (Please see Question 1, Workgroup 1).

The influence of Th1/Th2 and CD4+ regulatory t cells of mesenteric lymph nodes on systemic lipopolysaccharide.

Our aims were to study the influence of the mesenteric lymph nodes (MLN) of rats on systemic lipopolysaccharide and to identify the factor that affects the intestinal endotoxin translocation. Ninety-six male Wistar rats were randomly divided into a sham-operation group (S group) and a cecal ligation and perforation group (CLP group). Twenty-four hours after modeling, we tested the Th1/Th2 ratio and percentage of CD4+CD25+Foxp3+ Treg in the MLN. At the same time, the lipopolysaccharide (LPS) in the abdominal aortic blood was detected. In the CLP group, the Th1/Th2 ratio was obviously lower than in the S group. Otherwise, the percentage of CD4+CD25+Foxp3+ Treg of the CLP group was significantly higher than the S group. In the abdominal aortic blood, the LPS level of the CLP group was also higher than in the S group. Through correlation analysis, we found that the level of LPS was positively correlated with the percentage of CD4+CD25+Foxp3+ Treg, and negatively correlated with the Th1/Th2 ratio. This model reveals that the immune suppression of the MLN might affect the level of LPS in the abdominal aortic blood, which might play a certain role in affecting the endotoxin translocation.

A New Vascularized Cervical Lymph Node Transplantation Model

Vascularized lymph node transfer is of high interest for the treatment of lymphedema. Currently, there are few experimental small animal models of vascularized lymph node transplantation. In this article, our aim was to describe a new vascularized cervical lymph node transplantation model in rats. Ten male Sprague-Dawley rats weighing 200 to 250 g were used in this study. The anatomic features of the neck lymph nodes in rats were explored. Anatomic neck dissections were performed, and lymph node flaps were harvested. The common carotid artery and the jugular vein were used as the vascular pedicles of the lymph node flap. Methylene blue dye was injected into the arterial pedicle. Lymph nodes were identified, and their structure was confirmed by histological evaluation. Laser-assisted indocyanine green angiography was used to confirm perfusion of the lymph node flap. An adequate perfusion was observed in the lymph node flap. The dye disseminated evenly within the lymph nodes, indicating that the flap had a well-established vascular network and an adequate blood supply. Macroscopically, perfusion of 5 to 6 lymph nodes was observed. Histological examination of tissue samples confirmed well-defined lymph nodes. After indocyanine green administration, fluorescence was observed throughout the lymph node flap and within the venous pedicle of the flap. To the best of our knowledge, this is the first report describing vascularized lymph node flap in the head and neck region of a rat. Our lymph node flap preparation technique confirmed the presence of 5 to 6 lymph nodes within the flap. The presented vascularized lymph node flap can be applied to transplantation studies, lymphedema studies, and to studies on immunological mechanism of tolerance and rejection.

Microbiome Composition by Pyrosequencing in Mesenteric Lymph Nodes of Rats with CCl4-Induced Cirrhosis

Background: The cross talk between the gut microbiota and the immune system, which is essential to maintain homeostasis, takes place at the intestinal lymphoid tissue such as the mesenteric lymph nodes (MLNs). Here, we investigated the presence of bacterial DNA in MLNs of control and cirrhotic rats and its relationship with inflammatory responses. Methods: The MLN microbiome of cirrhotic rats with ascites, which was induced by carbon tetrachloride (CCl₄), was compared to that of control rats using quantitative real-time PCR and pyrosequencing of the 16S rRNA gene. Cytokines in blood samples were assessed by ELISA. Results: Unexpectedly, sequence analysis revealed a high microbial diversity in the MLNs of both control and cirrhotic rats with Proteobacteria as one of the most dominant phylum. CCl₄-induced liver injury was not associated with a change in bacterial load, but it was linked to a decrease in microbial diversity (p < 0.05) and alterations in the microbial community in MLNs. A high proportion of Bifidobacterium animalis was also positively correlated with elevated interleukin-10 expression (p = 0.002, false discovery rate = 0.03, r = 0.94). Conclusions: For the first time, the high microbial diversity observed in MLNs of both controls and CCl₄-induced cirrhotic rats provides evidence that bacterial translocation is more than a mere dichotomic phenomenon.

Accumulation and biodistribution of gold nanoparticles in the mesenteric lymph nodes at oral administration

The aim: to conduct analysis of the accumulation and biodistribution of gold nanoparticles in the structural and functional areas of mesenteric lymph nodes of white rats conducted with regard to the size and duration of the oral administration. The methods of dark field microscopy with Leica DM 2500 microscope were used to assess biodistribution of gold nanoparticle. The histochemical reaction of silver amplification with silver lactate by Danscher was used to confirm its identification. Results: It was found that the gold nanoparticles of 1-3 nm were not detected by these methods in any of the areas of the lymph nodes regardless of the duration of administration, gold nanoparticles of sizes 15 and 50 nm were detected in the form of clusters in the cytoplasm of macrophages and lymphocytes mainly in the mantle zone of lymphoid follicles and brain strands. Conclusion: It was established that the gold nanoparticles of sizes 15 and 50 nm were detected in the form of clusters in the cytoplasm of macrophages and lymphocytes

The developing schistosome worms elicit distinct immune responses in different tissue regions

Schistosome parasites follow a complex migration path through various tissues, changing their antigenic profile as they develop. A thorough understanding of the antibody response in each tissue region could help unravel the complex immunology of these developing parasites and aid vaccine design. Here we used a novel strategy for analysing the local antibody responses induced by Schistosoma japonicum infection at each site of infection. Cells from rat lymph nodes draining the sites of larval migration (the skin and lungs), the liver-lymph nodes where adults reside and the spleens were cultured to allow the in vivo-induced antibody-secreting cells to release antibody into the media. The amount and isotype of antibodies secreted in the supernatants differed significantly in the different lymph nodes and spleen, corresponding with the migration path of the schistosome worms. In addition, there were significant differences in binding specificity, as determined by surface labelling, western blots and by screening a glycan array. Through capturing the local antibody response, this study has revealed dramatic differences in the quality and specificity of the immune response at different tissue sites, and highlighted the existence of stage-specific protein and carbohydrate antigens. This will provide a valuable tool for the isolation of novel vaccine targets against the larval stages of schistosomes.

Lymph Flow and Contractile Activity of Mesenteric Lymph Nodes in Rats with Toxic Hepatitis Effects of Antioxidants

We studied contractile function of isolated mesenteric lymph nodes in rats with toxic hepatitis. We observed suppression of spontaneous and stimulated contractile activity of mesenteric lymph nodes and changes in biochemical composition of the lymph. We propose a method of correction of these dysfunctions with antioxidant α-tocopherol and Selen-Active.

Saquinavir-NO inhibits S6 kinase activity, impairs secretion of the encephalytogenic cytokines interleukin-17 and interferon-gamma and ameliorates experimental autoimmune encephalomyelitis

NO-hybridization of the HIV protease inhibitor Saquinavir generates a new chemical entity named Saq-NO, that retains the anti-viral activity and exerts lower toxicity. We show that Saq-NO inhibited the generation of various cytokines in ConA-stimulated unfractionated murine spleen cells and rat lymph nodes stimulated with ConA as well as in purified CD4+ T cells in vitro and reduced the circulating levels of cytokines in mice challenged with anti-CD3 antibody. Furthermore, Saq-NO reduced IL-17 and IFN-γ production in myelin basic protein (MBP)-specific cells isolated from rats immunized with MBP. These findings translated well into the in vivo setting as Saq-NO ameliorated the course of the disease in two preclinical models of multiple sclerosis. Our results demonstrate that Saq-NO exerts immunomodulatory effects that warrant studies on its application in autoimmune diseases.

Next-Generation Sequencing Identifies MicroRNAs that Associate with Pathogenic Autoimmune Neuroinflammation in Rats

MicroRNAs (miRNAs) are known to regulate most biological processes and have been found dysregulated in a variety of diseases, including multiple sclerosis (MS). In this study, we characterized miRNAs that associate with susceptibility to develop experimental autoimmune encephalomyelitis (EAE) in rats, a well-established animal model of MS. Using Illumina next-generation sequencing, we detected 544 miRNAs in the lymph nodes of EAE-susceptible Dark Agouti and EAE-resistant Piebald Virol Glaxo rats during immune activation. Forty-three miRNAs were found differentially expressed between the two strains, with 81% (35 out of 43) showing higher expression in the susceptible strain. Only 33% of tested miRNAs displayed differential expression in naive lymph nodes, suggesting that a majority of regulated miRNAs are EAE dependent. Further investigation of a selected six miRNAs indicates differences in cellular source and kinetics of expression. Several of the miRNAs, including miR-146a, miR-21, miR-181a, miR-223, and let-7, have previously been implicated in immune system regulation. Moreover, 77% (33 out of 43) of the miRNAs were associated with MS and other autoimmune diseases. Target genes likely regulated by the miRNAs were identified using computational predictions combined with whole-genome expression data. Differentially expressed miRNAs and their targets involve functions important for MS and EAE, such as immune cell migration through targeting genes like Cxcr3 and cellular maintenance and signaling by regulation of Prkcd and Stat1. In addition, we demonstrated that these three genes are direct targets of miR-181a. Our study highlights the impact of multiple miRNAs, displaying diverse kinetics and cellular sources, on development of pathogenic autoimmune inflammation.

Prevention of Mammary Tumor Development through Neuroimmunomodulation in the Spleen and Lymph Nodes of Old Female Sprague-Dawley Rats by L-Deprenyl

Background: Development of mammary tumors is an age-associated phenomenon that is likely due to deficits in the neuroendocrine-immune interactions. Previously, we demonstrated that <smlcap>L</smlcap>-deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, can enhance immune responses and restore noradrenergic (NA) innervation in the spleens of rats with carcinogen-induced and spontaneously developing mammary tumors. Objectives: To investigate whether (1) treatment of early middle-aged female rats would prevent the spontaneous development of mammary tumors accompanied by restoration of immunity in the spleen and draining lymph nodes (DLN) and sympathetic NA innervation in the spleen and (2) deprenyl can influence the proliferation of estrogen receptor (ER)-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 and Hs 578T) human breast cancer cells. Methods: Early middle-aged (8- to 9-month-old) female Sprague-Dawley rats were treated with 0, 1.0 or 2.5 mg of deprenyl/kg body weight (BW) daily i.p. for 12 months. Cells of ER-positive (ER+) and ER-negative (ER-) human breast cancer cell lines were incubated with media or 10^-3 to 10^-8<smlcap>M</smlcap> deprenyl for 1, 2, 4 or 6 days to examine the proliferation of cells. Results: Tumor incidence increased in saline-treated old female rats, while deprenyl treatment significantly reduced the incidence of mammary tumors in these rats. Saline-treated tumor-bearing rats exhibited reduced splenic NA innervation and norepinephrine (NE) content, splenic interleukin (IL)-2 and interferon (IFN)-γ levels and NK cell activity as well as DLN IL-2 and IFN-γ levels compared to young female rats without tumors. In contrast, treatment with 2.5 mg/kg of deprenyl enhanced IL-2 and IFN-γ production in both the spleen and DLN as well as splenic natural killer (NK) cell activity. Deprenyl treatment also increased concanavalin A (Con A)-induced proliferation of T lymphocytes in the DLN. Deprenyl-induced changes in immune responses were accompanied by enhanced NA innervation and NE content in the spleen. In vitro incubation of various concentrations of deprenyl with ER+ human breast cancer cell lines partly inhibited the proliferation of cells, while it had no effect on the ER- breast cancer cells. Conclusions: These results suggest that (1) development of mammary tumors is mediated through the loss of immunity and sympathetic NA nerve fibers accompanied by reduced NE levels in the spleen, (2) the prevention of mammary tumor development by deprenyl may involve the reversal of the tumor-associated decline in sympathetic NA activity and cell-mediated immune responses in the spleen and DLN and (3) the antitumor effects of deprenyl may be partially mediated through ER-dependent intracellular signaling pathways.

Metabolites of PPI-2458, a Selective, Irreversible Inhibitor of Methionine Aminopeptidase-2: Structure Determination and In Vivo Activity

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, [(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only ∼10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ([(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

Synthesis of ligand-functionalized water-soluble [18F]YF3 nanoparticles for PET imaging

We report a simple, efficient synthesis of novel (18)F-labeled imaging agents based on YF3 nanoparticles. Targeting ligands and antitumor drug molecules can be introduced onto the YF3 nanoparticles in a one-pot synthesis. The (18)F-labeling reaction proceeds in aqueous solutions at room temperature with excellent radiolabeling yields (>80%) in a very short time (5-10 min). (18)F-labeled YF3 nanoparticles displayed high stability in mouse and human serum, and their application for mapping lymph nodes in live rats after local injection has also been demonstrated.

Effect of surgically implanted root-end filling materials on the structure of draining lymph nodes of male albino rats

Introduction The success of endodontic surgery depends on the histocompatibility of the root-end filling material. Applications of nanotechnology improve their performance. Aim of the work Aim of the work was to compare the effect of a mineral trioxide aggregate (MTA)and a bioceramic nanoparticulate bioaggregate (BNB) on the histological structure of draining axillary lymph nodes of adult male albino rats after their surgical implantation into the skin. Materials and methods Forty adult male albino rats were divided into control and experimental groups.The latter was subdivided into MTA and BNB surgically implanted subgroups. After 7 days, the rats were sacrificed. Paraffin sections from both the proximal part of the dorsal skin and draining axillary lymph nodes were processed for H&E staining.Lymph node sections were further subjected to silver reticulin, Verhoeff’s Van Gieson stains as well as kappa light chains. Quantitative assessments and statistical analysis of the results were carried out. Results There was an increase in mononuclear inflammatory cells infiltrating the skin in the MTA subgroup. Lymph nodes of the MTA subgroup showed a marked decrease in the lymphocyte content of lymphatic nodules, wide lymph sinuses, multinucleate giant cells, and many macrophages. In the BNB-treated subgroup, lymphatic nodules had wide corona and small germinal centers. Reticular and collagen fibers were increased in the MTA subgroup. Kappa light chains’ immunoreactions were strong positive in MTA and mild positive in BNB subgroups. A highly significant increase in the mean area% of all fibers and kappa light chain immunoexpression of lymph nodes of the MTA subgroup were observed. Conclusion and recommendation MTA had less biocompatibility. BNB showed limited signs of acute inflammation. BNB is an up-to-date alternative to the currently used root-end filling materials. The chronic effects caused by BNB may require further study.

Genotoxicity of cadmium in rat lung cells assessed by an alkaline comet assay and the possible protective role of selenium

Introduction The success of endodontic surgery depends on the histocompatibility of the root-end filling material. Applications of nanotechnology improve their performance. Aim of the work Aim of the work was to compare the effect of a mineral trioxide aggregate (MTA)and a bioceramic nanoparticulate bioaggregate (BNB) on the histological structure of draining axillary lymph nodes of adult male albino rats after their surgical implantation into the skin. Materials and methods Forty adult male albino rats were divided into control and experimental groups.The latter was subdivided into MTA and BNB surgically implanted subgroups. After 7 days, the rats were sacrificed. Paraffin sections from both the proximal part of the dorsal skin and draining axillary lymph nodes were processed for H&E staining.Lymph node sections were further subjected to silver reticulin, Verhoeff’s Van Gieson stains as well as kappa light chains. Quantitative assessments and statistical analysis of the results were carried out. Results There was an increase in mononuclear inflammatory cells infiltrating the skin in the MTA subgroup. Lymph nodes of the MTA subgroup showed a marked decrease in the lymphocyte content of lymphatic nodules, wide lymph sinuses, multinucleate giant cells, and many macrophages. In the BNB-treated subgroup, lymphatic nodules had wide corona and small germinal centers. Reticular and collagen fibers were increased in the MTA subgroup. Kappa light chains’ immunoreactions were strong positive in MTA and mild positive in BNB subgroups. A highly significant increase in the mean area% of all fibers and kappa light chain immunoexpression of lymph nodes of the MTA subgroup were observed. Conclusion and recommendation MTA had less biocompatibility. BNB showed limited signs of acute inflammation. BNB is an up-to-date alternative to the currently used root-end filling materials. The chronic effects caused by BNB may require further study.

L-Deprenyl Reverses Age-Associated Decline in Splenic Norepinephrine, Interleukin-2 and Interferon-γ Production in Old Female F344 Rats

Aging in female rats is associated with cessation of reproductive cycles, development of mammary cancer, and increased incidence of autoimmune diseases. Previously, we demonstrated an age-related decline in sympathetic noradrenergic (NA) innervation in the spleen and lymph nodes of female F344 rats accompanied by significantly reduced natural killer cell activity, interleukin (IL)-2 and interferon (IFN)-γ production, and T- and B-cell proliferation, suggesting possible links between sympathetic activity and immunosenescence. Objectives: The aim of this study is to investigate the effects of <smlcap>l</smlcap>-(–)-deprenyl, a monoamine oxidase-B inhibitor, on the sympathetic nervous system and cell-mediated immune responses in old female rats. Methods: Low doses of <smlcap>l</smlcap>-deprenyl (0.25 or 1.0 mg/kg body weight, BW) were administered intraperitoneally to 19- to 21-month-old female F344 rats for 8 weeks. To assess the stereoselectivity of the effects of deprenyl on splenic sympathetic activity and immune responses, the <smlcap>d</smlcap>-enantiomer (<smlcap>d</smlcap>-(+)-deprenyl; 1.0 mg/kg BW) was also included in the studies. Norepinephrine (NE) concentration and content, and mitogen-induced T-cell proliferation and cytokine production were assessed in the splenocytes after deprenyl treatment. Results: Treatment with <smlcap>l</smlcap>-deprenyl reversed the age-related decrease in NE concentration and content and IFN-γ production, and increased IL-2 production in the spleen while <smlcap>d</smlcap>-deprenyl did not affect the age-associated reduction in splenic NE levels or cytokine production. Conclusions: These findings demonstrate that <smlcap>l</smlcap>-deprenyl exerts neurorestorative and immunostimulatory effects on the sympathetic nervous system and cell-mediated immune responses during aging and provides evidence for a causal relationship between some aspects of immunosenescence and the age-related decline in sympathetic nerves in the spleens of female F344 rats.

In Vitro Expansion of Regulatory T-Cells in the Rat

Introduction: CD4+CD25highFoxP3+ regulatory T-cells (Tregs) are of central importance in the immunological adaptation process following transplantation. Transfusing Tregs in order to induce tolerance represents a new therapeutical attempt to reduce or completely replace immunosuppressive treatment after transplantation. Thus, transplantation experiments in animal models (e.g. rat) currently are conducted to optimize this approach. However, the existing human protocols for in vitro expansion of Tregs cannot be applied directly transferred due to species specific differences. The aim of this study hence was the establishment of a method to expand rat regulatory T-cells from very small samples which would enable subsequent in vivo transplantation experiments - even using autologous cells. Methods: CD4+CD25high Tregs were isolated from lymph nodes of Lewis rats via cell sorting. Tregs were then cultured in RPMI-based medium in the presence of mAb to CD3 and CD28. Under the influence of different additional stimuli such as supplementation of interleukin-2 (IL-2), proliferation rates and viability of the cells were investigated. The influence of the applied culture conditions on the suppressive activity and phenotype of expanded Tregs was examined. Results: Following 7/14 days of expansion, the addition of IL-2 (n=15) led to approximately 3.3±3.1/3.4±2.2-fold expansion of Tregs, retaining a viability of 74.1±13.9%/32.2±8.9%, respectively. When replacing IL-2 with supernatant of a ConA-stimulated splenocyte culture (n=13), Tregs showed increased expansion rates of 11.4±8.8/8.3±4.2-fold (day 7/14) but at the same time a decrease in viability was evident (65.2±14.8% /24.7±10.4). Therefore the combination of both stimuli was tested as a third group (n=9), thus enhancing proliferation (9.4±3.5-fold) and cell viability (77.5±11.0%) which, in addition, remained stable until culture day 14 (9.8±7.4-fold expansion, 30.1±12.0% viability). The use of ConA-supernatant led to decreased FoxP3-expression as measured on day 7 (IL-2: 73.5±9.4%, ConA-supernatant: 27.6±2.2%, alternating stimuli: 43.4±22.5%) but interestingly, Tregs of all groups were functional and efficiently suppressed expansion of responder T-cells (IL-2: 94.1%, ConA-supernatant: 84.0%, alternating stimuli: 93.2%). Conclusion: The expansion of regulatory T-cells in the rat is significantly more difficult than in humans. However, the newly established protocol (IL-2 + supernatant of ConA-stimulated splenocytes) allows the efficient expansion of Tregs which maintain their strong suppressive functionality despite the partial loss of FoxP3-expression.

Crosstalk between Immune Cells and Mesenchymal Stromal Cells in a 3D Bioreactor System

Introduction Mesenchymal stromal cells (MSC), known for their high immune modulatory capacity are promising tools for several cell-based therapies. To better mimic the in vivo situation of MSC interactions with immune cells, we applied an artificial lymph node (ALN)-bioreactor culture system combining a miniaturized perfusion bioreactor with a 3D matrix-based cell culture of immune competent cells forming micro-organoids. Methods Rat lymph node cells and allogeneic bone marrow-derived MSCs were seeded in a 20:1 ratio within the agarose matrix of the ALN-reactor. Lymphocytes were pre-incubated with Concanavalin A (ConA) and then co-cultured with MSC in the matrix with additional ConA in the perfusing medium. Live/dead staining showed survival of the co-cultures during the 8-day ALN-reactor run. Paraffin sections of bioreactor matrices were analyzed by proliferating cell nuclear antigen (PCNA)-specific staining to determine MSC proliferation. Immune modulatory capacity was defined by daily analysis of cytokine secretion profiles (TNFα, IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p40/p70, GM-CSF). Results Cytokine peak secretion at day 2 was significantly inhibited by MSCs for TNFα (96.8 ± 4.8%) and IFNγ (88.7 ± 12.0%) in 3D co-cultures. In contrast, other cytokines (IL-1, IL-6, IL-12) were induced. Furthermore, we detected a significantly higher (58.8%) fraction of proliferating MSCs in the presence of immune cells compared to control bioreactors loaded with MSCs only. Conclusions In the future, this system might be an excellent tool to investigate the mechanisms of MSC-mediated immune modulation during simulated in vivo conditions.

Expression of HLA-B27 causes loss of migratory dendritic cells in a rat model of spondylarthritis.

In rats transgenic for human HLA-B27 and β(2) -microglobulin (B27-transgenic rats), colitis and peripheral inflammation develop spontaneously. Therefore, B27-transgenic rats provide a model of spondylarthritis. Because inflammation in these rats requires CD4+ T lymphocytes and involves intestinal pathology, we hypothesized that dendritic cells (DCs) that migrate from the intestine and control CD4+ T cell differentiation would be aberrant in B27-transgenic rats. Migrating intestinal lymph DCs were collected via thoracic duct cannulation from B27-transgenic and control (HLA-B7-transgenic or nontransgenic) rats. The phenotypes of these DCs and of mesenteric lymph node DCs were assessed by flow cytometry. The ability of DCs to differentiate from bone marrow precursors in vitro was also assessed. Lymph DCs showed increased activation and, strikingly, lacked the specific DC population that is important for maintaining tolerance to self-antigens. This population of DCs was also depleted from the mesenteric lymph nodes of B27-transgenic rats. Furthermore, in vitro culture of DCs from bone marrow precursors revealed a defect in the ability of B27-transgenic rats to produce DCs of the migratory phenotype, although the DCs that were generated induced enhanced interleukin-17 (IL-17) production from naive CD4+ T cells. We describe 2 different mechanisms by which HLA-B27 may contribute to inflammatory disease: increased apoptotic death of B27-transgenic DCs that normally function to maintain immunologic tolerance and enhanced IL-17 production from CD4+ T cells stimulated by the surviving B27-transgenic DCs.