Development of a Porcine Lymph Drainage Model to Study Particle Migration.

Background: Intra-articular wear particulate migration from the knee joint has been studied in various animal models as well as postmortem in patients who received total knee joint replacement. However, there still exists a need for a simple, yet analogous animal model for tracking the migration of wear debris from the knee joint, especially through the draining lymph nodes. Methods and Results: To fill this need, a proof-of-concept porcine model was developed for particle migration from the knee joint into the surrounding lymphatic system. Vitreous carbon particles were deposited both intra-articularly and extracapsularly in a bilateral manner to the hind limbs in pigs (n = 6). The regional/draining lymph nodes were qualitatively assessed weekly by a veterinarian by manual palpation to detect any enlargement or change in consistency when compared to the initial assessment before the surgical procedure. At 6 weeks, the draining lymph nodes were harvested and processed for histology. Microscopic evaluation showed carbon particle migration from the knee into 100% of the iliac lymph nodes, 50% of the inguinal lymph nodes, and 0% of the popliteal lymph nodes. Discussion: Overall, this study established a needed animal model for evaluating carbon particle migration to the draining lymph nodes from the knee joint.

Identification of Patients With Documented Pathologic Complete Response in the Breast After Neoadjuvant Chemotherapy for Omission of Axillary Surgery.

Importance: A pathologic complete response (pCR; no invasive or in situ cancer) occurs in 40% to 50% of patients with HER2-positive (HER2+) and triple-negative (TN) breast cancer. The need for surgery if percutaneous biopsy of the breast after neoadjuvant chemotherapy (NCT) indicates pCR in the breast (hereinafter referred to as breast pCR) has been questioned, and appropriate management of the axilla in such patients is unknown. Objective: To identify patients among exceptional responders to NCT with a low risk for axillary metastases when breast pCR is documented who may be eligible for an omission of surgery clinical trial design. Design, Setting, and Participants: This prospective cohort study at a single-institution academic national comprehensive cancer center included 527 consecutive patients with HER2+/TN (T1/T2 and N0/N1) cancer treated with NCT followed by standard breast and nodal surgery from January 1, 2010, through December 31, 2014. Main Outcomes and Measures: Patients who achieved a breast pCR were compared with patients who did not based on subtype, initial ultrasonographic findings, and documented pathologic nodal status. Incidence of positive findings for nodal disease on final pathologic review was calculated for patients with and without pCR and compared using relative risk ratios with 95% CIs. Results: The analysis included 527 patients (median age, 51 [range, 23-84] years). Among 290 patients with initial nodal ultrasonography showing N0 disease, 116 (40.4%) had a breast pCR and 100% had no evidence of axillary lymph node metastases after NCT. Among 237 patients with initial biopsy-proved N1 disease, 69 of 77 (89.6%) with and 68 of 160 (42.5%) without a breast pCR had no evidence of residual nodal disease (P < .01). Patients without a breast pCR had a relative risk for positive nodal metastases of 7.4 (95% CI, 3.7-14.8; P < .001) compared with those with a breast pCR. Conclusions and Relevance: Breast pCR is highly correlated with nodal status after NCT, and the risk for missing nodal metastases without axillary surgery in this cohort is extremely low. These data provide the fundamental basis and rationale for management of the axilla in clinical trials of omission of cancer surgery when image-guided biopsy indicates a breast pCR.

Primo Vessel Stressed by Lipopolysaccharide in Rabbits.

For tracking the primo vascular system, we observed the primo vessels in vivo in situ using the lipopolysaccharide (LPS) response in the lymphatic vessels of a rabbit. Injection of LPS (200 µg/kg) into the lymph nodes resulted in greatly stained primo vessels, which were swollen in some cases. We were able to obtain comparative images through alcian blue and diaminobenzidine staining, which clearly showed different morphologies of the primo vessels. The mechanism causing the response of the primo vessels to the injected LPS is still unclear; however, these results might be a first attempt at giving an explanation of the function of the primo vascular system and identifying the changes in the structure and function of the primo vascular system in response to an external stimulus such as an injection of LPS.

Involvement of α-adrenoceptors to the implementation of the contractile effects in the capsule of mesenteric lymph nodes in response to electrostimulation.

We studied changes in the contractile function of smooth muscle cells in bovine mesenteric lymph node capsule caused by electrical stimulation of nerve fibers in vitro. It was found that electrostimulation increased tonic tension and frequency of smooth muscle contractions in the node capsule. Tetrodotoxin prevented the stimulatory effect of electrical stimulation on the smooth muscle cells. Phentolamine, prazosin, and yohimbine significantly reduced the capsule response to electrical stimulation and norepinephrine application. It was concluded that excitation of nerve fibers in the capsule of bovine mesenteric lymph nodes upon electrical stimulation is realized, at least in part, via activation of α1-adrenoceptors and, to a lesser extent, via α2-adrenoceptors located on the membrane of smooth muscle cells.

Significance of para-esophageal lymph nodes in food or aeroallergen-induced iNKT cell-mediated experimental eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder driven by food hypersensitivity; however, the specific foods and mechanisms involved are unclear. In patients with EoE, we have found that hypersensitivities to corn and peanuts are the most common. Accordingly, we sensitized and exposed mice either intranasally or intragastrically with corn or peanut extract or saline. Esophageal eosinophilia, the genes of eosinophil-directed cytokines, and allergen-induced antibodies were examined in mice challenged with corn or peanut extract or saline. A high number of esophageal lamina propria eosinophils as well as eosinophilic microabscesses, intraepithelial eosinophils, extracellular eosinophilic granules, thickened and disrupted epithelial mucosa, and mast cell hyperplasia were observed in the esophagus of peanut or corn allergen-challenged mice. Mechanistic analysis indicated that para-esophageal lymph nodes might be critical in the trafficking of eosinophils to the esophagus and in EoE association to airway eosinophilia. Furthermore, experimentation with gene-targeted mice revealed that peanut allergen-induced EoE was dependent on eotaxin and invariant natural killer T (iNKT) cells, as CD1d and eotaxin-1/2 gene-deficient mice were protected from disease induction. Thus we provide evidence that para-esophageal lymph nodes are involved in food- or aeroallergen-induced eosinophilia and patchy EoE pathogenesis, likely a mechanism dependent on eotaxins and iNKT cells.

Integrated lymphography using fluorescence imaging and magnetic resonance imaging in intact mice.

We assessed lymph drainage in living mice by an integrated imaging method using fluorescence imaging (FLI) and magnetic resonance imaging (MRI). Mice were subcutaneously injected with quantum dots and gadofluorine 8 into the right rear footpad. They were fixed on a transparent flat plate and underwent FLI and MRI successively. Small markers were attached to the mouse surface for spatial coregistration, and image fusion of FLIs and MRIs was performed. Two-dimensional fluorescence reflectance imaging was used for FLI. FLI and MRI provided generally consistent results and demonstrated lymphatic flow to the popliteal, sacral, and iliac lymph nodes in most mice and to the renal, inguinal, and lumbar-aortic lymph nodes in some mice. On the fusion images, the locations of the lymph nodes in the mouse trunk were in good agreement between FLI and MRI, indicating successful spatial registration even for the deep structures. The popliteal node tended to be visualized a little farther caudally in FLI than in MRI, presumably because the overlying tissues were thicker in the cranial portion. Integrated FLI/MRI lymphography with image fusion appears to be a useful tool for analysis of the murine lymphatic system.

Structure and function of the lymphatic system: an overview.

The lymphatic system is often overlooked, but is a vital part both of the circulation and the immune system. This article provides a basic overview of the structure and function of the lymphatic system as a grounding for understanding the development of chronic oedemas, notably lymphoedema.

Chronic intermittent psychosocial stress (social defeat/overcrowding) in mice increases the severity of an acute DSS-induced colitis and impairs regeneration.

Ulcerative colitis is a multifactorial disease, with immunological, genetic, and environmental factors playing an important role in its pathogenesis. Here we investigated the consequences of exposure to chronic psychosocial stress on the severity of a dextran sulfate sodium (DSS)-induced colitis in male C57BL/6 mice. Chronic stress was induced by repeated exposure to social defeat (SD, 2 h) and overcrowding (OC, 24 h) during 19 consecutive days. SD/OC mice showed a diminished body weight gain, thymus-atrophy, and adrenal hypertrophy, but similar light-phase plasma corticosterone concentrations, compared with unstressed mice. In contrast, the rise in dark-phase corticosterone concentration was significantly attenuated in SD/OC mice, whereas plasma ACTH concentrations and hypothalamic CRH mRNA expression did not differ between stressed and nonstressed groups. Additionally, adrenal cells from SD/OC mice showed a decreased in vitro response to ACTH stimulation. Subsequent treatment with 1% DSS for 7 d resulted in a more severe intestinal inflammation in SD/OC mice, as reflected by an increase in body weight loss, histological damage scores, and secretion of IL-6, TNFalpha, and interferon-gamma from mesenteric lymph node cells and by decreased colon length. The impaired health status of stressed mice was also reflected by a significantly lower survival rate after termination of the DSS treatment. In conclusion, the present findings demonstrate that chronic intermittent exposure to a psychosocial stressor before the induction of acute DSS-colitis results in adrenal insufficiency, increases in the severity of the acute inflammation, and impairs the healing phase.

The effects of dietary lipids on adrenergically-stimulated lipolysis in perinodal adipose tissue following prolonged activation of a single lymph node.

The effects of feeding beef suet (mostly saturated and monoenoic fatty acids), sunflower oil (rich in n-6 fatty acids) and fish oil (rich in n-3 fatty acids) on the response of mesenteric, omental, popliteal and perirenal adipocytes to experimentally-induced local inflammation were studied in adult guinea pigs. After 6 weeks on the experimental diets, the animals were fed standard chow, and lipopolysaccharide was injected unilaterally daily for 4 d to induce swelling of one popliteal lymph node. Basal lipolysis in the perinodal adipocytes of all depots studied was higher in the sunflower oil-fed animals than in the controls fed on standard chow, and lower in those fed on suet or fish oil. Dietary lipids altered rates of lipolysis during incubation with l0(-5) M noradrenaline in all samples studied from the locally-activated popliteal depot, but only in adipocytes within 5 mm of a large lymph node in the other depots. The fish-oil diet attenuated the spread of increased lipolysis within the locally-activated popliteal adipose tissue, and from this depot to other node-containing depots. These experiments show that n-6 polyunsaturated fatty acids promote and n-3 fatty acids suppress the spread of immune activation to adipocytes within and between depots, and alter the sensitivity of perinodal adipocytes to noradrenaline. Dietary effects are reduced or absent in adipocytes in sites remote from lymph nodes, and thus such samples do not adequately represent processes in perinodal adipose tissue. These results are consistent with the hypothesis that perinodal adipocytes interact with adjacent lymphoid cells during immune responses.

Chronic lymph flow responses to hyperproteinemia.

The long-term responses of lymph flow, lymph protein transport, and the permeability-surface area (PS) product to hyperproteinemia have been studied in conscious dogs. Plasma protein concentration (PPC) was increased by daily intravenous infusion of previously collected autologous plasma for 9 days. Lymph flow was determined by collecting lymph chronically from a lymphatic afferent to the popliteal node in the hind leg. Compared with the average value during the normal-PPC period, the following changes occurred during 10 days of high PPC: lymph flow decreased from 12.3 +/- 1.1 to 3.8 +/- 0.6 microl/min, lymph protein transport decreased from 241 +/- 24 to 141 +/- 21 microg/min, PS product decreased from 4.7 +/- 0.5 to 3.0 +/- 0.5 microl/min, PPC increased from 7.1 +/- 0.1 to 8.8 +/- 0.4 g/dl, lymph protein concentration increased from 1.9 +/- 0.1 to 3.8 +/- 0.1 g/dl, plasma colloid osmotic pressure increased from 18. 6 +/- 0.8 to 24.2 +/- 2.1 mmHg, and lymph colloid osmotic pressure increased from 4.8 +/- 0.2 to 10.4 +/- 0.7 mmHg. In conclusion, long-term hyperproteinemia in dogs resulted in chronic decreases in lymph flow, lymph protein transport, and the PS product and chronic increases in lymph protein concentration and lymph colloid osmotic pressure. The marked decrease in lymph flow during hyperproteinemia decreased lymph protein transport and thus contributed to the increase in lymph protein concentration. In addition, the decreases in PS product and lymph protein transport suggest that transcapillary protein flux decreases during hyperproteinemia.

Time-lapse quantitative computed tomography lymphography: assessing lymphatic function in vivo.

RATIONALE AND OBJECTIVES: Immobility and massage produce different local limb lymph flow rates. We studied their influence on accumulation of radiopaque nanoparticulates in regional lymph nodes of normal rabbits. METHODS: Quantitative lymphography at 10-min intervals was used to follow the transport of subcutaneous (s.c.) nanoparticulates produced from insoluble esters of diatrizoic acid. In one design, both hindpaws received 0.5 ml of nanoparticulate s.c., and one hindpaw was massaged. In a second design, one hindpaw was injected and massaged while imaging the popliteal, presacral, and paraaortic nodes every 10 min. RESULTS: Gentle massage rapidly increased popliteal node accumulation in comparison with the immobile limb. On the massaged side, mean Hounsfield (HU) units, maximum Hounsfield units, and calculated iodine were significantly greater at 10 min and all subsequent times. In the node transfer experiments, it took 12, 30, and 45 min, respectively, to obtain 100-HU mean attenuation; 200-HU maximum attenuation thresholds were achieved at 20, 47, and 69 min, respectively. CONCLUSION: Quantitative computed tomography lymphography reflects local lymph physiology. Gentle massage of the s.c. injection site is a powerful lymphotropic stimulus.

Reentry of T cells to the adult thymus is restricted to activated T cells.

To seek information on the capacity of mature T cells to migrate to the thymus, mice were injected with Thy-1-marked populations enriched for resting T cells or T blast cells; localization of the donor cells in the host thymus was assessed by staining cryostat sections of thymus and by FACS analysis of cell suspensions. With injection of purified resting T cells, thymic homing was extremely limited, even with injection of large doses of cells. By contrast, in vivo generated T blast cells migrated to the thymus in substantial numbers. Thymic homing by T blasts was greater than 50-fold more efficient than with resting T cells. Blast cells localized largely in the medulla and remained in the thymus for at least 1 mo post-transfer. Interestingly, localization of T blasts in the thymus was 10-fold higher in irradiated hosts than normal hosts. Thymic homing was especially prominent in mice injected with T blasts incubated in vitro with the DNA precursor, 125I-5-iodo-2'deoxyuridine (125IDUR); with transfer of 125IDUR-labeled blasts to irradiated hosts, up to 5% of the injected counts localized in the host thymus. These data suggest that thymic homing by T blasts might be largely restricted to cells in S phase. The physiological significance of blast cell entry to the thymus is unclear. The possibility that these cells participate in intrathymic tolerance induction is discussed.

[ A comparative lympho-scintigraphic evaluation of manual lymphatic drainage and pressotherapy in edema of the arm following treatment of a breast tumor]. / Evaluation comparative lymphoscintigraphique du drainage lympathique manuel et de la pressothérapie sur l'oedème du membre supérieur secondaire au traitement d'une tumeur mammaire.

Manual lymphatic drainage (MLD) and pressure-therapy are part of the therapeutical gears used in physiotherapy for reducing edema. The solution to the problem of the reduction fo lymphedema of the extremities rests with the evacuation of the liquid phase and the resorption of the stagnant proteins in the interstitial compartment not being collected by the lymphatic system. The injection of a 99 Technetium-labelled colloid substance provides a functional approach to the problem of lymphatic stasis.

[Changes in the functional activity of macrophages of the skin and regional lymph node after exposure of albino rats to total deep hyperthermia]. / Izmeneniia funktsional'noi aktivnosti makrofagov kozhi i regionarnogo limfouzla posle vozdeistviia na organizm belykh krys obshchei glubokoi gipotermii.

In the experiments performed on white male rats (150-170 g of body mass) effect of total deep hypothermia has been studied in macrophages of the skin and regional suprascapular lymph node of various localization: Langerhans cells of the epidermis, histiocytes of the derm and hypoderm, macrophages of medullary sinuses, interdigitating cells of T-zones in the lymph node. After narcotization the animals are cooled with the rate 1 degree C per 5 min up to the rectal temperature of 18 degrees C. They are kept at this temperature for 2 h, and then are warmed with the same rate up to 37 degrees C. Actively phagocytizing macrophages of the skin and lymph node are revealed by their adsorption of trypan blue. The Langerhans and interdigitating cells of the lymph node are revealed by means of the ATP-ase method. After the cooling effect functional activity of macrophages with various localization increases. For the Langerhans cells it is manifested as a greater amount of the cells and their processes, for the interdigitating cells--as an elevated ATP-ase activity in 7 and 30 days after the experiment. Dermal histiocytes and macrophages of the lymph node sinuses respond to the cooling with an increasing adsorption of tripan blue. Amount of the cells, that adsorb the dye also increases. A conclusion is made that after the hypothermal effect protective-barrier properties of the dermal region increase.

Modification of lymphocyte migration by sulfated polysaccharides.

The role of sulfated polysaccharides in lymphocyte migration has been analyzed in vivo using lymphocytes labeled with an intracellular DNA-binding fluorochrome Hoechst 33342. The influence of a panel of sulfated polysaccharides on entry (by injecting the sulfated polysaccharide prior to the labeled cells) and displacement from lymphoid organs (by injecting the sulfated polysaccharide after the labeled cells have localized) indicated that different sulfated polysaccharides have selective effects on entry and displacement, and furthermore positioning of subpopulations within organs. Additional experiments suggested that receptors for sulfated polysaccharides on high endothelial venules may interact with complementary structures on lymphocytes. The data supporting this conclusion were: (a) the normal localization behavior of lymphocytes preincubated with sulfated polysaccharides; (b) an inverse relationship between the expression of lymphocyte surface receptors for sulfated polysaccharides and the ability of the lymphocytes to enter lymphoid organs and (c) the selective binding of sulfated polysaccharide-coupled fluoresceinated beads to high endothelial venules. In this case only the beads coupled with the sulfated polysaccharides that inhibited entry bound to the high endothelial venules. These findings are discussed in terms of a fundamental cellular recognition system utilizing sulfated polysaccharides.

Afferent lymph and lymph borne cells: their influence on lymph node function.

In AO rats the afferent lymphatics to the right cervical lymph nodes (LN) were interrupted and the LN were encased in silicone rubber tubes to prevent reunion of the lymphatics. At regular intervals over the next 12 weeks the following were measured in comparison with the intact contralateral LN - LN weight, influx of lymphocytes from the blood, blood flow, the incorporation of 125IUdR and the incorporation of 35S-sulphate into high endothelial venules (HEV). Systematic histological observations are also reported. One day after deafferentization lymphocyte influx was significantly reduced although blood flow was unchanged and a temporary increase in LN weight was associated with crowding of the lymphatic sinuses with small lymphocytes. The subsequent decline in lymphocyte influx was biphasic and quicker than the decline of other parameters--being undetectable by 6 weeks. Flattening of HEV and diminished secretion of 35S-sulphate was noted at 1 week and progressive degeneration and eventual disappearance of the HEV network was seen by 6-12 weeks. Doubtlessly because of lack of antigenic stimulation 125IUdR incorporation, and numbers of lymphoblasts, plasma cells and finally germinal centres were progressively reduced. The numbers of macrophages and interdigitating cells (IDC) were greatly reduced by 3 weeks and very few were present at 6 weeks probably because most or all arrive in afferent lymph and have a limited life span in the LN. At 12 weeks the LN was difficult to recognize as such since only stromal cells and occasional small lymphocytes remained. In supplementary experiments u.v. irradiation of the LN at the time of deafferentization reduced lymphocyte influx without affecting blood flow suggesting that a u.v. sensitive cell like the IDC may influence lymphocyte influx. In conclusion the involution of the deafferentized LN is partly due to the lack of antigen but progression to the complete loss of specialized structure and function is probably due to lack of other factors including non-lymphoid cells that normally arrive in afferent lymph.