Regional graft vs host reaction to H-Y antigen (immunological mechanisms)

Structural changes in the regional popliteal lymph nodes have been studied in C57Bl/6 male mice at the peak of the reaction "graft-versus-host" to H-Y antigen. Morphological and morphometrical investigations have been carried out in three groups of males (10 animals in each group). The first group includes intact animals (the first control group). To the males of the second group (the second control group) lymphoid cells are inoculated from intact C57Bl/6 females. To the males of the third group (experimental group) lymphoid cells are inoculated from H-Y antigen immunized C57Bl/6 females (anti-H-Y effector lymphocytes). The popliteal lymph nodes of the male mice from the third group twice increase in their size comparing to those in the control (the first and the second groups). Miotic activity increases in them 4.5 times, amount of cellular blast forms in medullary cords--4 times and 10 times--in the light zone of the cortical substance. Ratio of macrophages and eosinophils in structural components of the lymph nodes studied changes; this is, evidently, connected with massive destructive progresses, that take place in the lymph nodes of the animals from the third group. The results of the morphological investigations are in agreement with the hypothesis suggested, explaining the mechanism of development of the regional reaction "graft-versus-host" to H-Y antigen, basing on idiotype-antiidiotype interaction (the idiotypic network in the immune system).     

Morphology of mesenteric lymph nodes after balneologic procedures

Strong and weak sulfate baths produce certain increase in amount and in area of the lymphoid noduli with the germinative center, in the deep cortex and a decrease in the relative area of the medullary substance, while iodobromine baths facilitate to an outgrowth of the medullary cords and to ectasia of sinus lumens. After the balneoprocedures lymphoid noduli appear in the deep cortex; amount of destructive cells in the medullary sinuses, in the medullary cords and in the intermedullary zone decreases; amount of mast cells, eosinophiles and neutrophiles increases (slightly). Blast transformation of small lymphocytes and increase in number of middle lymphocytes are observed in the germinative centers.     

Structural changes in the lymph node sinuses in acute blood loss]

By means of light and scanning electron microscopy sinuses of the popliteal lymph nodes have been studied in 50 Wistar male rats. After modelling of an acute hemorrhage in the animals (in 15, 120 min and 1 day) volume of the medullary and marginal sinuses increases essentially, while blood stream intensity decreases in peripheral tissues. Amount of fenestrae in the sinusal lining decreases, that is, perhaps, caused by edema of cytoplasm in the lining cells and accompanied with formation of numerous cytoplasmic protrusions into the sinusal lumen. In 3 days the blood stream level in the peripheral tissues restores, structural organization of the littoral cells in the lymphatic sinuses normalizes. The data obtained demonstrate that during the first day after the acute hemorrhage decrease of hematolymphatic exchange intensity in lymph nodes and enhancement of transitory lymph flow in them take place.     

Morpho-functional status of the lymph nodes of rats during rehabilitation after exposure to vapors of dimethyl sulfate]

By means of histological methods inferior tracheobronchial lymph nodes have been investigated in 48 Wistar rats in 2 weeks and 3 months after cessation of inhalation of DNSV at maximum possible dose (0.1 mg/m3) for 2 and 14 days. Cell composition of the lymph nodes and cross sections of structural components have been compared. The cross section of the cortex decreases, while that of the medulla increases in comparison with corresponding parameters of the acute experiment. In 2 weeks and 3 months after DMSV effect for 2 days the part (%) of poorly differentiated cells and middle lymphocytes increases, while content of plasma cells in the medullary cords decreases. During rehabilitation after DMSV effect for 14 days a low level plateau is noted (as in the acute experiment). However, in lymphoid nodules++ within content of the poorly differentiated cells and middle lymphocytes in the cortical germinative centers in 2 weeks and 3 months the number of the poorly differentiated and reticular cells increases essentially, as well as mitotically dividing cells (in comparison to the acute experiment). In the medullary cords of the lymph nodes after 3 months of rehabilitation content of immature plasma cells is essentially higher than in 2 weeks of rehabilitation and than in the acute experiment. During rehabilitation the level of cells destruction in the lymph nodes noticeably decreases in comparison to the acute experiment.     

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.     

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.     

Structural changes in popliteal lymph nodes in the graft vs. host reaction to H-Y antigen in male C57BL/6-strain mice

Regional popliteal lymph nodes in intact, control and experimental (I, II, III groups, respectively) have been studied by means of the morphometric method in male C57Bl/6 line mice at the pick of the reaction produced by injection of spleen cells and mesenteric lymph nodes obtained from syngenic females and repeatedly immunized to H-Y antigen (10 animals in each group). Injection of the cell suspensions from the immunized and intact females of the C57Bl/6 mice result nearly in two-fold increasing mass of the regional popliteal node at the expense of enlarged size of all its zones. Changes in cytoarchitectonics of the node structural components result from redistribution of certain cellular elements. The essential changes in the cell composition of the lymph nodes in the II and III male groups are accompanied with an increasing part of the stromal reticular cells. Simultaneously, content of small lymphocytes decreases significantly. In the III group of mice there is a sharp increase in the content of young forms of the lymphoid line cells in all structural components of the node, as well as in eosinophilic granulocytes in medullary cords. In the dark cortical zone of the nodes (III group) there occur tissue basophils (mast cells), that, together with increasing number of acidophilic granulocytes and appearance of neutrophilic cells, demonstrates that there is an inflammatory reaction in the organ studied as a response to the lymphocytic suspension injected. In the experimental group of the animals a complete disappearance of plasma cells is noted in the node cortex, but some increase of their part takes place in the medullary cords.     

Limb lymph node response to bone fracture

In previous clinical studies, dilation of afferent lymphatics and enlargement of inguinal lymph nodes (LN) were observed in lymphoscintigrams from patients with persistent posttraumatic edema of lower extremities after fractures and trauma of soft tissues. In this study, changes in rat popliteal and iliac lymph nodes draining lymph from the site of tibial fracture and adjacent soft tissue injury were investigated. The observed parameters were lymph node weight, cell number, phenotype frequency, cell cytokine expression, and reactivity to mitogens. The key observations included: a) increase in the weight and total cell number of the lymph nodes; b) increased autotransformation rate and responsiveness of lymph node cells to mitogen; c) decreased frequency of ED1 macrophages and activated OX8 cytotoxic cells in flow cytometry analysis; d) high expression of OX6 class II-positive, OX7 (stem cells), OX62 (migrating dendritic cells), ED1 (macrophages), and OX12 (B cells) on immunohistochemical sections of LNs with some few HIS48 (granulocytes); e) high expression of NOS3 and TGF beta by lymph node lymphocytes and endothelial cells. In summary, local lymph nodes reacted to internal wounds, such as bone fracture and injury to adjacent tissues, through mobilization of cells from the blood circulation, along with activation of cellular subsets. The molecular mechanism that provides the signal for this reaction remains unknown. The absence of major changes in the frequency of lymph node cell subpopulations indicates that lymph nodes are constitutively prepared for influx of antigens from damaged tissues and react only with increase in cell number and cell activation. The nature of the reaction, including lack of immunization against autoantigens, remains unclear. Further elucidation will require studies on the mechanism of cross-tolerance to self-antigens during wound healing.     

Functional morphology of the lymphatic system of the rat uterus during pregnancy

In the regional lymph nodes of the uterus the comparative volume of the paracortical zone significantly increases, especially within the period of the 13th-17th days of pregnancy. In the popliteal lymph node similar effect is not discovered. From the 7th up to the 11th day edema, vasodilatation, infiltration with special leucocytes are revealed. Endothelium of the postcapillary venules is hypertrophied, contains many migrating lymphocytes, which accumulate around the vessels mentioned. The volume of the microcirculatory bed is moderately increased. By the 17th day plasmoblasts, plasmocytes, Motta's cells, monocytes and especially macrophages appear in the paracortical zone. In B-zones and in medullary sinuses blasts, plasma cells, monocytes, macrophages, mitotically deviding cells increase in number. The part of the reticular cells decreases. The tensometric method demonstrates an increasing pressure of lymph in the iliac lymph node at pregnancy. Collateralies appear in the ovarian vein system, in the broad ligament of the uterus, in the lumbar area. The uterine vascular system is supposed to participate in adaptation to pregnancy. In genesis of the regional lymph node changes, discirculatory shifts, predominating during placental organogenesis, combine with phenomena of cell migration and proliferation (clearly revealed by the time when formation of the placenta is completed).     

Morphocytochemical reaction of the lymph nodes in dogs to the administration of lysozyme

By means of morphological, morphometrical and histochemical methods pelvic and tracheobronchial lymph nodes have been studied in dogs and concentration of lysozyme has been estimated in blood serum, in lymph and the lymph nodes after a single intramuscular injection of lysozyme (2 mg/kg of body mass). In the material investigated total concentration of lysozyme reaches its maximal values in 6 h after injection, then it gradually decreases and in 48 h reaches its control level. Morphometrically changes in cell composition are revealed predominantly of immune-competent cells in T- and B-dependent zones of the lymph nodes. Thus, the volumetric part of lymphoblasts in the germinative centers of the lymphoid nodules reaches its maximal indices by 48 h after lysozyme injection, while plasmatization of the paracortical zone and of medullary cords increases up to the 7th day. By the 14th day the volumetric part of lymphoblasts, immunoblasts and plasmocytes decreases gradually, and in 21 days after injection of the drug contents of the blast forms of the cells in the structural-functional zones of the lymph nodes does not differ from that in the control. The data obtained demonstrate the immunomorphological rearrangement of the lymph nodes in response to the exogenic lysozyme administration.     

Cellular composition of structural components of the lymph nodes of rats during rehabilitation after dimethyl sulfate poisoning

By means of microanatomical methods the inferior tracheobronchial lymph nodes have been investigated in 48 Wistar rats in 2 weeks and 3 months after discontinuance of inhalation of dimethylsulfate (DMS) vapours for 2 and 14 days by the animals in concentration 2.0 mg/m3, that is to say during rehabilitation period. Comparison of relative parameters of the structural components areas and cell composition of the lymph nodes has been carried out. During rehabilitation period after DMS inhalation for 2 days the cortical and medullary areas in histological preparations do not essentially differ from corresponding parameters of an acute experiment (2 days, 2.0 mg/m3, without rehabilitation). Amount (%) of cells with mitotic figures in the lymphoid nodules++ increases in 2 weeks and in 3 months. Contents of poorly differentiated cells during rehabilitation periods increase in the cortical plateau, but keeps nearly at the same low level as during the acute experiment in the lymphoid nodules++. In 2 weeks after DMS influence for 14 days, the cortical and medullary area in the histological preparations reach the control levels. In the lymphoid nodules++ a relative amount of reticular, poorly differentiated, mitotically dividing cells increases, and in the medullary cords contents of middle and small lymphocytes become greater in comparison with the acute experiment (14 days, 2.0 mg/m3, without rehabilitation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Degree of chromatin activity and the RNA level in lymphocytes of popliteal lymph nodes of dogs after administration of an antigen]

In the experiment performed on 127 dogs by means of cytospectrofluorometric analysis, using fluorochrome acridine orange in dynamics up to 1 year, changes in the level of chromatin activation and RNA content have been studied in lymphocytes of the germinative centers and the crown of lymphoid nodules, in the paracortical zone and medullary cords of the regional and contralateral popliteal lymph nodes, after subcutaneous injection of antigen (BCG vaccine, 0.2 mg/kg) into the lateral area of the foot of the left pelvic extremity. The immune response is accompanied with a periodical increase in the level of chromatin activation and RNA content in populations of lymphocytes in the regional and contralateral popliteal lymph nodes with maximum in 6 h, 3-7 days, 1-3 months after the antigen injection. The intensity of these processes has an unequal level in lymphoid cells of various structural components; it is higher in lymphocytes of the contralateral lymph node.     

Macrophages homing to metastatic lymph nodes can be monitored with ultrasensitive ferromagnetic iron-oxide nanocubes and a 1.5T clinical MR scanner

Background

Due to the ability of macrophages to specifically home to tumors, their potential use as a delivery vehicle for cancer therapeutics has been suggested. Tracking the delivery and engraftment of macrophages into human tumors with a 1.5T clinical MR scanner requires the development of sensitive contrast agents for cell labeling. Therefore, this study aimed to determine whether intravenously injected macrophages could target a primary tumor as well as metastatic LNs, and whether these cells could be detected in vivo by MRI.

Methodology

Peritoneal macrophages were obtained from BALB/c nude mice. The viability, phagocytotic capacity and migratory activity of the macrophages were assessed. MR imaging was performed using a clinical 1.5 T MR scanner and we estimated the T2* of the labeled macrophages. Metastatic lymph nodes were produced in BALB/c nude mice. We administrated 2×10⁶ macrophages labeled with 50 µg Fe/mL FIONs intravenously into the mice. In the 3D T2* GRE MR images obtained one day after the injection of the labeled macrophages or FION solution, the percentages of pixels in the tumors or LNs below the minimum normalized SI (signal intensity) threshold were summated and reported as the black pixel count (%) for the FION hypointensity. Tumors in the main tumor model as well as the brachial, axillary and inguinal lymph nodes in the metastatic LN models were removed and stained. For all statistical analyses, single-group data were assessed using t test or the Mann-Whitney test. Repeated measurements analysis of variance (ANOVA) with Tukey–Kramer post hoc comparisons were performed for multiple comparisons.

Conclusions

The FION-labeled macrophages, which could be non-invasively monitored using a 1.5 T clinical MR scanner, targeted both the main tumors and LN metastases. Overall, the results of this study suggest that the use of macrophages may have many future applications in the clinic for vectorizing therapeutic agents toward main tumors as well as LN metastases.     

Changes in the functional activity of macrophages of the skin and regional lymph node after exposure of albino rats to total deep hyperthermia

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.     

Comparative study of the changes in microanatomical organization of lymph nodes located and functioning in the area of venous congestion

By the morphometric method the microanatomical organization of the popliteal and kidney lymph nodes of the type Vi was studied in venous congestion. In the first experimental series (lymph nodes localize in venous congestion area) the venous congestion in the popliteal lymph nodes was created by the ligature of caudal vena cava. In the second experimental series (lymph nodes, which clean the venous congestion area--the kidney vena was tied up. The small and short time increase of the marginal sinus volume and hypertrophy of the medullary cords, which seemed about medullary substance B-zone stimulation were characteristically for the lymph nodes, localize in the venous congestion area. The sharp increase of the marginal sinus volume during the long experimental time and paracortical T-zone hyperplasia were characteristically for the lymph nodes, which clean the venous congestion area.     

Cellular composition of various structural zones of the iliac and mesenteric lymph node of pregnant rats

The dynamic of cellular reactions demonstrates certain changes in functional activity of all structures of the node during pregnancy. A similar trend of processes in the iliac (regional for the uterus) and mesenteric lymph nodes has been defined. At early stages of pregnancy, lymph nodule are the most active, this is demonstrated as an increasing portion of lymphoblasts, macrophages and dividing cells. During this period, cell composition of the cortical plateau is relatively stable. For the paracortical zone of the mesenteric lymph nodes a rather significant decrease in the portion of middle lymphocytes and reticular cells is characteristic. There is not any significant change in the relative amount of the cells in the same functional zone of the iliac lymph nodes during the same period of pregnancy. The medullar cords demonstrate an increasing number of blast forms and young plasmocytes. However, as the pregnancy develops, the structure of the paracortical zone undergoes an essential change--progressively increases the portion of lymphoblasts and large lymphocytes. The blastic reaction in the mesenteric lymph nodes is proved to depend, to some extent, on that in the iliac lymph nodes of the same animal. Mature plasma cells become the dominating cellular element in the medullary cords. At the end of the pregnancy a relative amount of the reticular cells increases in all structural zones of the node.     

The effect of parathormone on the reactivity of renal blood flow to vasopressin in normotensive and spontaneously hypertensive rats

It has been shown that after parathyroid hormone (PTG) administration vasopressin increases the falling rate of the renal cortical blood flow and decreases the falling rate of the renal medullary blood flow in normotensive rats. In spontaneously hypertensive rats (SHR) the PTG decreases the falling rate of the cortical and medullary blood flow after vasopressin administration. PTG-induced hypercalcemia in SHR followed by vasopressin administration leads to the renal blood flow redistribution reaction due to which the medullary blood flow dominates over the cortical one.     

Blood vasculature of the lymph node in the dog: anatomical evidence for participation of extrahilar arterial vessels in the blood supply of the cortex.

The organization of the arterial vessels of dog lymph nodes (LN) was studied using methods of visualization of the vasculature by systemic injection of different tracers (colloidal carbon, Micropaque resin and methylmethacrylate) followed by observation of the samples by light microscopy (after clearing of the thick sections of LN) or scanning electron microscopy (corrosion casts). LN from all of the three groups of nodes studied (tracheobronchial, paratracheal and popliteal) showed an extensive network of arterial vessels encircling the capsule of the organ. We found that branches of these capsular arteries penetrated deeply into the cortical domain of LN. The capsule-originating vessels appeared to have a significant participation in the blood supply of the LN parenchyma at the cortical domains of the organs. Our findings are in contrast with current views on the angiology of the LN that consider that virtually all of the arterial capillaries of the LN parenchyma come from hilar arteries. We propose, therefore, that important segments of the LN cortex receive their blood supply from capsular arteries rather than from hilar vessels.     

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions^1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens^3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network^2,6-12. Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ^12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures^11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal''s environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.     

Interrelationships of B- and T-cell areas in peripheral immune organs of the albino rat after hind limb autotransplantation

Using 68 3-month-old male albino rats, it was established that the pattern of the changing interrelationships of B- and T-cell areas in the spleen and in the popliteal, inguinal and medial iliac lymph nodes regional for the experimental limb during the initial stage after hind limb autotransplantation, with or without sciatic nerve alloplasty, represents a universal type of initial response of the peripheral immune organs to external challenge which takes place in three steps: 1) an increase in the number and size of the lymph nodules, 2) enlargement of T-cell areas, 3) an increase in the number of structures containing antibody-forming cells (the medullary cords in the lymph nodes and the splenic cords). Sciatic nerve alloplasty gives rise to expansion of the medullary cords in the lymph nodes and the marginal zone and cords in the spleen, with parallel significant enlargement of T-cell areas.