Immunoelectron microscopy of the hormonogenic sites of the thyroglobulin molecule

Hog thyroglobulin was treated with the monoclonal anti-thyroglobulin antibody, 16.3.2, which recognizes the thyroxine residues of thyroglobulin. Size-exclusion high-performance liquid chromatography of the immune complexes indicated that there were 3.5 immunoreactable thyroxine residues/mol of 19 S thyroglobulin. The immune complexes were negatively stained and observed under an electron microscope. Under antigen excess conditions, the complexes were demonstrated as branched chains in which thyroglobulin molecules were linked by antibody molecules. When the antibody was in excess, we found many single thyroglobulin molecules each connecting to 2-3 antibody molecules. Based on the morphology of the immune complexes, we conclude that thyroxine residues are formed preferentially on the molecular surface and localized near both ends of the elongated molecule of 19 S thyroglobulin and possibly at one side of the middle of the molecule.     

High concentration of bilirubin in post-nodal lymph associated with red blood cell catabolism in lymph nodes of the sheep.

Qualitative and quantitative analysis of post-nodal lymph of the sheep has shown that the distinct yellow colour of this fluid pool is due to the presence of relatively large amounts of bilirubin. In efferent lymph from thepopliteal, prefemoral, prescapular, renal and intestinal lymph nodes total bilirubin concentrations were 3-8 times higher than the corresponding concentrations in blood plasma. In contrast the total bilirubin concentrations in afferent lymph from the lower leg and kidney were less than the corresponding concentrations in blood plasma. Histological examination of several popliteal and mesenteric lymph nodes revealed the presence of free iron and bilirubin in the cytoplasm of cells located near the lymphatic sinuses of the node. In addition, the concentration of bilirubin in efferent lymph from the popliteal node was observed to increase following an induced rise in the number of red blood cells reaching the node by way of the afferent lymphatic duct. These latter observations suggest that the bilirubin in post-nodal lymph is associated with the catabolism of extravascular red cells by reticulo-endothelial cells within the lymph nodes.     

Significance of extranodal extension of regional lymph nodes in surgically resected non-small cell lung cancer

STUDY OBJECTIVES: Regional lymph node (LN) involvement affects the prognosis of patients with surgically resected non-small cell lung cancer (NSCLC). The significance of extranodal extension in these groups of patients was prospectively studied to determine its clinicopathologic relationships and its influence on patient survival. METHODS: A total of 199 NSCLC patients who were proved to have regional LN involvement after resection were included. Histologic examinations including tumor cell type, grade of differentiation, vascular invasion, regional LN metastasis emphasizing the number and station of LN involvement, the presence or absence of extranodal extension, and the immunohistochemistry of p53 expression were obtained. The relationships between extranodal extension and histologic type, grade of differentiation, vascular invasion, tumor size, pathologic stage, p53 expression, or patient survival were analyzed. RESULTS: Extranodal extension was significantly higher in women, adenocarcinoma, advanced stage, tumors with vascular invasion, or p53 overexpression. The total number and positive rate of resected LNs with extranodal extension were significantly correlated with advanced stage, tumors with vascular invasion, or p53 overexpression. By multivariate analysis of survival, the presence or total number of LNs with extranodal extension, tumor stage, and p53 expression were significant prognostic factors. The 5-year survival rate of stage IIIA patients without extranodal extension (30.4%) was significantly better than that of stage II patients with extranodal extension (16.8%). No survival difference between extranodal positive stage II and IIIA patients was noted. CONCLUSIONS: Extranodal extension of regional LNs is an important prognostic factor in patients with surgically resected NSCLC.     

Cytoclasmosis in central lymph production.

Electron microscopic observations and ancillary data are reviewed to show that lymphatic organs produce quantities of central lymph by releasing cytoplasm-depleted cells into colloidal hydrosols produced by cell fragmentation (cytoclasmosis).     

Phase microscopy studies of Hodgkin's disease lymph nodes in relation to histogenesis of the Sternberg-Reed cell

1. The free reticulum cell in Hodgkin’s disease undergoes progressive cytologicalterations leading to the formation of Sternberg-Reed cells.

2. The other free cellular components of Hodgkin’s disease nodes are cytologically normal.

     

Blood and lymph node T lymphocytes in cutaneous T cell lymphoma: evaluation by light microscopy.

Cytology of peripheral blood and lymph node lymphocytes from a group of unselected patients with cutaneous T cell lymphoma (CTCL) was studied by light microscopy. Twenty of 45 patients had circulating lymphocytes with convoluted nuclei recognized in routine Wright-Giemsa-stained peripheral blood smears. Cytocentrifuge preparations of E-rosetted lymphocytes showed that greater than 10% of the T cells had convoluted nuclei in each of 16 patients with positive blood smears and in six of 17 whose blood smears were negative or inconclusive. Peripheral blood involvement with greater than 10% convoluted T cells was most frequent in patients with erythroderma (100%) including those with normal of decreased lymphocyte counts, and was not uncommon in patients with mycosis fungoides in the plaque or tumor phase (42%). The light-microscopic morphology of the abnormal cells found in the patients with the plaque or tumor phase of mycosis fungoides was not distinguishable from that of the erythrodermic patients. Increased percentages (less than 15%) of T cells having convoluted nuclei were also found in the lymph node cell suspensions from CTCL patients with adenopathy (18 of 25 patients). These results suggest that a high frequency of extracutaneous involvement occurs in patient with CTCL, the clinical significance of which remains to be determined.     

Molecular and particulate depositions for regional myocardial flows in sheep

The deposition of microspheres in small tissue regions is not strictly flow dependent. In comparison with the soluble flow marker 2-iododesmethylimipramine (IDMI), deposition of 16.5-microns microspheres was mildly but systematically biased into high flow regions of rabbit hearts (Bassingthwaighte JB, Malone MA, Moffett T-C, King RB, Little SE, Link JM, Krohn KA. Am J Physiol 1987;253 (Heart Circ Physiol 22):H184-H193). To examine the possibility of bias in larger hearts, a similar study was undertaken in sheep. 141Ce- and 103Ru-labeled 16.5-microns microspheres in one syringe and 125I- and 131I-DMI in another syringe were injected simultaneously into the left atrium of five open-chest sheep while obtaining reference blood samples from the femoral artery. In six other sheep, one microsphere type and one IDMI were used. Hearts were removed 1 minute after injection, cut into approximately 254 pieces averaging 217 mg, and regional deposition densities calculated for each tracer from the isotopic counts. Correlations in the five animals between the two differently labeled IDMIs and between the two microspheres were both greater than or equal to 0.98. In all 11 sheep, scatter plots of microsphere deposition densities versus IDMI densities showed that differences between microspheres and IDMI had substantially more scatter (0.84 less than r less than 0.98) but were not random. Microsphere depositions tended to be lower than IDMI depositions in low flow regions and higher in high flow regions, in accord with the expected bias that at a bifurcation a microsphere is most likely to enter the branch with higher flow. There was less bias ascribable to endomyocardial/epicardial maldistribution. Thus, while microsphere depositions appear to err systematically with respect to flow when the regions of interest are small enough that the diameters of their arterioles are only a few times those of the microspheres, microspheres are, in sheep as in rabbits, adequate for estimating regional flows.     

Subunit stoichiometry and juxtaposition of the photosynthetic coupling factor 1: Immunoelectron microscopy using monoclonal antibodies

Monoclonal antibodies specific to the α subunits of the photosynthetic coupling factor 1 (CF₁) were used as marker molecules in an electron microscopic analysis of the subunit organization of this enzyme. Immune complexes were obtained by incubation of CF₁ with saturating amounts of anti-α-subunit IgG, isolated by gel filtration, and visualized by electron microscopy. The maximum number of antibodies bound to a CF₁ molecule was three, the angle defined by a neighboring pair of antibodies characteristically being 120°. These results are interpreted as direct evidence for the presence of three α subunits in the CF₁ complex, the relative orientation of them being described by 3-fold rotary symmetry. Our observations thus favor an overall subunit stoichiometry of α₃β₃γδε.     

Equilibration of intravascular albumin with lung lymph in unanesthetized sheep

In 16 unanesthetized sheep with chronic lung lymph fistulas we measured pulmonary vascular pressures, lymph flow, lymph and plasma total protein and albumin concentration. We determined the rate of equilibration of radioiodinated albumin between plasma and lung interstitial fluid (lung lymph) in three steady-state conditions; baseline (n = 14), increased pulmonary microvascular pressure (n = 9) and increased microvascular permeability (n = 4). The tracer protein equilibration proceeded according to single compartment wash-in kinetics in all experiments. Lung lymph flow averaged 5.3 +/- 2.8 (S.D.)ml/h under baseline conditions, 16.1 +/- 10.6 ml/h during increased pressure and 37.3 +/- 29.4 ml/h during increased permeability. The half time of equilibration averaged 2.9 +/- 1.0 h, 2.2 +/- 1.0 h and 0.7 +/- 0.2 h, respectively. Lung interstitial fluid equilibrates with plasma proteins more rapidly than most other organs. The marked difference between increased permeability and the other conditions demonstrates the sensitivity of this method. No evidence was obtained that any tracer protein entered lung lymph within the caudal mediastinal lymph node.     

Thoracic duct lymph flow and its driving pressure in anesthetized sheep

We examined the relationship between thoracic duct lymph flow (TDF) and its driving pressure (DP) in six anesthetized sheep. DP was determined as the thoracic duct pressure (TDP) minus the innominate vein pressure (VP). TDF was measured using an ultrasound transit-time flow meter, placing a flow probe beside the caudal mediastinal lymph node. TDP was measured with a fine needle inserted near the flow probe. TDP increased linearly together with an increase in VP after balloon inflation in the cranial vena cava with a TDP/VP ratio of 0.79. DP decreased, therefore, with an increase in VP and this decrease in DP correlated directly with a fall in TDF. After rapid i.v. fluid infusion, TDF increased but DP varied among the six sheep. Nonetheless, after balloon inflation with expanded volume (i.e., i.v. fluid infusion), DP and TDF were positively correlated. We conclude that DP is the main factor determining TDF when VP rises in conjunction with increased lymph production.     

Abdominal lymph flow response to intraperitoneal fluid in awake sheep

Lymphatic vessels are important in draining excess fluid from the abdominal space and preventing ascites. In sheep, diaphragmatic lymph vessels draining the abdominal space run to the caudal mediastinal lymph node and efferent vessels from the node drain into veins in the neck. To estimate the lymph flow response to excess intraperitoneal fluid in sheep, we cannulated a caudal mediastinal node efferent lymphatic in 5 sheep. After the sheep recovered from the surgery, the lymph flow (QL) was 154 +/- 161 (SD) microliters/min and the lymph protein concentration (CL) was 3.7 +/- 9 g/dl. Lymph flow decreased linearly with increases in lymphatic outflow pressure greater than 6 cmH2O. From this linear QL vs. outflow pressure relationship, we estimated the effective pressure driving lymph flow as the outflow pressure at which QL = 0. At baseline, the driving pressure was 24.7 +/- 14.0 cmH2O. After we infused Ringers solution (10% body weight) into the abdominal space, QL increased significantly to 7.0 +/- 4.1 times baseline and CL decreased significantly to 0.7 +/- 0.6 g/dl. Although the abdominal pressure increased significantly from 10.6 +/- 2.8 cmH2O to 15.8 +/- 2.1 cmH2O, we found no increase in lymphatic driving pressure.     

Corticosteroids inhibit endotoxin-induced lung lymph neutrophil stimulating activity in sheep

The Adult Respiratory Distress Syndrome (ARDS) most frequently is the result of sepsis. Accumulation of neutrophils in lung interstitium is a well-documented phenomenon, but the nature of their presence remains obscure. We hypothesized that endotoxin causes the release of substances into lung lymph that activate neutrophils and that methylprednisolone may prevent sequestration and activation of neutrophils. We used the sheep lung lymph fistula-endotoxin model of ARDS to test this hypothesis. Unanesthetized animals were given either 0.5 microgram/kg of E. coli endotoxin intravenously alone or, on a different experimental day, an identical dose of endotoxin preceded by a 1 gm bolus of methylprednisolone plus a 1 gm/hr continuous infusion. Endotoxin infusion caused the release of substances into lung lymph that were capable of stimulating normal sheep neutrophils to aggregate, migrate, and release superoxide. This activity appeared within 1 hour of endotoxin and persisted for at least 4 hours. Pretreatment by methylprednisolone did not prevent the early activity but did significantly reduce such activity 3-4 hours after endotoxin, when the permeability defects caused by endotoxin are most pronounced. We speculate that endotoxin-stimulated production of humoral neutrophil-activating substances in the lung may play a role in the pathogenesis of acute lung injury.     

The effect of anaesthesia and surgery on lymph flow, protein and leucocyte concentration in lymph of the sheep.

Anaesthesia and the trauma of surgery, associated with the cannulation of lymphatic ducts in various regions of the body of the sheep, had a profound effect on lymph flow, protein concentration and leucocyte concentration of lymph. In general lymph flow was depressed and the protein concentration elevated in lymph collected at the time of cannulation, or within the first 24 hours of recovery from surgery. The changes in protein concentration in lymph draining the peripheral regions of the body appeared to be due to surgical interference in the region of drainage. The greatest changes in lymph flow were observed in lymph draining peripheral regions (skin, tendon, muscular areas) while lymph draining soft tissues in central regions (kidney, liver) was less affected by the anaesthesia and surgical stress. A neutrophilia was observed in venous blood collected under anaesthesia while the overall numbers of lymphocytes in three sources of efferent lymph were depressed. It is suggested that corticosteroid hormones may play a role in the changes in leucocyte migration observed during anaesthesia and surgical stress. Changes observed in the cellular content of afferent lymph appeared to be due to a low grade inflammation associated with surgical interference in the region of lymphatic drainage.     

Spleen and lymph node cell populations, In vitro cell proliferation and interferon-y production during the primary immune response to Toxoplasma gondii

An animal model for the study of transient lymphadenopathy-splenomegaly during toxoplasmosis is presented. Injection of CBA/J mice with the low virulent, cyst-forming strain of Toxoplasma gondii (Pe strain) induces a three to four fold increase in weight and cellularity of spleen and lymph nodes with peak changes at 30-50 days after infection. The spleen displays marked haemopoiesis, a 30 fold increase in mononuclear phagocytes, and a two fold increase in Lyt2+ lymphocytes. Lymph nodes show a five fold increase in mononuclear phagocytes and a four and a half fold increase in Lyt2+ T cells. The increase in mononuclear phagocytes significantly alters T cell/macrophage ratios and this is associated with decreases in in vitro cell proliferation to mitogen and toxoplasma antigen. The relationship between alterations in cell balance of mononuclear phagocytes and T cell subsets and the expression of transient immune dysfunction can now be examined by modulating changes in these cell types.     

Giardia muris-induced depression of the primary immune response in spleen and mesenteric lymph node cell cultures to sheep red blood cells

Primary in vitro plaque-forming cell (PFC) responses to sheep red blood cells (SRBC) were examined for spleen and mesenteric lymph node (MLN) cell populations from susceptible (A/J) and resistant (B10.A) mice during the infection with Giardia muris. Spleen and MLN cells isolated from mice during the acute phase of the infection were less responsive to SRBC in vitro than those from uninfected mice. Depressed anti-SRBC PFC response was detected earlier and was more pronounced in MLN cell cultures when compared to the response of spleen cell cultures. Spleen and MLN cells from donors infected with G. muris for 15 days had the capacity of depressing PFC response to SRBC of cells isolated from uninfected mice. This suppressor activity was localized in the plastic-adherent fraction of spleen cell populations isolated from A/J and B10.A mice. Since G. muris is a gastro-intestinal infection of mice, lower capacity of the MLN cells to respond to an antigenic stimulation in vitro may explain, in part, the proliferation of the trophozoites during the acute phase of the infection.     

Immunoelectron microscopy of the bone marrow mononuclears labelling with rabbit anti-mouse brain serum using peroxidase-anti-peroxidase method

Summary Ultrastructural features of mouse bone marrow mononuclears (BMM) labelled with peroxidase-anti-peroxidase (PAP) complex using rabbit anti-mouse brain serum (RAMBS) and rabbit anti-mouse brain serum, absorbed with thymocytes (RAMBS_T) were investigated. After incubation of cells with RAMBS_T which have high activity against mouse pluripotent hemopoietic stem cells (CFU_s) about 0.6% of BMM were labelled. According to their fine structure these cells were similar to those described earlier as presumptive pluripotent hematopoietic stem cells (PHSC). After incubation with RAMBS 1.9% of BMM were labelled, including cells marked by RAMBS_T and typical small lymphocytes with ultrastructure of T-lymphocytes.Abbreviations PAP Peroxidase-anti-peroxidase - RAMBS Rabbit-anti-mouse brain serum - RAMBS_T Rabbit-anti-mouse brain serum, absorbed with thymocytes - NRS Normal rabbit serum - PHSC Pluripotent hemopoietic stem cells - BMM Bone marrow mononuclears - N/C Nuclear/cytoplasmic ratio - GER Granular endoplasmic reticulum