Efferent Lymph Research Articles

A method for tracking the migration of blood lymphocytes.

A method has been devised for labeling whole blood with the fluorescent dye fluorescein isothiocyanate (FITC) so the migration of blood lymphocytes can be studied in the sheep. Although lymphocytes can be purified from blood using density gradient media or elutriation it is difficult to obtain a large number of cells, because many cells are usually lost during the purification steps. It is desirable to label at least 10(8)-10(9) lymphocytes for lymphocyte tracking studies, because a smaller number is difficult to subsequently detect and quantitate in blood and lymph even using flow cytometry. Also, it is desirable to minimize the in vitro manipulation of lymphocytes, because dead or damaged lymphocytes will not recirculate. By labeling all the cellular components of a sample of whole blood rather than first purifying the lymphocytes we have been able to satisfy both of these criteria. Although labeled blood cells of all types are reinjected into the animal, the lymphocytes are easily distinguishable from other cells using a flow cytometer. In these studies between 2.4-12.4 x 10(8) lymphocytes were injected intravenously, and they were detectable in the blood and lymph for at least 10 days. The recovery of FITC-labeled (FITC+) lymphocytes in efferent lymph is comparable to that of lymphocytes labeled with other fluorescent or radioactive markers. The presence of labeled non-lymphoid cells in the animal makes this technique impractical for studies of lymphocyte localization within histologic sections. However, it is useful for studies in animals in which lymphatic vessels can be cannulated and the blood-to-lymph recirculation of labeled lymphocytes monitored, and it also may be applicable for studies in which lymphoid organ suspensions are analyzed using flow cytometry.

Analysis of the expression and secretion of isotypes of sheep B cell immunoglobulins with a panel of isotype-specific monoclonal antibodies

Monoclonal antibodies to sheep light chain, IgM and IgG were produced and used to assess total immunoglobulin (Ig) synthesis by sheep B cells in culture and antibodies to specific antigens. By using these antibodies in a dual fluorescence-activated cell sorting analysis of sheep efferent lymph B lymphocytes the percentage change in surface Ig isotype of B lymphoblasts from IgM to IgG after the antigenic stimulation of the local lymph node was measured. An extension of this analysis to paired blood and afferent or efferent lymph B cells made it possible to investigate the recirculation characteristics of B cells expressing different Ig isotypes.

Organization and cytology of lymphoid tissue in the cynomolgus monkey conjunctiva

AbstractBackground: Knowledge of the structure of conjunctival lymph follicles is limited in comparison with those of other mucosa. Consequently a thorough morphological study of the follicles was undertaken to see if they are similar to others or whether they have any unique features and to provide an adequate background for future experimental studies.Methods: Normal lymphoid tissue of the conjunctiva of one side of nine young adult cynomologus monkeys was examined using a whole mount method and light and electron microscopy.Results: Only the peripheral tarsal and orbital regions of the upper and lower palpebral conjunctiva contained lymph follicles. Lymphocytes, reticulocytes, macrophages including tingible body macrophages, putative follicular dendritic cells, and plasmacytes were indentified in the follicles and M‐cells were present in the follicle associated epithelium. High endothelial venules (HEV) displaying evidence consistent with lymphocyte extravasation were present in both primary and secondary follicles. A majority were secondary follicles with a germinal center composed largely of lymphoblasts. Mitoses were found in secondary follicles, mostly confined to the germinal centers, and they were numerous in many. Increased cell density and virtual elimination of intercellular space adjacent to some high endothelial venules suggested a possible association of these vessels with germinal center development. Chains of follicles were linked by lymph vessels, an approaching lymphatic dividing to embrace a follicle and recombining beyond it and passing to the next in line; many conjunctival follicles there‐fore have both afferent and efferent lymphatics. The vessels were in direct contact with the follicle perimeters; lymph capillaries were not present within the substance of most follicles. Numerous valves indicated drainage radially from the tarsal conjunctiva then laterally or medially from the orbital conjunctiva through the follicle chains towards the nearest canthus.Conclusions: Conjunctival lymph follicles are fundamentally similar in structure to those found elsewhere in the body. However, the beads on a chain‐like arrangement of lymph vessels and follicles may be unique. The possible association of HEV with follicle foramtion could be a general characteristic of mucosal follicles. ©1995 Wiley‐Liss, Inc.

The Neuropeptide Substance P does not Influence the Migration of B, T, CD8+ and CD4+ (‘Naive’ and ‘Memory’) Lymphocytes from Blood to Lymph in the Normal Rat

Thoracic duct lymphocytes (TDL) continuously patrol through the body, facilitating immune responses at most sites. The neuropeptide Substance P might regulate immune responses by influencing the migration of TDL. Therefore, it was investigated whether Substance P affects the migration of thoracic duct B, T, CD8+ and CD4+ ('naive' and 'memory') lymphocytes from blood to lymph in vivo. Labelled TDL were either incubated with Substance P and then injected into normal rats, or incubated without Substance P and then injected into rats continuously receiving Substance P intravenously. The numbers of labeled B, T, CD8+ and CD4+ ('naive' and 'memory') lymphocytes were determined in blood and thoracic duct lymph for 1 and 5 days, respectively. Neither the in vitro incubation with Substance P nor its in vivo application influenced the disappearance of any lymphocyte subset from the blood or its reappearance in the lymph. In addition, continuous intravenous application of the Substance P antagonist CP 96.345 did not alter the volume or the lymphocyte number of the efferent lymph. The present study indicates that the nervous system does not influence immune responses via Substance P by altering the migration pattern of B, T, CD8+ and CD4+ ('naive' and 'memory') lymphocytes.

THE GUT

The aim of this study was to test the hypothesis that the gut is capable of becoming a cytokine-generating organ following either a lethal or nonlethal inflammatory insult. Adult male rats were given an intraperitoneal challenge with saline, or with a nonlethal (.1 mg/g) or LD50 (.5 mg/g) dose of zymosan. Mesenteric lymph nodes, efferent mesenteric lymph, liver, spleen, and blood (portal and systemic) were obtained at 2, 4, 6, 8, or 10 h post challenge. Organs, lymph, and blood were tested for bacterial translocation (BT); blood and lymph were assayed for tumor necrosis factor (TNF) and IL-6. After .1 mg/g zymosan, BT was limited to the mesenteric lymph node complex only; .5 mg/g zymosan promoted BT to blood, mesenteric lymph, and organs (p < .05 vs. control or .1 mg/g zymosan). The magnitude of portal bacteremia was greater than systemic bacteremia (p < .003). Serum TNF peaked at 2 h (p < .05 vs. control), and serum IL-6 peaked at 4-6 h (p < .05 vs. control) post zymosan challenge. Portal and systemic bioactivity was similar for either cytokine, and serum bioactivity did not correlate with zymosan dose. TNF bioactivity was increased in the mesenteric lymph at 2 h post challenge with .5 mg/g zymosan only (p < .05 vs. control or .1 mg/g zymosan). IL-6 bioactivity was increased in the mesenteric lymph at 4 through 10 h post zymosan challenge (p < .05 vs. control), but was similar with either dosage of zymosan. In conclusion, the gut may be capable of producing cytokines in response to an inflammatory stimulus, even in the absence of portal or systemic spread of bacteria. The magnitude of the cytokine response does not correlate with the magnitude of bacterial translocation.

Kinetics of interferon gamma production In vivo during infection with the S48 vaccine strain of Toxoplasma gondii

The kinetics of interferon gamma (IFN gamma) production in vivo was examined in sheep during a primary and secondary infection with the sheep vaccine strain (S48) of Toxoplasma gondii. Efferent lymph plasma from a node draining the site of inoculation was tested for anti-viral activity which could be neutralized with monoclonal antibodies against IFN gamma. Within 2 to 5 days of primary infection IFN gamma was detected in each of five sheep and persisted for 6 to 9 days. Accelerated production of IFN gamma occurred after secondary infection, the cytokine being detected in the first 24 h, and persisting in lymph for a further 4-5 days. From day 6 onwards after primary infection, efferent lymph cells produced IFN gamma when stimulated in vitro with a crude T. gondii antigen. These results show that IFN gamma is induced in sheep after infection with the S48 strain of T. gondii.

Sequence and Repeat Structure Variants in the Long Terminal Repeat of Maedi-Visna Virus EV1

Diversity in the LTR of maedi-visna virus strain EV1 has been examined by PCR-based gene amplification using DNA from infected cells both in vitro and in experimentally infected animals. In vitro, several variant structures were found in the U3 regions of the LTR which contained repeats of sequences including presumed AP-1 and AP-4 binding sites. Although these repeat variants formed a minor fraction of the LTRs present in the proviral population, they were neither produced nor lost at a significant rate when PCR was performed on cloned viral DNA and so were unlikely to be artefacts of the isolation procedure. When LTRs were isolated from two experimentally EV1 infected sheep, repeat variant structures were found to be present in efferent lymph by 14 days postinfection (p.i,) (although not seen at 9 days p.i.). They were also present at later times and in blood. Overall sequence diversity at 9 days p.i. was reduced compared both with the infecting virus and with later times of infection. When a number of the variant LTR structures were used to drive CAT reporter gene constructs in chondrocytes, all were found to be active, although consistent differences of up to fourfold in activity were seen. However, there is no evidence from these data for strong selective pressure operating on the LTR in vivo.

An analysis, using monoclonal antibodies, of the role of interferon‐γ in ovine immune responses

A mAb (IFN-9) which neutralizes biologically active ovine and bovine IFN-gamma was used to deplete levels of the cytokine in vivo in sheep and examine the consequences for immune responses to adjuvanted antigen and skin reactivity to Bacillus-Calmette-Guerin (BCG). Groups of sheep were immunized with ovalbumin in the adjuvants, Quil A or dextran sulphate (MW 500,000; DXS), both of which elicit production of IFN-gamma. MAb anti-IFN-gamma or an isotype control mAb (anti-carbonic anhydrase positive particles) were inoculated i.v. during primary and/or secondary responses. Reactions monitored in efferent prefemoral lymph indicated that anti-IFN-gamma effectively depleted levels of IFN-gamma in lymph but had no effects on the magnitude and kinetics of lymphocyte and lymphoblast traffic, or total or isotypic titres of specific Ig. When incubated in vitro with ovalbumin, antigen-reactive cells from anti-IFN-gamma treated sheep did not produce IFN-gamma, suggesting an on-going modification to cytokine production. In contrast, skin reactions to purified-protein derivative in sheep immunized with BCG were reduced by < 40% by anti-IFN-gamma. The results indicate that IFN-gamma production may not be obligatory for delayed-type hypersensitivity reactions or for the adjuvant action of Quil A or DXS, and that specific mAb can alter the profile of cytokines produced by antigen-reactive cells in sheep.

Induction of CD4+ and CD8+ T cell responses in efferent lymph responding to Toxoplasma gondii infection: analysis of phenotype and function

The kinetics of induction of T cell responses were examined in efferent lymph from a node draining the site of a primary inoculation of Toxoplasma gondii. The numbers of T cells increased after infection, due initially to an expansion of the CD4+ T cell population followed by an increase in the number of CD8+ T cells which coincided with the peak lymphoblast response. Proliferative responses of CD4+ T cells to T. gondii antigen were detectable from day six after infection and immune efferent lymph cells inhibited the intracellular multiplication of T. gondii in vitro. Optimum inhibition was achieved using CD8+ T cells restimulated in vitro, and the effector function appeared to be directed preferentially against the autologous rather than the allogeneic infected target cell. The results provide unique information on the induction of immune responses to T. gondii in vivo and provide evidence that both CD4+ and CD8+ T cells are necessary for the development of protective immunity induced by the S48 strain of T. gondii which is used as a live vaccine in sheep.

Initial lentivirus-host interactions within lymph nodes: a study of maedi-visna virus infection in sheep

Reactive changes occurring within lymph nodes draining the subcutaneous site of acute infection with maedi-visna virus (MVV) were studied, and the appearance of infected cells correlated with the immune response. Cells infected with virus were detected in the node by cocultivation from day 4 postinfection (p.i.), with maximum numbers being seen between days 7 and 14, but even then infected cells were rare, with a maximum frequency of 23 50% tissue culture infective doses (TCID50) in 10(6) lymph node cells. At later times, infected cells were still detected, but their numbers fell to 1 to 2 TCID50 per 10(6) cells. Virus-specific CD8+ cytotoxic T-cell precursors (CTLp) were isolated from infected nodes from day 10 p.i. onwards, and T-cell proliferative responses to MVV were first detected on day 7 and consistently detected after day 18. Histological analysis showed a vigorous immune response in the node. There was a marked blast reaction in the T-cell-rich zones, which was greatest at the time when the number of virally infected cells was at its height. At this stage, large numbers of plasma cells were seen in the medullary cords, indicating that extensive T-cell-dependent B-cell activation was occurring in the T-cell-rich zones. Germinal centers were prominent shortly after the onset of the T-zone response and were still present at 40 days p.i. Phenotype studies of isolated lymph node cells failed to detect major changes in the proportion or phenotype of macrophages, CD1+ interdigitating cells, and CD4+ or CD8+ T cells despite the fact that CD8+ lymphoblasts form a major population leaving the node in efferent lymph. This suggests that there is a balanced increase in the number of all cell types in response to the virus within the node and selective migration of CD8+ lymphoblasts containing virus-specific CTLp from the node. Virus-specific immune responses are therefore present within the node when infectious virus isolation is maximal, but cellular immunity may act to control the level of infection from day 18 onwards.

Response of the regional lymph node to bluetongue virus infection in calves

Bluetongue virus (BTV) infection of cattle is characterized by prolonged cell-associated viremia. In an effort to further evaluate the antiviral response of BTV-infected cattle, the role of the regional lymph node (LN) in the immune response of calves to BTV was characterized. Calves were inoculated with BTV in the skin of the neck in an area drained by the superficial cervical LN. Calves were euthanized at regular intervals after inoculation and both BTV-challenged and contralateral (control) superficial cervical LNs were harvested. In addition, some calves had cannulation of the superficial cervical efferent lymphatics prior to inoculation. Lymphocyte subpopulation analysis was done on LN cell suspensions and lymph cells using a panel of cell-specific monoclonal antibodies. There was a significant increase in the proportion of B cells in the challenged LN after inoculation as compared with the control LN. In addition, BTV-specific antibodies were detected in efferent lymph plasma from the challenged LN in one cannulated calf by 9 days after inoculation (DAI), as determined by competitive enzyme-linked immunosorbent assay, whereas BTV-specific antibodies were not detected in serum from this calf through 12 DAI. Analysis of lymph cells revealed a sustained increase in cell output from the challenged LN due to an increase in lymphoblasts and CD8 + T cells. In contrast, the cell output from the control LN dropped markedly by 8 DAI and there was no significant increase in any specific cell population. Double label analysis characterized lymphoblasts as activated CD8 + cells, as determined by expression of MHC Class II antigens (CD8 + MHC II +). These cells were only transiently present as CD8 + MHC II + cells were not identified in lymph from the challenged LN at 14 DAI. Few CD8 + MHC II + cells were identified at any time in lymph from the control LN or in lymph from a mock infected calf. The data indicate that B cell proliferation in the challenged LN and release of activated CD8 + cells from this LN were specific responses to BTV infection. The rapid expansion of activated CD8 + T cells indicates that these cells may limit early viral spread. It is concluded that the regional LN draining inoculated skin is critical to the immune response of calves to BTV infection.

Endotoxin-mediated dendritic cell release from the intestine. Characterization of released dendritic cells and TNF dependence.

Dendritic cells (DC) acquire Ag in peripheral tissues and transport it to lymph nodes where they efficiently activate resting T cells. We have shown that i.v. endotoxin causes increased release of intestinal DC into lymph. In this paper we further characterize the release of DC and the properties of the released cells. A total of 50 micrograms of endotoxin injected i.v. causes an increase in DC output within 6 h that peaks between 12 and 24 h, with a maximum output of 8 to 15 times normal. At the same time lymphocyte output is markedly decreased. The increased output of DC is followed by a decrease to subnormal levels. The stimulated release of DC is almost totally blocked by a monoclonal anti-TNF-alpha Ab. A second injection of TNF-alpha does not result in further DC release. DC are not released from lymph nodes into efferent lymph by endotoxin. DC collected from lymph after endotoxin treatment show increased expression of the p55 IL-2 receptor and the OX48 Ag but otherwise resemble normal lymph DC. In functional assays they show no significant differences from normal in their ability to stimulate a MLR or to present Ags to sensitized T cells. Immunocytochemistry with the use of MRC OX62 suggests that the DC are released into lymph from the lamina propria of the small intestine. The stimulated release of DC mediated by TNF-alpha may be important in regulating Ag presentation in lymph nodes draining inflammatory sites.

Recombinant ovine interferon gamma inhibits the multiplication of Chlamydia psittaci in ovine cells

The local production of interferon-gamma (IFN-gamma) in sheep in response to Chlamydia psittaci was measured by cannulation of the efferent lymph duct draining the site of challenge inoculation. Peak production of IFN-gamma (256 U/ml) was detected 24 h after challenge. Based on these physiological data, functional studies were carried out in vitro to determine the effect of recombinant ovine (rOv) IFN-gamma on the multiplication of C. psittaci in ovine fibroblasts. IFN-gamma inhibited the multiplication of C. psittaci in ovine cells over a range of concentrations (250 U/ml to 2.5 U/ml) in a dose-dependent manner. The inhibition of chlamydial multiplication was most pronounced when cells were treated with rOvIFN-gamma for 24 h before infection. The addition of exogenous L-tryptophan (500 micrograms/ml) to cultures within 48th of infection abrogated the anti-chlamydial effect of rOvIFN-gamma thus suggesting that tryptophan deprivation is an anti-chlamydial mechanism induced by rOvIFN-gamma in these ovine cells.

Kinetics of the local and systemic antibody response to primary and secondary infection with S48 Toxoplasma gondii in sheep

Vaccination of sheep with live tachyzoites of Toxoplasma gondii, strain S48, affords protection against subsequent challenge with the parasite, but the mechanisms of immunity have not been fully determined. To understand better the nature of the antibody response the kinetics of both local and systemic antibody production were monitored in vaccinated sheep by means of an enzyme-linked immunosorbent assay and Western blotting. Local specific IgG production was analysed in efferent lymph obtained from the cannulated pre-femoral lymph node draining the site of infection. Antibody in efferent lymph plasma and peripheral blood serum from animals vaccinated with S48 tachyzoites was monitored and compared with IgG production in vaccinated sheep given a secondary tachyzoite challenge. Secondary challenge resulted in a clear immunological memory response, antibody being detected in the lymph 3 to 4 days after infection as compared with 7 to 8 days after a primary infection. IgG production was dominated by antibody recognizing a protein with an apparent molecular weight of 30 kDa, but other antigens (32, 24 and 11 kDa) were also readily detected.

Early stages of infection with Trypanosoma congolense: Parasite kinetics and expression of metacyclic variable antigen types

Trypanosoma congolense develops in the skin of sheep at the site of inoculation of metacyclic trypanosomes, forming a chancre containing large numbers of parasites. By cannulating the afferent and efferent lymphatic ducts draining the skin and regional lymph node, the progressive development and migration of trypanosomes from the chancre was monitored and the expression of metacyclic antigen types (M-VATs) was determined. The kinetics of development of parasitosis in the afferent and efferent lymph was similar. Trypanosomes were detected in lymph 5 to 6 days after the inoculation of cultured metacyclic trypanosomes, at the same time as the chancre first appeared in the skin. The numbers of trypanosomes in the lymph reached their peak levels 8 to 10 days post infection and thereafter numbers fell, although there were still many parasites in the lymph after the chancre had regressed. Trypanosomes in the afferent lymph expressed mainly M-VATs and the absolute numbers of four M-VATs which were monitored increased up to 9 days post infection. There was a fall in numbers by day 10, but 92% of the trypanosomes in the afferent lymph continued to express M-VATs. In contrast, trypanosomes from the efferent lymph were found not to express M-VATs suggesting that a major switch in VAT expression occurs in the lymph node. Specific antibody responses, measured by neutralization tests, were evident 16 to 20 days after infection in afferent lymph but only low levels of antibodies were found in efferent lymph.

Primary and secondary responses of the ovine lymph node to Toxoplasma gondii: Cell output in efferent lymph and parasite detection

Efferent lymphatic cannulation was used to study the dissemination of strain S48 of Toxoplasma gondii and the cell output from the prefemoral lymph node, after infection of both "naive" and vaccinated sheep. In the former the mean cell output decreased for 3 days before reaching a peak at 11 and 12 days, but in vaccinated ewes a similar drop in cell output and subsequent peak occurred significantly earlier, at 24 h and 5 days, respectively. The cellular response in both types of sheep was largely due to a marked increase in blast cells. The detection of live toxoplasms and parasite DNA by mouse inoculation and the polymerase chain reaction, respectively, gave similar results; the parasite was demonstrated in lymph from days 3 to 12 during a primary infection but with a sharp cut-off after day 9 coinciding with the peak blast cell response. Very little evidence of T. gondii was found in lymph of vaccinated sheep after challenge. Immunity, which is thought to be largely T-cell mediated and is sustained without continuous antigenic stimulation, suppresses dissemination of the parasite in the lymph and therefore to other sites, which might include the gravid uterus.

Development of a sandwich immunoassay for the detection of soluble ovine IL-2Rα chain

Following T cell activation with antigen or mitogens, there is an up-regulation of interleukin-2 receptorα (IL-2Rα) chain expression. A high proportion of the IL-2Rα chain is shed from the surface of the T cell in a soluble form following proteolytic cleavage, and thus determination of soluble IL-2Rα (sIL-2Rα) chain is an excellent measure of lymphocyte activation. A sandwich immunoassay for the detection of ovine sIL-2Rα chain has been developed. Three monoclonal antibodies (mAbs) with specificity for the IL-2Rα chain, demonstrated by immunoprecipitation of a 50 kDa protein from an ovine IL-2Rα chain cDNA transfected Chinese hamster ovarian (CHO IL-2R) cell line, were analysed for additive and competitive binding to CHO IL-2R cells and Concanavalin A (Con A) activated ovine lymphocytes, respectively. Two non-competitive ovine IL-2Rα chain specific mAbs were then used in a sandwich immunoassay to detect native sIL-2Rα chain in the supernatant (SN) of Con A activated ovine lymphocytes and recombinant sIL-2Rα chain in the SN of CHO IL-2R cells. Soluble IL-2Rα chain could also be detected in complex biological fluid. In the efferent lymph of a cannulated ovine popliteal lymph node (LN), an increase in the level of sIL-2Rα chain following local alloantigen LN activation was observed. This increase correlated with an increase in the output of activated T cell blasts.

Circulating cytotoxic T lymphocyte precursors in maedi-visna virus-infected sheep

Maedi-visna virus (MVV)-specific cytotoxic T lymphocytes (CTL) were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in the efferent lymph and peripheral blood of sheep chronically infected with MVV. Cytotoxicity was mediated by CD8+ lymphocytes and was specific for particular strains of MVV. These precursor CTL were detected in the blood between day 23 and day 100 after infection via the skin. In one out of seven persistently infected sheep MVV-specific cytotoxicity was seen in uncultured peripheral blood cells. Again the effector population consisted of CD8+ lymphocytes. The only other viral infections in which CTL have been detected in peripheral blood mononuclear cells prior to secondary stimulation are those caused by the simian and human immunodeficiency viruses.

Does Silicone Gel Migrate Via Lymphatics After Subcutaneous Injection?

PP Narini, JL Semple, JB Hay, SJ Lugowski, D Smith. Does silicone gel migrate via lymphatics after subcutaneous injection? Can J Plast Surg 1994;2(2):67-70. Reports have documented the presence of elemental silicon, evidence of silicone elastomer, or silicone polymers (gel) in lymph nodes and other sites distant from implanted prostheses. It has been suggested that this occurs via the lymphatic system; however, the mechanism of spread or migration of silicone has not been previously studied. This study investigated the possible role of lymphatics in the migration of silicone gel. In the sheep model, it is possible to obtain continuous samples of both afferent and efferent lymph by cannulating lymphatic vessels. The drainage areas of subcutaneous lymph nodes in the sheep have previously been studied. In our model, the efferent lymphatic vessel from the prefemoral lymph node was cannulated to obtain samples of lymph (efferent and ‘pseudoafferent’). After baseline samples were collected, 3 to 5 mL of free silicone gel was injected subcutaneously in the drainage area of this node. Samples (5 to 10 ml.) of lymph were continuously collected (for up to 50 days), sealed, stored at −20°C, and then submitted as a group for trace element analysis to quantitate the levels of elemental silicon. No statistically significant increase was seen in baseline levels of elemental silicon after silicone gel injections. Statistically significant higher levels of silicon were found in afferent (mean 799 ± 22 part per billion [ppb]) compared with efferent lymph (mean 607 ± 19 ppb). This experimental study did not identify significant increases in elemental silicon levels in lymphatic vessels after the subcutaneous introduction of free silicone gel. Higher levels were found in afferent versus efferent lymph. This implies that migration of silicone gel does not occur soon after the exposure of a subcutaneous space to free silicone gel, or that the migration of silicone gel may occur by a cellular mechanism that can bypass the lymphatic vessels.

Dynamics of viral spread in bluetongue virus infected calves

The kinetics of viremia and sites of viral replication in bluetongue virus (BTV) infected calves were characterized by virus isolation, serology and immunofluorescence staining procedures. In addition, the role of the regional lymph node and lymphatics draining inoculated skin in the pathogenesis of BTV infection was determined by analyzing efferent lymph collected from indwelling cannulas. Viremia persisted for 35 to 42 days after inoculation (DAI) and virus co-circulated with neutralizing antibodies for 23 to 26 days. Virus was first isolated from peripheral blood mononuclear (PBM) cells at 3 DAI, after stimulation of PBM cells with interleukin 2 and mitogen. BTV was frequently isolated from erythrocytes, platelets and stimulated PBM cells but never from granulocytes and rarely from plasma during viremia. Virus was consistently isolated from erythrocytes late in the course of veremia. Interruption of efferent lymph flow by cannulation delayed the onset of viremia to 7 DAI. BTV was infrequently isolated from lymph cells, and few fluorescence positive cells were observed after lymph and PBM cells were labelled with a BTV-specific monoclonal antibody. Virus was isolated from spleen by 4 DAI and most tissues by 6 DAI, whereas virus was isolated from bone marrow only at 10 DAI. Virus was not isolated from any tissue after termination of viremia. It is concluded that primary viral replication occurred in the local lymph node and BTV then was transported in low titer to secondary sites of replication via infected lymph and PBM cells. We speculate that virus replication in spleen resulted in release of virus into the circulation and non-selective infection of blood cells which disseminated BTV to other tissues. Virus association with erythrocytes likely was responsible for prolonged viremia, although infected erythrocytes eventually were cleared from the circulation and persistent BTV infection of calves did not occur.