Cytokine mRNA expression in leukocytes of efferent lymph from stimulated lymph nodes in pigs.

To test the hypothesis that characteristic cytokine responses occur in stimulated porcine lymph nodes (LNs), lymph node efferent ducts were surgically cannulated. Efferent lymph (EL) leukocytes were collected before and after stimulation of LNs with mitogens [bacterial lipopolysaccharide (LPS) or phytohemagglutinin-P(PHA-P)] and antigens [hen egg white lysozyme (HEWL) or purified protein derivative of tuberculin (PPD)]. Cytokine mRNA expression was evaluated by quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). Interleukin (IL)-1alpha was predominantly produced after all stimuli except for HEWL after which tumour necrosis factor (TNF)-alpha message was dominant. None of the stimuli induced message for IL-2, IL-4 or IL-8. Other cytokine mRNAs were produced in variable amounts and percentage of overall production of each cytokine message was in the following descending rank: LPS: IL-1alpha, TNF-alpha, interferon (IFN)-gamma, IL-10, IL-12-p35, IL-6, IL-12-p40 and TNF-beta; PHA-P: IL-1alpha, TNF-alpha, IL-10, IFN-gamma, IL-12-p40 and TNF-beta; HEWL: TNF-alpha, IL-1alpha, IFN-gamma, IL-10, IL-6, IL-12-p40, TNF-beta and IL-12-p35 and PPD: IL-1alpha, IFN-gamma, TNF-alpha and IL-10. Time course response of cytokines revealed early (IL-1alpha, 10, TNF-alpha) and intermediate (IL-12-p40, TNF-beta, IFN-gamma) responses for PHA-P and early (IL-1alpha, 6, 10, IL-12-p35, IL-12-p40, TNF-alpha), intermediate (TNF-beta, IFN-gamma) and late (IL-1alpha, 6) for LPS. Cytokine mRNA response induced by HEWL was early (IL-alpha, IFN-gamma), intermediate (IL-10, IL-12-p40, TNF-beta), late (IL-1alpha, IL-12-p35) and very late (IL-1alpha, 6, 10, IL-12-p40, TNF-alpha). In Bacillus Calmette-Guérin (BCG) sensitized pigs, stimulation of LNs with PPD induced message for IL-1alpha, 10, TNF-alpha and IFN-gamma which peaked at 24h. Cytokine mRNAs varied by stimulus and differed for antibody and cell-mediated immune response.

Cytokines in Mycoplasma hyorhinis-induced arthritis in pigs bred selectively for high and low immune responses.

Yorkshire pigs were bred selectively for high and low immune responses (H and L pigs, respectively) based on multiple antibody (Ab) and cell-mediated immune response traits. In a previous experiment, generation 4 (G4) pigs of each line were infected with Mycoplasma hyorhinis. High responders had a more rapid and higher Ab response and less polyserositis, but arthritis was more severe in H pigs than in L pigs. To test the hypothesis that line differences were attributable to differential expression of cytokines, M. hyorhinis infection was induced in pigs of G8. Arthritis was more severe clinically (P,

Immune-endocrine interactions in agricultural species: chromium and its effect on health and performance.

The purported performance-enhancing effects of supplemental Cr, as elaborated in studies focusing on beef and dairy cattle models of agricultural stress, affect both immune and endocrine pathways. Furthermore, interactions between the immune and endocrine systems, associated with the actions of insulin and cortisol, may be coordinated through the production of regulatory cytokines. Unlocking the mechanism of action of Cr as a useful farm animal management tool may provide further understanding of the health and performance ramifications of immune-endocrine interactions in agricultural species.

A novel type of adhering junction in an epithelioid tumorigenic rat cell culture line.

Two major types of plaque-bearing adhering junctions are commonly distinguished: the actin microfilament-anchoring adhaerens junctions (AJs) and the desmosomes anchoring intermediate-sized filaments (IFs). Both types of junction usually possess the common plaque protein, plakoglobin, whereas the other plaque proteins and the transmembrane cadherins are mutually exclusive. For example, AJs contain E-, N-, or P-cadherin in combination with alpha- and beta-catenin, vinculin and alpha-actinin, whereas in desmosomes, desmogleins and desmocollins are associated with desmoplakin and one or several of the plakophilins (PP1-3). Here we describe a novel type of adhering junction comprising proteins of both AJs and desmosomes and the tight junction (TJ) plaque protein, ZO-1, in a newly established, liver-derived tumorigenic rat cell line (RMEC-1). By immunofluorescence microscopy, cell-cell contacts are characterized by mostly continuous-appearing lines which are usually resolved by electron microscopy as extended arrays of closely spaced small plaque subunits. These plaque-covered regions are positive for plakoglobin, alpha- and beta-catenin, the arm-repeat protein p120, vinculin, desmoplakin and protein ZO-1. They are positive for E-cadherin in cultures early on in passaging, but tend to turn negative for all known cadherins in densely grown cultures. On immunoblotting SDS-PAGE-separated proteins from dense-grown cell monolayers, "pan-cadherin" antibodies have reacted with a band at approximately 140 kDa, identified as N-cadherin by peptide fingerprinting of the immunoprecipitated protein, which for reasons not yet clear is modified or masked in immunolocalization experiments. The exact histological derivation of RMEC-1 cells is not known. However, the observations of several endothelial markers and the fact that all cells are rich in IFs containing vimentin and/or desmin, while only subpopulations also reveal IFs containing CKs 8 and 18, is suggestive of a mesenchymal, probably endothelial origin. We discuss the molecular relationship of this novel type of extended junction with other types of adhering junctions.

Hemostatic factors in rabbit limb lymph: relationship to mechanisms regulating extravascular coagulation.

Mechanisms regulating extravascular coagulation in interstitial fluids of peripheral tissues are poorly understood, since measurements of hemostatic factors in these fluids are unavailable. Because lymph from a body region reflects the composition of its interstitial fluid, we measured hemostatic factors in limb lymph of rabbits both as activity and as antigen. Mean lymph-to-plasma activity ratios were the following: fibrinogen, 0.28; prothrombin, 0.26; factor X, 0.27; factor VII, 0.17; and factors V and VIII, 0.08. All lymph fibrinogen was clottable; fibrin degradation products were absent. Lymph von Willebrand factor antigen was < 10% of plasma antigen and consisted primarily of lower molecular weight multimers. Mean lymph-to-plasma activity ratio for antithrombin was 0.38 and for tissue factor pathway inhibitor the ratio was 0.40. Low levels of antithrombin-factor Xa were measurable in lymph. The data are compatible with a basal factor VIIa-tissue factor-catalyzed extravascular activation of factor X that is prevented from progressing to generation of fibrin in limb interstitial fluid and lymph by low levels of factor VIII and factor V and by the inhibitory activity of antithrombin and tissue factor pathway inhibitor.

Kaposi's sarcoma, vascular permeability, and scientific integrity.

On March 13, 1992, Nakamura et al published an article in the journal Science reporting that sulfated polysaccharide peptidoglycan (SP-PG) inhibited the growth and vascular hyperpermeability characteristics of Kaposi's sarcoma-related cells and lesions in nude mice. While examining their key composite Fig 3, A through E, and related Table 2, we were surprised by several photographic features and other irregularities in the figures, which we explored further through a series of experiments. We were unable to confirm some of the pivotal findings. We communicated our concerns to Science but our letter was rejected. After submission of additional analysis, the matter was reopened by Science, but again our correspondence was rejected. Despite extensive review, the salient points raised in our initial correspondence remain unanswered or only tangentially addressed. The original conclusions by Nakamura et al are still not only highly dubious, but the validity of the peer review process and self-correcting nature of scientific inquiry are also called into question.

Alcohol, immunomodulation, and disease.

Recent research findings point to a spectrum of alcohol-induced immune dysfunctions in animal models and humans. Use of alcohol in vivo causes abnormalities in the function and/or structure of a broad array of cells involved in humoral and cellular immunity, including lymphocytes, Kupffer cells and other macrophages, as well as the endothelium of blood vessels and lymphatics. Regulatory cytokines and neuroendocrine factors can mediate some of these immunomodulatory effects which may be further re-phased, exaggerated or unbalanced by other drugs of misuse. A variety of animal models is available to study acute and chronic alcoholism, non-alcohol drug misuse, AIDS as well as other opportunistic infections, and neoplasias, which hold promise of clarifying the role of alcohol as an immunomodulator.

AIDS, alcohol, endothelium, and immunity.

Analogies are drawn between important unknowns in AIDS and alcohol research, related to underlying common pathogenetic mechanisms, immunodysregulation, cofactors, and prominent vascular manifestations. The central role of the blood and lymphatic vasculatures and specifically their endothelial lining in many facets of the immune response is reviewed. Evidence is presented that both alcohol and HIV (as well as other coinfecting viruses in AIDS) target and alter endothelial cells and the angiogenic process. These concepts are further illustrated by a serendipitous viral epidemic among rats on continuous long-term alcoholic and control nonalcoholic diets, where synergism between alcohol and virus appeared to underlie multiple vascular proliferative lesions in the liver. In AIDS and alcoholism/alcoholic liver disease (ALD), the prominent features of dysregulated angiogenesis point to the endothelium as a key player in pathogenesis of these seemingly disparate disorders and potentially in immunomodulation.

Modification of lymphocyte subsets in the intestinal-associated immune system and thymus by chronic ethanol consumption.

Modification of the mucosa-associated intestinal immune system of female C57BL/6 mice was studied during consumption of the Lieber-DeCarli diet supplemented with 5% v/v ethanol or laboratory chow with ethanol (20% w/v) in the drinking water. All groups received ethanol for 11 weeks. Mice fed the Lieber-DeCarli diet had fewer CD8+ cells/villus than the chow-fed controls. Mice that received ethanol in the drinking water had fewer IgA-containing cells and CD8+ cells than controls. There were no differences in the number of cells in the mesenteric lymph nodes between ethanol-treated mice and their respective controls. Nevertheless, chow-fed control mice had more cells than those fed the Lieber-DeCarli control diet. Although no differences were detected in the percentages of CD4+, CD8+, LECAM-1+, and LECAM-1+ CD4+ cells, there was a decrease in the percentage of LECAM-1+ CD8+ cells in ethanol-fed mice when compared with their Lieber-DeCarli controls. Mice receiving ethanol in the drinking water showed alterations in the CD4 CD45RC subsets and in the CD8 CD45RC subsets. Similar results were observed in mice receiving Lieber-DeCarli diets alone or supplemented with ethanol. The low dose, chronic exposure of dietary ethanol in the Lieber-DeCarli-fed mice did not significantly affect the numbers of various thymocyte subsets. But, a decrease in the percentage of CD4- CD8+ cells was observed in the thymus of mice receiving ethanol in the drinking water. Chronic ethanol consumption caused significant decreases in the number of CD8+ and IgA+ cells in the intestinal lamina propria, important in mucosal immune defenses.

Endothelial transdifferentiated phenotype and cell-cycle kinetics of AIDS-associated Kaposi sarcoma cells.

The nature of Kaposi sarcoma (KS) (vascular malignancy vs. discordant angiogenesis) and lineage of the progenitor cell remain unclear. Therefore, AIDS-KS enzyme isolate cultures were prepared from excised skin lesions. Endothelial marker positivity for Factor VIII related antigen (F8RAg), Ulex europaeus agglutinin (UEA), and angiotensin-converting enzyme (ACE) were determined by fluorescence microscopy (FM) and flow cytometry (FCM). DNA cell-cycle analysis was performed using FCM. KS lesions showed large thick-walled channels (F8RAg and UEA strongly +), narrow vascular slits and thin-walled lakes (F8RAg and UEA weakly +), and non-prominent spindle cells (F8RAg and UEA almost uniformly negative). KS cultures yielded heterogenous populations of spindle, stellate, and flattened endothelial-like cells, displaying positivity for F8RAg (64 +/- 3%; mean +/- SE), UEA (40 +/- 9%), and ACE (81 +/- 9%). When injected subcutaneously in the nude mouse these cells failed to produce tumors. During contact inhibition induced quiescence, KS cultures exhibited a high G2M (18 +/- 3%) compared to non-KS (7 +/- 4%; p < 0.04), evidence of an altered proliferative potential consistent with a transdifferentiated or transformed phenotype.

Effective stabilization of ethanol levels in multiple-well tissue culture plates.

Underestimation of ethanol (EtOH) volatility in vitro is a potential source of experimental error. EtOH (0-5% in culture medium) was added to 24- or 96-well tissue culture plates with standard low evaporation lids and incubated at 37 degrees C in humidified 7.5% CO2 and 92.5% air. After 72 hours, approximately 70% of the initial EtOH had disappeared from the aqueous phase of the plate (EtOH volatilization). EtOH concentrations gradually decreased in high-concentration wells (1-5%) and increased in low-concentration wells (0-0.1%) over time. This temporal redistribution of EtOH (EtOH diffusion) was detected after only 1 hour of incubation. Parafilm, Blenderm surgical tape, and ELISA plate-sealing tape barriers inconsistently or inadequately prevented EtOH volatilization and diffusion, but a newly designed plate-sealing clamp (PSC) apparatus inhibited this phenomenon. Rat hepatic sinusoidal endothelial cells cultured with the PSC apparatus maintained intact cell membranes for 72 hours and stable levels of monolayer permeability for at least 48 hours. By stabilizing in vitro EtOH concentrations, the PSC apparatus eliminates a potential source of major experimental error.

Alcohol, hepatic sinusoidal microcirculation, and chronic liver disease.

According to the "intact cell hypothesis," ethanol (EtOH) primarily targets nonparenchymal hepatic sinusoidal and perisinusoidal cells, thereby promoting sinusoidal capillarization, which impairs microcirculatory exchange of nutrients and wastes, promotes tissue fibrosis, and only indirectly damages hepatic parenchyma. To test this hypothesis, sinusoidal ultrastructure and hepatic lymph flow and protein composition were examined in rats up to 16 weeks after intragastric EtOH (36% calories)-high fat infusion (Tsukamoto-French model) (TF). The findings were compared to dietary controls and interpreted in light of restricted transsinusoidal protein movement observed in patients with alcoholic cirrhosis. In vitro, alterations in rat hepatic sinusoidal endothelial cell (RSE) morphology, proliferative index, and transendothelial macromolecular permeability (Evans blue-albumin uptake into microcarrier beads) were determined after acute and more chronic exposure to 0.1%-5 vol% EtOH. TF displayed 75% increased liver size, perisinusoidal collagenosis, and basal lamina deposition, ascitic fluid, and doubling of hepatic lymph liquid and protein flux. In vitro, 1% EtOH retracted RSE cell margins, enhanced transendothelial Evans blue-albumin flux and suppressed proliferative index. Thus, high EtOH concentration, clinically attainable in the portal blood during an alcoholic binge, both in vivo and in vitro, promotes early structural and functional alterations in sinusoidal endothelium, which over time may be responsible for progressive restriction of free intrahepatic exchange of liquid, macromolecules, and migrating immune cells.

Vascular abnormalities in experimental and human lymphatic filariasis.

Whereas clinical descriptions of grotesque lymphedema and standard light microscopy in human filariasis have elucidated the natural progression of this disease, the link between the nematode and vascular abnormalities including elephantiasis remains poorly understood. Accordingly, we examined the nature and distribution of lymphatic and blood vascular derangements in a variety of tissues and organs from 37 ferrets acutely and chronically infected with Brugia malayi and in 15 patients with Wuchereria bancrofti or Brugia malayi infestation (resected skin, subcutaneous tissue, and lymph nodes) using light and transmission electron microscopy, immunohistochemistry, and in vivo microscopy. In ferrets, eosinophilic abscesses and epithelioid and giant cell granulomas with fragmented worms in various stages of disintegration were found in multiple organs. Blood microvasculopathy consisted of endothelial hyperplasia, focal thickening and stenosis, vessel obliteration with marked perivascular infiltration of lymphocytes, plasma cells, eosinophils, and numerous large macrophages laden with a coarse golden-brown pigment. Endothelial ballooning and swelling, pavementing, denuding, scarring, and sludge formation were seen along with high endothelium in atypical locations. Dilated lymphatics were most prominent near adult worms and showed plump endothelium, thickened walls and valves, thrombus formation, and often perilymphangitis and adjacent tissue fibrosis. In vivo microscopy showed wriggling live adult worms in dilated incompetent sludge-filled groin lymphatics even when microfilaremia and peripheral edema were absent. In human tissues, in addition to "pachyderm" skin changes (keratosis, papillomatosis, acanthosis and collagen deposition), there was blood vessel and lymphatic vasculopathy similar to ferrets (angiocentric inflammation, congestion, vasculitis, thrombosis, thickened vessel walls, dilated lymphatics, lymphangitis, reactive lymph nodal hyperplasia and nodal fibrosis). These changes reflect generalized endothelial damage due to worm products, physical injury to valves and vessel walls from lymphatic-dwelling live worms, and host immune reactivity. Whereas adult worms target the lymphatic apparatus, their offspring and the host immune response primarily affects the blood microvasculature.

A quantitative lymphocyte localization assay.

Multiple lesions (up to 100 sites) were induced in the skin of sheep using either allogeneic lymphocytes or, in BCG-sensitized animals, tuberculin. Cells recovered from an indwelling lymph catheter draining a prescapular lymph node were labeled with 111-indium, returned to venous blood, and allowed to circulate for 3 hours. Sheep were killed, and the skin lesions and lymph nodes were removed and counted in a gamma spectrometer. High levels of radioactivity (up to 38,000 cpm/lesion) were recovered from lesions, and only a few hundred cpm were recovered from comparable normal skin sites. Dose-response relationships and time kinetics were demonstrated for these lesions, and the radioactivity on blood and lymph cells was measured. The contribution of cell-free radioactivity was negligible. Using replicate injection sites, analytical, internally controlled studies can now be initiated to study the induction, promotion, and suppression of lymphocyte traffic.

Colonic mucus in human fetal development.

The labelling behaviour of specimens of human fetal colon at different stages of development was examined using eight fluorescently labelled lectins with preferences for different carbohydrate structures. Peanut agglutinin (PNA) binding can be demonstrated in specimens from fetal colons between the 4th and 5th week and the 5th and 6th lunar month. Since CEA may bind to similar structures as PNA, an answer was sought to the question whether these structures are in fact identical. While there is evidence in favour of non-identity, it is not entirely conclusive. PNA-binding to fetal tissues is of interest since PNA binds to adult transformed colonic tissues, but not to normal ones.