Hypothermic perfusion of rabbit livers: effect of perfusate composition (Ca and lactobionate) on enzyme release and tissue swelling

Rabbit livers were preserved by continuous hypothermic (5 degrees C) perfusion at a flow rate of 1 ml/min-1 g-1 for as long as 72 hr. Cell swelling (total tissue water, TTW) and the rate at which intracellular enzymes were released into the perfusate were measured. Livers perfused with a simple NaCl-based solution containing hydroxyethyl starch as a colloid released relatively large amounts of aspartate aminotransferase (AST, 442 +/- 224 u/liter-1 100 g-1) and lactic dehydrogenase (LDH, 1580 +/- 688 u/liter-1 100 g-1) into the perfusate during 72 hr of perfusion. The addition of Ca (0.5 mmol/liter) to the perfusate reduced the leakage of enzymes into the perfusate (AST, 70 +/- 30 u; LDH, 450 +/- 50 u) and reduced cell swelling (TTW, 3.1 kg/kg dry mass vs 4.4 kg/kg dry mass without added Ca). But the use of a higher concentration of Ca (1.5 mmol/liter) caused membrane damage (AST, 4000 +/- 1500 u; LDH, 10,000 +/- 2222 u) and increased cell swelling (TTW, 3.7 kg/kg dry mass). The release of intracellular enzymes caused by continuous perfusion with a chloride-based perfusate also could be reduced by replacing the chloride with lactobionate (AST, 100 +/- 30 u; LDH, 400 +/- 100 u, at 72 hr). In the lactobionate-containing perfusate, the addition of Ca (0.5 or 1.5 mmol/liter) did not alter the rate at which intracellular enzymes were released. There was no tissue swelling after 72 hr of preservation with the lactobionate-containing perfusate, and the TTW (2.1 kg/kg dry mass) was similar to the TTW of freshly harvested rabbit livers.(ABSTRACT TRUNCATED AT 250 WORDS)     

The self-directed T cell repertoire against mouse lysozyme reflects the influence of the hierarchy of its own determinants and can be engaged by a foreign lysozyme.

The T cell repertoire is shaped by the processes of positive and negative selection. We have previously shown that mice are tolerant to a native self-Ag, mouse lysozyme (ML), but they respond vigorously when challenged with different ML peptides ("cryptic" self-determinants). In this study, we have addressed the issue of the physiological significance of both the hierarchy (dominance/crypticity) of self-determinants within ML and the anti-cryptic, self (ML)-directed T cell repertoire. Our results demonstrate that there are several ML peptides that bind well to MHC but are totally nonimmunogenic when tested for proliferative T cell response and cytokine secretion: a subset of these peptides presumably represent the originally dominant self-determinants of ML, which have rendered the T cells tolerant during thymic selection. Other ML peptides, which bind well to MHC and are immunogenic, correspond to the cryptic determinants of ML: T cells against cryptic ML determinants escape tolerance induction. Thus, the mature T cell repertoire against ML bears the direct imprint of the hierarchy of self (ML)-determinants. Interestingly, hen egg white lysozyme could prime T cells in vivo that were cross-reactive with certain cryptic ML determinants, and vice versa, without requiring any coimmunization with the foreign lysozyme and ML peptide(s). Moreover, repeated, deliberate priming and expansion of T cells by hen egg white lysozyme immunization concomitantly enhanced T cell response to such cross-reactive ML determinants. This reciprocal self-foreign determinant cross-reactivity may play a previously unrecognized, but crucial, role in the expansion and diversification of self-reactive clones in the autoimmune response.     

Th2 polarization enhanced by oral administration of higher doses of antigen

Although oral administration of a soluble proteinantigen can induce various immune responses, theeffect of the dosage of oral antigen on thepredominance of Th2-type cytokine and antibodyresponses has not been well clarified yet. In thepresent study, we fed T cell receptor (TCR) transgenic(tg) mice various amounts of chicken ovalbumin (0.1,5, and 250 mg) and examined the resulting immuneresponses to this antigen. In these TCR tg mice, theresponses of antigen-specific T cells were greatlyamplified concomitantly with significantantigen-specific cytokine secretion. We found that ahigh dose (250 mg) of antigen significantlyupregulated the serum antigen-specific IgG1 and IgAantibody responses in mice later intraperitoneallyinjected with antigen plus adjuvant. The miceadministered the same oral dose but not immunizedshowed upregulation of Th2-type IL-4 and IL-5secretion and downregulation of Th1-type IL-2 andIFN-. This enhancement of Th2-type cytokineand antibody responses was more marked when largerdoses of antigen orally administered. These resultsdemonstrated that antigen feeding induces thedevelopment of T cells secreting Th2-type cytokines ina dose-dependent manner and that these T cells have ahelper function for the production of antibodies ofthe Th2-type isotypes. This experimental system shouldbe useful to screen foods and other substances thatcan modulate Th2-type responses relating to allergy.     

Effects of short-term hypothermic perfusion and cold storage on function of the isolated-perfused dog kidney

The isolated-perfused dog kidney was used as a model to measure the effects of short-term hypothermic preservation on renal function and metabolism. Kidneys were cold-stored in Collins' solution, hypotonic citrate, or phosphate-buffered sucrose for 4 and 24 hr, or were continuously perfused for 4 and 24 hr with a synthetic perfusate. Following preservation kidneys were perfused with an albumin-containing perfusate at 37 degrees C for 60 min for determination of renal function. The results indicate that many of the effects of short-term preservation on renal function in dog kidneys are similar to results reported for rat and rabbit kidneys. Cold storage for 4 hr resulted in a large decrease in GFR (57%), but only a small decrease in Na reabsorption (from 97 to 87%). Cold storage for 24 hr caused a further decline in renal function (GFR = 95% decrease, Na reabsorption = 49-64%). Results were similar for all cold storage solutions tested. Perfusion for 4 hr was less damaging to renal function than cold storage. The GFR decreased only 14% and urine formation and Na reabsorption were practically normal. After 24 hr of hypothermic perfusion, the GFR was reduced by 79%, urine flow was normal, and Na reabsorption was 78%. There were no obvious biochemical correlates (adenine nucleotides, tissue edema, or electrolyte concentration) with the loss of renal function during short-term preservation. The results suggest that the isolated-perfused dog kidney can be used to test the effects of preservation on renal function, and yields results similar to those obtained using small animal models.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium localization in the choroid plexus

Summary The localization of sodium ion in the cat choroid plexus was studied by use of potassium pyroantimonate. The precipitates formed by the potassium pyroantimonate occur mostly on the plasma membrane in the epithelial cell and occasionally in the perivascular space. The precipitates in the epithelial cell are most numerous at the apical surface, particularly on the microvilli, and least in number at the basal and lateral surfaces. In the endothelial cell, the dense precipitates are situated on the plasma membrane as well as on the limiting membrane of the pinocytotic vesicle. Although the dense precipitates are sometimes situated on the external surface of the plasma membrane of the epithelial cell, most of them are localized on the internal surface of the plasma membrane. A similar localization of the precipitates is to be seen on the plasma membrane of the erythrocyte. When the cerebrospinal fluid/plasma ion ratio and potential gradients across the choroid plexus are considered, the precipitates on the plasma membrane would suggest a localization of sodium needed for the activation of ATPase.

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Zusammenfassung Die Lokalisation des Natriumions im Plexus chorioideus der Katze wurde mit Hilfe von Kaliumpyroantimonat untersucht. Die durch Kaliumpyroantimonat gebildeten Niederschläge treten meistens an der Plasmamembran in den Epithelzellen und gelegentlich im perivaskulären Raum auf. In den Epithelzellen kommen die Niederschläge am zahlreichsten an der apikalen Oberfläche vor, besonders an den Mikrovilli, am geringsten an den basalen und lateralen Oberflächen. In der Endothelzelle liegen die dichten Niederschläge an der Plasmamembran und an der Grenzmembran der Pinozytosebläschen. Einige der dichten Niederschläge befinden sich an der äußeren Oberfläche der Plasmamembran der Epithelzellen, die meisten aber an der inneren Oberfläche der Plasmamembran. Eine ähnliche Lokalisation der Niedersschläge wurde an der Plasmamembran des Erythrozyten festgestellt. Wenn man das Liquor Plasma-Ionenverhältnis und die Potentialgradienten am Plexus chorioideus in Betracht zieht, liegt es nahe, die nachgewiesene Lokalisation des Natriums auf eine Aktivierung von ATPase zu beziehen.

     

Changes in fecal microflora induced by intubation of mice with Bacillus subtilis (natto) spores are dependent upon dietary components.

We examined changes in mouse fecal microflora after various dietary components and Bacillus subtilis (natto) spores were delivered by intubation. The administration of intact spores of Bacillus subtilis (natto) did not affect fecal Enterobacteriaceae and Enterococcus spp. in all three diet groups; on the other hand, it did affect fecal Bacteroidaceae and Lactobacillus spp., depending upon the diets fed. The administration of autoclaved spores did not alter fecal microflora. In vitro cultures of Lactobacillus murinus obtained from mouse feces, together with Bacillus subtilis (natto) under aerobic conditions as a mixed culture, revealed that the growth of L. murinus was enhanced by the addition of intact spores of Bacillus subtilis (natto). This enhancement of growth was displayed only in media containing either sucrose, glucose, maltose, or fructose but not in media containing cornstarch, soluble starch, or microcrystalline cellulose. From these results it was evident that some metabolites of Bacillus subtilis (natto) produced during germination and (or) outgrowth of spores of this strain, requiring monosaccharides or oligosaccharides, participated in the enhancement of the growth of L. murinus.     

Cutting edge: introduction of an endopeptidase cleavage motif into a determinant flanking region of hen egg lysozyme results in enhanced T cell determinant display

The choice of which determinants of a whole Ag will be presented on cell surface MHC class II molecules after uptake and processing by APC is the result of the interplay between structural characteristics of the Ag and the processing machinery of the APC. In this study, we demonstrate that introduction of a dibasic motif adjacent to a subdominant determinant enhances the presentation of this determinant from the whole molecule. This is the first report showing that a single amino acid substitution in a whole Ag, designed to introduce an endopeptidase recognition site, enhances display of class II-restricted determinants, most likely by creating a peptide chain cleavage in the antigenic molecule. Our findings have important implications for the understanding of immunodominance and for vaccine design.     

Antigen presentation by Peyer's patch cells can induce both Th1- and Th2-type responses depending on antigen dosage,but a different cytokine response pattern from that of spleen cells

The Th1 and Th2 preference induced by cells from the Peyer's patch (PP) and spleen (SPL) with various doses of an antigen was examined. The same splenic T cell receptor-transgenic CD4+ T cells were first incubated with PP or SPL cells in the presence of various doses of an antigen, and the cytokine response was observed after secondary stimulation. A Th2-type pattern was only obtained for primary stimulation at 10 microM of the antigen with PP cells, whereas a Th1 pattern was induced at both higher and lower concentrations. SPL cells in the presence of 0.1 to 1 microM of the antigen induced the secretion of Th2-type cytokines. Ten and 100 microM of the antigen plus SPL cells did not induce the release of a large quantity of cytokines. PP cells induced a different cytokine pattern at the antigen concentration that induced a similar level of T cell proliferation with SPL cells. Our findings suggest that the antigen-dose dependent development of Th1/Th2 cells is differentially modulated by the antigen-presentation function of cells in PP and SPL.