Peroxynitrite production, DNA breakage, and poly(ADP-ribose) polymerase activation in a mouse model of oxazolone-induced contact hypersensitivity.

Peroxynitrite-induced poly(ADP-ribose) polymerase activation has been implicated in the pathogenesis of various inflammatory conditions. Here we have investigated whether peroxynitrite and poly(ADP-ribose) polymerase may play a role in the pathophysiology of the elicitation phase of contact hypersensitivity. We have detected nitrotyrosine, DNA breakage, and poly(ADP-ribose) polymerase activation in the epidermis of mice in an oxazolone-induced contact hypersensitivity model. As tyrosine nitration is mostly mediated by peroxynitrite, a nitric-oxide-derived cytotoxic oxidant capable of causing DNA breakage, we have applied peroxynitrite directly on mouse skin and showed poly(ADP-ribose) polymerase activation in keratinocytes and in some scattered dermal cells. We have also investigated the cellular effects of peroxynitrite in HaCaT cells, a human keratinocyte cell line. We found that peroxynitrite inhibited cell proliferation and at higher concentrations also caused cytotoxicity. Peroxynitrite activates poly(ADP-ribose) polymerase in HaCaT cells and poly(ADP-ribose) polymerase activation contributes to peroxynitrite-induced cytotoxicity, as indicated by the cytoprotective effect of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. The cytoprotective effect of 3-aminobenzamide cannot be attributed to inhibition of apoptosis, as apoptotic parameters (caspase activation and DNA fragmentation) were not reduced in the presence of 3-aminobenzamide in peroxynitrite-treated cells. Moreover, poly(ADP-ribose) polymerase inhibition by 3-aminobenzamide dose-dependently reduced interferon-induced intercellular adhesion molecule 1 expression as well as interleukin-1beta-induced interleukin-8 expression. Our results indicate that peroxynitrite and poly(ADP-ribose) polymerase regulate keratinocyte function and death in contact hypersensitivity.

Changes of virulence factors accompanying the phenomenon of induced fluconazole resistance in Candida albicans.

We investigated a fluconazole-sensitive (MICflu = 5 micrograms ml-1) clinical isolate and a fluconazole-resistant (MICflu > 80 micrograms ml-1) laboratory mutant Candida albicans strain developed from the sensitive one. We studied putative virulence factors including germination, adherence ability to either buccal epithelial cells or acrylate surface, the secreted aspartic proteinase, and the extracellular phospholipase activity of the two strains as well as their growth. The fluconazole-resistant strain proved to be superior to the original strain in all the virulence traits tested. The higher virulence of the fluconazole-resistant strain was also supported by a mouse model. These results suggest that the development of fluconazole resistance can be accompanied by serious morphological and physiological changes: several putative virulence traits, moreover the in vivo virulence can increase simultaneously.

The relief of biliary obstruction in experimental rats by diverting the hepatic lymph into the duodenum.

Twelve days after their common bile ducts had been ligated rats were icteric and their hepatic lymph nodes and lymph ducts were two or three times their normal size. Cannulation of the hepatic lymph ducts of these rats yielded bile-stained lymph which flowed at nine times its normal rate. As the lymph flowed, so the concentration of bilirubin in the blood declined; after 5 days the rats were no longer jaundiced, and by day 11 there were normal amounts of bilirubin in the blood. In another series it proved possible on seven occasions to insert the free end of the hepatic lymph cannula into the duodenum at the time that the bile was obstructed. Five of these animals showed no increase in serum bilirubin and remained in good condition until the experiments were terminated up to 61 days later. It seems that the effects of biliary obstruction can be mitigated by shunting the hepatic lymph into the intestine.

IgA antibodies in the bile of rats. V. Primacy of the GALT as a source of IgA.

In each of a series of rats the common bile duct and the thoracic duct (cisterna chyli) were cannulated so that both bile and thoracic duct lymph could be collected quantitatively for several hours. The concentrations of IgA in samples of lymph and bile were measured by radioimmunoassay so that the output of IgA per unit time could be calculated. Although the output of IgA in the lymph did not decline significantly, the output in the bile fell so that by 2 hr it had been reduced to less than 20% of the peak value. Similar experiments in rats which had been immunized actively by injecting antigens into the GALT showed a corresponding rapid decline in titres of specific biliary antibodies after fistulation of the thoracic duct. The low levels of IgA in the bile of rats that had been drained of thoracic duct lymph were restored quickly to normal values by the intravenous infusion of a volume of thoracic duct lymph equal to that which had been lost; this restoration was transient, and the concentration of IgA in the bile soon declined again after the infusion ceased.

A quick method for obtaining lymph-borne dendritic macrophages from rats.

After conventional cannulation of the thoracic duct (cisterna chyli) of rats, the mesenteric nodes were frozen solid by the application of solid carbon dioxide and a metal probe that had been cooled to the temperature of liquid nitrogen. This procedure destroyed the functional integrity of the node so that peripheral intestinal lymph, rich in dendritic macrophages flowed, unaltered, into the thoracic duct. In this way peripheral intestinal lymph could be collected immediately and the time-consuming and expensive two-stage procedure of conventional surgical lymphadenectomy was avoided.

Some properties of dendritic macrophages from peripheral lymph.

Peripheral lymph was collected from the skin and liver of sheep, and from the intestine of rats. The dendritic macrophages contained in it were isolated by centrifuging the lymph over a layer of 'Nycodenz'. Similar cells were produced by culturing mononuclear cells from venous blood, but the yields were very small. The numbers of dendritic cells in the lymph from the legs of sheep increased five-fold after xylene had been applied to the skin. Dendritic macrophages displayed abundant class II histocompatibility antigens on their surfaces, as well as immunoglobulins. Although the latter were probably acquired passively, they remained present for several days on cells cultured in vitro. When in vitro, dendritic cells could be shown to phagocytose marker particles, such as latex beads, but their performance was unimpressive compared to macrophages from the peritoneal cavities of rats. In contrast, their ability to phagocytose rapidly T4 phage or influenza viruses unequivocal and striking.

Immunity to a syngeneic sarcoma induced in rats by dendritic lymph cells exposed to the tumour either in vivo or in vitro.

Rats were prepared surgically so that peripheral intestinal lymph could be collected from them while a syngeneic tumour (the HSN sarcoma) was growing in each major Peyer's patch of the small intestine. Dendritic lymph cells were isolated from the lymph and injected i.p. into naive, syngeneic rats. Each of the 16 recipients received just under 10(6) such cells and was challenged 10 days later with a subcutaneous dose of 10(4) viable HSN cells. Six weeks after this challenge only 7 of the recipients had a tumour and these were small (mean weight 1.8 g), while 17 controls (which had each been treated with 10(6) thoracic duct lymphocytes from the same donors, and given the same challenge) all had large tumours (mean weight 8.8 g). The remaining 9 test rats were still free of tumours when they were killed and autopsied 4 months after challenge. Dendritic lymph cells from normal rats were 'sensitised' by incubating them overnight on a monolayer of HSN cells. They were then transferred to 5 naive recipients which received the usual challenge. Six weeks later they all had tumours (mean weight 1.3 g) but these were much smaller than those in the 5 controls (mean weight 9.3 g).

The shedding of viable cells into the local lymph by tumours growing in the gut of rats.

Suspensions of syngeneic sarcoma cells were injected into the Peyer's patches of rats from which the mesenteric nodes had been removed. By later cannulating the thoracic duct of such rats it was possible to collect peripheral intestinal lymph that had come directly from the tumour bearing area without being filtered through a regional node. The number of viable tumour cells in the lymph coming from the tumours was monitored by culturing the whole lymph cells in a limiting dilution assay. The tumours grew to a diameter of approximately 1 cm in 25 days and during this time tumour cells were present in the lymph at a ratio of approximately 1 tumour cell per 10(5) lymph cells. In euthymic rats this number declined as the immune response developed. In athymic rats the number increased by approximately 10 fold during the experiments. It was concluded that the shedding of viable cells parallels the linear, not the volumetric dimensions of the tumour.

The production of hybridomas from the gut associated lymphoid tissue of tumour bearing rats. I. Mesenteric nodes as a source of IgG producing cells.

Rat X rat hybridomas secreting antibodies with tumour specificity have been prepared using cells from the mesenteric nodes of rats bearing syngeneic sarcomata in their Peyer's patches. The antibodies obtained, which embraced all of the major immunoglobulin classes, varied in cellular reactivity from the individually tumour specific to cross-reactive with both normal and tumour cells. Several IgA producing hybridomas were prepared using this protocol but IgG secretors were obtained more frequently and they accounted for about half of the specific hybridomas. Specific IgG producers were found to predominate also when hybridomas were prepared from the mesenteric nodes of a rat immunized by injection of horseradish peroxidase into the Peyer's patches. Comparison of these data with our earlier results using spleen cells taken from rats that were either hyperimmunized with, or were bearing the same tumours in the leg, show that mesenteric nodes draining a tumour growing in the Peyer's patches are a much better source specific IgG producing B cells.

The production of hybridomas from the gut associated lymphoid tissue of tumour bearing rats. II. Peripheral intestinal lymph as a source of IgA producing cells.

Peripheral intestinal lymph afferent to the mesenteric nodes has been collected from rats bearing syngeneic sarcomata in their Peyer's patches and the B cells used to produce rat X rat hybridomas. Analysis of the hybridoma supernatants by radioimmunoassay for the presence of immunoglobulins, showed that hybridomas secreting IgA predominated. Eleven out of the 15 hybridomas selected for antibody binding to cells of the immunising tumour secreted IgA antibodies, and six of these were tumour specific. Efferent mesenteric lymph (i.e. normal thoracic duct lymph), on the other hand, was found to be a poor source of B cells for hybridoma production and no specific IgA secreting hybridomas were obtained. The high yield of IgA secreting hybridomas obtained shows that peripheral intestinal lymph is a better source of IgA committed B cells than are the mesenteric nodes or thoracic duct lymph. We conclude that the IgA producing cells in the latter tissues are too far along the differentiation pathway to plasma cells to undergo successful somatic cell fusion.

Effect of alcohol on the integrity of the intestinal epithelium.

The absorption of macromolecules from the small intestine of rats was studied in terms of the amount of peroxidase activity that appeared in thoracic duct lymph after a 10 mg dose of horseradish peroxidase had been injected directly into the lumen of the duodenum. When the horseradish peroxidase was injected as a solution in saline no peroxidase activity was detected in the lymph. When ethyl alcohol was included in the dose at final concentrations of 12.5-16% the flow rate of the lymph increased markedly for an hour or so and during this time peroxidase activity was detected in the lymph. An electronmicroscope study of the duodenal epithelium that had been exposed to alcoholic solutions of horseradish peroxidase showed that the enzyme had penetrated between the enterocytes. It was concluded that the presence in the intestine of substantial amounts of alcohol temporarily destabilises the intercellular junctions of the epithelium and thus promotes the absorption of materials which are normally excluded.

IgA antibodies in the bile of rats. III. The role of intrathoracic lymph nodes and the migration pattern of their blast cells.

Eight weeks after rats had had their mesenteric lymph nodes (MLN) removed surgically, they were found to be still able to generate substantial titres of biliary IgA-antibodies after antigens were injected into their Peyer's patches. This suggested that systemically significant IgA production could be induced in extra-abdominal lymphoid tissue. It was found that the intrathoracic lymph nodes (ITLN) were an important source of IgA production. These nodes could be stimulated to produce biliary antibody by introducing antigen either into the peritoneal cavity or directly into the thorax. Cells forming IgA were identified in the ITLNs by haemolytic plaque assays and immunoperoxidase techniques. In spite of this, immunoblasts obtained from the ITLNs, and labelled with 125IUdR did not localize in the gut after i.v. injection to anywhere near the extent that immunoblasts from the MLN did. Instead they seemed to have a predilection for localizing in the lungs.

Production of IgA secreting hybridomas: a monoclonal rat antibody of the IgA class with specificity for RT1c.

We report a method for the production of rat hybridomas secreting IgA antibodies with biological activity. By fusing the rat myeloma Y3.AG.1.2.3 with mesenteric lymph node cells taken from animals that had been immunised via the Peyer's patches with allogeneic cells we have obtained a hybridoma secreting monoclonal antibodies of the IgA class with specificity for RT1c.

Comparative aspects of the transport of immunoglobulin A from blood to bile.

Polymeric and monomeric human IgA were isolated from the sera of patients with IgA myeloma; rat IgA polymer, monomer and IgG2 were isolated from the ascitic fluid or sera of Lou/Wsl rats bearing appropriate myelomata. The purified Ig preparations were labelled with 125I and injected intravenously into rats, rabbits, guinea-pigs or sheep that had had a cannula inserted into the common bile duct so that their bile could be collected quantitatively. Rats and rabbits transported 30% of the injected dose of both IgA polymers, but no other type of immunoglobulin, from blood to bile within 5--7 h. Sheep and guinea-pigs were unable to transport any of the immunoglobulin preparations from blood to bile, even though the injected material remained circulating in the blood.

Studies of the transport of polyclonal IgA antibody from blood to bile in rats.

Bile or thoracic duct lymph, collected from rats 7-9 days after suspensions of B. abortus, S. typhi or SRBC had been injected into the Peyer's patches, contained high titres of specific agglutinins. Samples of these fluids were injected i.v. into unimmunized, syngeneic recipients and the partitioning between blood and bile of the injected antibodies was studied and found to depend on the source and class of the antibody. IgA antibodies from lymph plasma disappeared rapidly from the recipients' blood and half of the dose was recovered in the bile within 2 h of its injection. IgA antibodies which had been collected from bile and so had previously traversed the liver and acquired secretory component, appeared in the recipients' bile much less rapidly so that less than half of the dose entered the bile over a period of 40 h. Passively administered IgG antibodies did not enter the recipients' bile to any significant extent and specific haemolysins never appeared in the bile after either passive or active immunization.

Levels of adenosine deaminase in some experimental animal tumours and the possible therapeutic effect of the ADA inhibitor 2-deoxy-coformycin.

The intracellular adenosine deaminase activities (ADA) in 12 different experimental animal tumours were measured. Unlike the leukaemic lymphoblasts of man, those of two spontaneous rat leukaemias did not have elevated levels of the enzyme. Very high levels were found in a rat plasma-cell tumour (IR 461) and an attempt was made to treat such tumours with the specific enzyme inhibitor, 2-deoxy-coformycin. The shortage of this drug prevented a systematic study, but a daily dose of 8 mg/kg had a significant inhibitory effect on the growth of tumours.

Microenvironmental arginine depletion by macrophages in vivo.

Since the tumour-selective cytotoxic activity of activated macrophages in vitro can be attributed to depletion of the culture medium of L-arginine by macrophage arginase, a series of experiments was designed to determine whether such a mechanism could operate in vivo. Extracellular fluid obtained from Gullino chambers within established tumours contained high levels of arginase, no detectable arginine and high levels of ornithine. When tumours were disaggregated into single-cell suspensions, arginase was readily detected within tumour macrophages but not within malignant cells. Inflammatory ascites induced in mice by Corynebacterium parvum was rich in arginase, depleted of L-arginine and cytotoxic in vitro to L5178Y and V79 cells. High levels of arginase in the ascites fluid were associated with resistance to challenge with syngeneic L5178Y cells. Lymph collected from the cisterna chyli in rats bearing a macrophage-rich sarcoma on the small bowel contained elevated levels of arginase, was depleted of arginine and contained increased concentrations of ornithine. We conclude that in sites of macrophage infiltration there is microenvironmental arginine depletion due to the action of arginase, and that arginase release could represent an important macrophage effector mechanism against a variety of targets, including malignant cells, virus-infected cells, fungi and parasites.