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Hydrogen sulphide (H2S), the third endogenous gaseous signalling molecule, has attracted attention in biochemical research. The selective detection of H2S in living systems is essential for studying its functions. Fluorescence detection methods have become useful tools to explore the physiological roles of H2S because of their real-time and non-destructive characteristics. Herein we report a near-infrared fluorescent probe, NIR-HS, capable of tracking H2S in living organisms. With high sensitivity, good selectivity and low cytotoxicity, NIR-HS was able to recognize both the exogenous and endogenous H2S in living cells. More importantly, it realized the visualization of endogenous H2S generated in cells overexpressing cystathionine β-synthase (CBS), one of the enzymes responsible for producing endogenous H2S. The probe was also successfully applied to detect both the exogenous and endogenous H2S in living mice. The superior sensing properties of the probe render it a valuable research tool in the H2S-related medical research.

Main-stay in treatment of leishmaniasis relies on chemotherapy but none of the current drugs combines high activity and low toxicity at affordable costs. Dinitroanilines are a new class of drugs with proved in vitro antileishmanial activity. However the development of their pharmaceutical formulations has been compromised by low water solubility and low accumulation in diseased organs. These limitations can be overcome by incorporation in lipid-based nanoformulations such as liposomes and solid lipid nanoparticles. In previous work this strategy was already followed with the incorporation of a dinitroaniline, oryzalin, resulting in the improvement of the biodistribution profile. The present work aims at demonstrating the in vitro and in vivo therapeutic activity of these oryzalin nanoformulations, and establishing a systematic comparison of both systems. After oryzalin incorporation suitable physicochemical properties for parenteral administration were obtained. Nanoformulations revealed reduced cytotoxicity and haemolytic activity when compared with free-oryzalin, while retaining the in vitro intracellular activity. Therapeutic activity, assessed in a murine model of visceral leishmaniasis, was evaluated in terms of number of administrations, dose-response and influence of the lipid excipient. Results demonstrate the superiority of both oryzalin nanoformulations on the reduction of parasitic burden in liver and spleen as compared to the control group (84 to 91%) and similar to Glucantime. A strong reduction in ED50 values (3 to 65 fold) as compared to free-oryzalin was also obtained, depending on the organ and nanoformulation used. Both oryzalin nanoformulations are potential candidates as therapeutic agents against visceral leishmaniasis.

Background: Photopatch testing is important for diagnosing photoallergic contact dermatitis. We aimed to evaluate the use of photopatch test at the National Skin Centre, Singapore., Methods: All patients who had photopatch tests done between 2007 and 2011 at the National Skin Centre were included., Results: Twenty-two patients were included. The mean age was 40.2. Female : male ratio was 3.4. The ethnic groups were Chinese (68%), Malay (4%), Indian (14%) and others (14%). Ten out of 22 patients (45.5%) had a positive photopatch test. There were 20 positive photopatch test reactions found in these 10 patients, and all 20 positive reactions were of current relevance. The frequencies of the positive photopatch test reactions were 2-hydroxy-4-methoxybenzophenone (oxybenzone) (n = 6), 2-hydroxymethoxymethylbenzophenone (mexenone) (n = 3), 2-ethylhexyl-4-dimethylaminobenzoate (n = 1), ketoprofen gel (n = 1) and the patient's own product (n = 9)., Conclusions: Our study suggests that sunscreen is the most common photoallergen to date as opposed to musk ambrette, which was the most common photoallergen in our earlier study in 1991-1993. This finding is similar to the recent European Multicentre Photopatch Test Study., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: The objective of this study was to evaluate the utility of plasma Inhibin B (InhB) as a biomarker of testicular injury in adult rats following short-term exposure to the known Sertoli cell toxicants mono-2-ethylhexyl phthalate (MEHP), 1,3 dinitrobenzene (DNB), or carbendazim (CBZ)., Methods: Following oral gavage administration of the compounds for 2 or 7 days, the rats were evaluated for clinical signs, body weight, food consumption, organ weights, plasma hormone levels, and gross and microscopic pathology., Results: MEHP, DNB, and CBZ produced a range of testicular toxicity characterized by minimal exfoliation of germ cells as demonstrated by increased cellular debris in the epididymis (MEHP) to more severe and dose/duration responsive Sertoli cell vacuolation, germ cell degeneration, and multinucleated giant cells of germ cell origin (DNB and CBZ). The slight to moderate Sertoli and germinal cell injuries did not correlate with significant changes in plasma InhB levels following 2- or 7-day exposures. However, more severe injury to germinal epithelium following up to 7 days of exposure, but not after 2 days of exposure, correlated with decreased plasma InhB levels and less consistently with increases in plasma follicle stimulating hormone., Conclusion: In conclusion, under the conditions of these studies, changes in InhB were not an effective early onset marker of testicular toxicity or an effective marker for slight to moderate levels of acute injury, and only reflected more severe disruption of spermatogenesis. Changes in plasma InhB and follicle stimulating hormone were poorly correlated except in some instances of moderate to marked testicular toxicity., (© 2013 Wiley Periodicals, Inc.)

Oryzalin (ORZ) is a dinitroaniline that has attracted increasing interest for the treatment of leishmaniasis. The possible use of ORZ as an antiparasitic agent is limited by low water solubility associated with an in vivo rapid clearance. The aim of this work was to overcome these unfavorable pharmaceutical limitations potentiating ORZ antileishmanial activity allowing a future clinical use. This was attained by incorporating ORZ in appropriate liposomes that act simultaneously as drug solvent and carrier delivering ORZ to the sites of Leishmania infection. The developed ORZ liposomal formulations efficiently incorporated and stabilised ORZ increasing its concentration in aqueous suspensions at least 150 times without the need of toxic solvents. The incorporation of ORZ in liposomes reduced the in vitro haemolytic activity and cytotoxicity observed for the free drug, while ORZ exhibits a stable association with liposomes during the first 24h after parenteral administration, significantly reducing ORZ blood clearance and elimination from the body. Simultaneously, an increased ORZ delivery was observed in the main organs of leishmanial infection with a 9-13-fold higher accumulation as compared to the free ORZ. These results support the idea that ORZ performance was strongly improved by the incorporation in liposomes. Moreover, ORZ liposomal formulations can be administrated in vivo in aqueous suspensions without the need of toxic solvents. It is expected an improvement in the therapeutic activity of liposomal ORZ that will be tested in future work., (Copyright © 2012 Elsevier B.V. All rights reserved.)

1,3-Dinitrobenzene (DNB) causes testicular injury, particularly to Sertoli cells, and induces apoptosis in the surrounding germinal cells in rodents; however, the mechanisms causing this toxicity are poorly understood. Our studies, using standard and molecular tools, were conducted to better understand the pathogenesis of the testicular effects. Four daily oral doses of 0.1-8mg/kg/day caused marked testicular lesions in rats from 4mg/kg/day. Global transcriptomics revealed cell cycle and cell death as the major biological processes affected with the expression of genes associated with cell cycle progression ("mitotic roles of polo-like kinase") being particularly altered. In a single dose time course study (4mg/kg), no adverse changes were recorded; however, in contrast to the data from the multiple dose study, plasma testosterone and testicular steroidogenesis-related gene expression were affected. These steroid hormone effects were confirmed in vitro using the H295R steroidogenesis assay. With this global approach we show that DNB not only induces apoptosis and interferes with cell cycle in the testes but that DNB can also modulate steroid hormone biosynthesis, suggesting an interference with the endocrine system. However, the contribution of the endocrine changes to the severe testicular lesions is presently unknown and requires further investigation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Rats were treated with a single oral dose of 10, 25 and 50mg/kg of 1,3-dinitrobenzene (DNB), and the testis was subjected to a GeneChip microarray analysis. A total of 186 and 304 gene probe sets were up- and down-regulated, respectively, by the DNB treatment, where spermatocyte death and Sertoli cell vacuolation in testis and increased debris of spermatogenic cell in epididymis were noted. The expression profile for four sets of genes were investigated, whose expressions are reported to localize in specific cell types in the seminiferous epithelium, namely Sertoli cells, spermatogonia plus early spermtocytes, pachytene spermatocytes and round spermatids. The data demonstrated that pachytene spermatocyte-specific genes elicited explicit down-regulation in parallel with the progression of spermatocyte death, while other gene sets did not show characteristic expression changes. In addition, Gene Ontology analysis indicated that genes associated with cell adhesion-related genes were significantly enriched in the up-regulated genes following DNB treatment. Cell adhesion-related genes, namely Cdh2, Ctnna1, Vcl, Zyx, Itgb1, Testin, Lamc3, Pvrl2 and Gsn, showed an increase in microarray and the up-regulation of Cdh2 and Testin were confirmed by real time RT-PCR. The gene expression changes of pachytene spermatocyte-specific genes and cell adhesion-related genes were thought to reflect a decrease in the number of spermatocytes and dysfunction of Sertoli-germ cells adhesion junction, and therefore these genes would be potential genomic biomarkers for assessing DNB-type testicular toxicity., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

2,4-dintitrotoluene (2,4-DNT) is an explosive frequently found in the soil of military installations. Because reptiles can be common on these sites, ecological risk assessments for compounds such as 2,4-DNT could be improved with toxicity data specific to reptiles. Western fence lizards, Sceloporus occidentalis, were used to develop a laboratory toxicity model for reptiles. A hierarchical approach was used; acute to subchronic studies were conducted to provide toxicity data relevant to short- and long-term exposures. First, a modified median lethal dose (LD50) study was conducted on male and female lizards using a stage-wise probit model. The LD50 was 577 mg/kg for female and 380 mg/kg for male lizards. Subsequently, a subacute experiment was conducted to further assess 2,4-DNT toxicity to male lizards and to define exposure levels for a longer term, subchronic study. The subchronic study was conducted for 60 consecutive days; male lizards were exposed to 0, 9, 15, 25, 42, 70 mg/kg/d. Dose-dependent mortality was observed in the three highest dose groups (25, 42, and 70 mg/kg/d); all other animals survived the study duration. Benchmark dose model calculations based on mortality indicated a 5% effect level of 15.8 mg/kg/d. At study termination, a gross necropsy was performed, organ weights were taken, and blood was collected for clinical and hematological analysis. Body weight, kidney weight, food consumption, postdose observations, and blood chemistries all were found to be significantly different from controls at doses above 9 mg/kg/d. Also, preliminary results suggest behavioral observations, and reduced food consumption may be a sensitive indicator of toxicity. The present study indicates Sceloporus occidentalis is suitable for evaluating toxicity of compounds to reptilian species.

We established a new and facile model to investigate allergic mechanism and assess the effect of antiallergic compounds. Male Wistar rats were actively or passively sensitized. Active sensitization was performed by injection of both dinitrophenylated-ovalbumin (DNP-OA) and Bordetella pertussis. Nine days later, DNP-OA was injected into the right hind footpad. This antigen challenge induced a biphasic footpad swelling that consisted of an early-phase (EPR) and a late-phase response (LPR). In rats passively sensitized with rat anti-DNP-OA serum, DNP-OA induced only EPR. The EPR was suppressed by disodium cromoglycate, a mast cell stabilizer, but not by cyclosporin A, an immunosuppressant, while the LPR was suppressed by cyclosporin A. Furthermore, to investigate these two allergic responses determined by the interactions between the hapten and the carrier proteins, two distinct haptenated antigens were created. DNP-Ascaris (DNP-As) induced a marked EPR and LPR in DNP-As-sensitized rats. However, DNP-As induced only EPR in DNP-OA-sensitized rats, indicating that the usage of the same carrier protein in both sensitization and challenge was necessary for induction of LPR. These data suggest that this actively sensitization model in which EPR and LPR are functionally distinguishable should be useful for evaluating the efficacy of antiallergic compounds.

Military activities associated with training, munitions manufacturing, and demilitarization has resulted in soil residues of munition compounds and their breakdown products. Two isomers of dinitrotoluene (2,4- and 2,6-) are often found in soil associated with those activities at considerable concentrations. Consequently, issues regarding the effects of exposure to birds that visit these habitats require evaluation. To provide data useful to a risk assessment approach, we followed a controlled dosing regime (gavage) using 2,4-dinitrotoluene (2,4-DNT) in the Northern Bobwhite (Colinus virginianus) for 60 days following a 14-day range-finding study and the determination of a LD50 using the up/down method. The LD50 was determined to be 55 mg/kg using corn oil as a vehicle. Individuals dosed exceeding this level were moribund or died within 60 h of exposure. Morbidity and death occurred during the 14-day range-finding study at dosing regimens of 35 and 55, but not at 15, 5, and 0.5 mg/kg-day. Compound-related morbidity/mortality occurred in the 60-day study during the first week of exposure at 25 and 15, but not at 5, 1, and 0 mg/kg-day. Overt signs of toxicity occurred with both sexes at the onset of exposure. Signs included weight loss, diarrhea, and lethargy. Dose-related changes in egg production, ovary, kidney, and brain mass, and body weight, but not feed consumption, were found. Changes in kidney mass and histological observations suggest accumulation of nitrogenous waste may be the cause of morbidity. These data suggest that oral 2,4-DNT exposures are more acutely toxic and has a different etiology than 2,4,6-trinitrotoluene in birds.

Mice lacking the homeodomain transcription factor Nkx2-3 are either asplenic or develop a spleen of significantly reduced size with poorly organized white pulp. In this report, we analyze the effect of this mutation on B lymphocyte development and differentiation. Follicular dendritic cells in spleen, but not lymph node, of Nkx2-3(-/-) mice fail to express a developmental Ag (follicular dendritic cell-M2) and show an abnormal association with B cells, despite essentially normal expression of several chemokine genes. Bone marrow reconstitution studies show the splenic disorganization and absence of marginal zone B cells to be of stromal rather than hemopoietic origin. Furthermore, Nkx2-3(-/-) mice show an excess of conventional B cells in mesenteric lymph node and peritoneal cavity, whereas transitional B cells are rare in spleen but overrepresented in bone marrow. Finally, immunization of Nkx2-3(-/-) mice with a T cell-dependent Ag elicits clusters of germinal center B cells, although these fail to develop to the same extent as in controls and there is no evidence of affinity maturation in serum Ab. Similarly, Ab-forming cells fail to aggregate into foci early in the response. Collectively, these data indicate a substantial role for Nkx2-3 in the correct association of lymphocytes and splenic stromal elements that is independent of chemokine expression.

This paper deals with the use of derivative potentiometric stripping analysis (dPSA) as a rapid and precise method to determine Cd(II), Cu(II), Pb(II), and Zn(II) levels in red and white wine samples from Sicily, Campania, and Tuscany and to investigate the possible connection between the content of these metals and the pesticide treatments used in vine-growing to control plant diseases and pests. dPSA allowed direct quantitation of heavy metals in acidified wines without any sample pretreatment. Mean recoveries of Cd(II), Cu(II), Pb(II), and Zn(II) ranged from 95.5 to 99.2% for white wine samples and from 96.1 to 100.0% for red wine samples. The obtained results showed that Cd(II) was not found in any sample and that Cu(II), Pb(II), and Zn(II) levels were always lower than the toxicity limits in both fungicide- and water-treated wines. Nevertheless, the contents of metals were increased in samples from organic and inorganic pesticides treatment with respect to the water-treated samples. In particular, quinoxyfen, dinocap-penconazole, and dinocap applications considerably increased Cu(II) and Zn(II) contents in white and red wines. The levels of lead were significantly raised by azoxystrobin and sulfur treatments.

Background: Patch testing is the confirmatory procedure for allergic contact dermatitis. The test requires the application of chemicals under occlusion for approximately 48 hours to maximize penetration, although it can also produce irritation. Photomechanical waves (PW) have been shown to render the stratum corneum transiently permeable and facilitate the delivery of macromolecules into the epidermis. This alternative might reduce prolonged occlusion of the skin to minimize irritancy, while retaining the sensitivity of the test., Objective: PW was used to facilitate the delivery of an allergen into the skin in vivo., Methods: The allergic skin reaction using PW delivery was compared with 5-minute and 21-hour occlusion in a sensitized hairless albino guinea pig model. The pigs were sensitized by intradermal injection of (0.01%) dinitrochlorobenzene (DNCB) and topical administration (0.1%, 1 week later) of the hapten. One month later, testing for the allergic response was performed by the administration with PW of 10 microL of 0.1% DNCB., Results: Our results show that there was an allergic reaction for the 24 hour occlusion or PW delivery of the antigen. In contrast, no response was observed for the 5-minute occlusion with the antigen., Conclusion: The rapid delivery of antigens with PW can improve the test for the diagnosis of contact dermatitis., (Copyright 2001 by W.B. Saunders Company)

As part of a collaborative work, male rats were administered 1,3-dinitrobenzene (1,3-DNB) daily at 0, 25 and 50 mg/kg/day from the age of 6 weeks for 4 weeks (4-week exp.), or at 25, 50 and 75 mg/kg/day from the age of 8 weeks for 2 weeks (2-week exp.). After the end of each administration period, all survivors were sacrificed, and their testes and epididymides were removed, weighed and examined histopathologically. The following results were obtained. In the 4-week exp.: At 50 mg/kg/day, the weights of testes and epididymides showed decrease with macroscopic atrophy. The testicular spermatogenic epithelium showed decrease in the number of sperm-spermatocytes, degeneration/necrosis, giant cell formation and vacuolation, reduction in sperm counts also being evident in the ducts of the epididymides. In the 2-week exp.: At 50 and 75 mg/kg/day, the weights of testes and/or epididymides showed decrease with macroscopic atrophy. Several histopathological changes in the testes and epididymides were essentially the same changes as in the group given 50 mg/kg/day in the 4-week exp., with a clear relation. These results indicate that a 2-week administration period is sufficient to detect testicular and epididymal histopathological changes induced by 1,3-dinitrobenzene in male rats.

Interstitial cystitis (IC) is a debilitating disease that has been adversely affecting the quality of women's lives for many years. The trigger in IC is not entirely known, and a role for the sensory nerves in its pathogenesis has been suggested. In addition to inflammation, increased mast cell numbers in the detrusor muscle have been reported in a subset of IC patients. Experimentally, several lines of evidence support a central role for substance P and neurokinin-1 (NK-1) receptors in cystitis. The availability of mice genetically deficient in neurokinin-1 receptor (NK-1R(-/-)) allows us to directly evaluate the importance of substance P in cystitis. An unexpected finding of this investigation is that NK-1R(-/-) mice present increased numbers of mast cells in the bladder when compared with wild-type control mice. Despite the increase in mast cell numbers, no concomitant inflammation was observed. In addition, bladder instillation of wild-type mice with a sensitizing antigen induces activation of mast cells and an acute inflammatory response characterized by plasma extravasation, edema, and migration of neutrophils. Antigen-sensitized NK-1R(-/-) mice also exhibit bladder mast cell degranulation in response to antigen challenge. However, NK-1R(-/-) mice are protected from inflammation, failing to present bladder inflammatory cell infiltrate or edema in response to antigen challenge. This work presents the first evidence of participation of NK-1 receptors in cystitis and a mandatory participation of these receptors on the chain of events linking mast cell degranulation and inflammation.

m-Dinitrobenzene is a multitarget toxicant. This study presents a concentration-time threshold model in m-dinitrobenzene (m-DNB)-induced neurotoxicity in F344 rats based on pharmacokinetic modeling and variable duration infusions with neuropathological end points. Pharmacokinetic parameters for m-DNB were determined after giving a single i.v. dose of 10 mg/kg m-DNB. Time dependency of the brain lesions was studied by either giving a single bolus i.v. dose of 30 mg/kg m-DNB or infusing this dose over 6, 12, or 24 h, or 2, 4, 6, 8, or 14 days. The results show that the 6-day infusion, in which the theoretical steady-state blood concentration was 2.0 microM, caused brain damage, whereas the 8- and 14-day infusions, in which the steady-state blood concentrations were 1.5 and 0.8 microM, respectively, did not induce apparent brain damage. When this dose was infused over 6 h, the peak blood concentration of m-DNB was 35 microM and the time (T(m)) for which m-DNB exceeded the 2-microM concentration threshold was 18.8 h, but no brain damage was observed. However, when the same total dosage was infused over periods of either 12 or 24 h, or 2, 4, or 6 days, the theoretical blood concentrations were from 21.9 to 2.0 microM and the T(m) was from 22. 7 to 144 h, and brain damage was produced. Hence a T(m) of 22.7 h was considered to be the time threshold for m-DNB-induced brain damage. It is concluded that a high concentration alone does not result in m-DNB-induced neurotoxicity and that in addition to a concentration threshold, there also exists a time threshold. Both apparently need to be exceeded before neurotoxicity is seen., (Copyright 1999 Academic Press.)

To test the hypothesis that glutathione (GSH) status in brain tissue plays an important role in the selective neurotoxicity of m-dinitrobenzene (DNB), the sensitivity to intoxication of three groups of male F344 rats were studied and correlated with brain tissue GSH levels. In Group I were young 6-8 week old rats with normal GSH levels, and in Group II were rats of the same age whose brain GSH levels had been reduced by intracerebroventricular (i.c.v.) injections of L-buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. In Group III were 6 month old rats that, as a result of normal aging, show GSH levels of 16-29% below those seen in younger animals. All three groups were subjected to a 1 to 4 dose schedule of dosing with DNB (7.5 mg/kg/day i.p.) and killed 1 day after the last dose of DNB. It was found that whereas Group I animals developed ataxia and brain stem lesions after 4 doses, Group III animals showed these changes after 3 doses, while Group II animals had brain stem lesions after only 2 doses of DNB. The timing of the onset of these changes correlated closely with the degree of reduction of brain tissue levels of GSH, this being greatest in those animals infused i.c.v. with BSO. This demonstration indicates that GSH status in brain tissue is likely to be an important factor in determining regional sensitivity to gliovascular damage from this agent.

A mixture toxicity investigation was conducted using the bioluminescent marine bacterium Vibrio harveyi as the test organism for dual combinations of nitrobenzene and dinitrobenzene. Change in bioluminescence was used for determination of toxicity. Combination toxicity was evaluated using statistical comparisons, isopleths (isobologram and isobole plot), an additive index, and a mixture toxicity index. Both isopleths and mixture toxicity indices suggest that various combinations are additive, while the additive index value suggests antagonism. All evaluations were conducted as equitoxic mixtures. Statistical determination was performed using the z test. Numerous comparisons were different at the 1% level. Slope of line associated with isobole plot was suggested to be an important factor, resulting in statistical differences among comparisons. Distribution, using the Shapiro-Wilk test, was determined for both individual combination groups and solution composition in isopleths. All distributions evaluated were normal. These results suggest that the V. harveyi toxicity test is useful for mixture toxicity studies.

A rapid, accurate method was developed for monitoring employee absorption of dinitrotoluene (DNT). The method reduces DNT and its metabolites in urine to primary arylamines, diazotizes them with nitrous acid, then couples the diazo compounds with N-(1-Naphthyl)ethylenediamine, producing a colored complex. Spectrophotometric analysis of the colored complexes at 550 nm provides a measure of DNT absorption. The chemistry prevents interferences from all but primary arylamines and compounds reduced to primary arylamines. A six-month monitoring program of employees at a DNT manufacturing facility was conducted. Control samples from individuals not exposed to DNT were used to define an exposure indication level. The exposure indication level was used to correlate DNT exposure with job description or individual activity and was defined as apparent DNT and metabolite concentrations greater than 38 micrograms/ml. Group exposure also was indicated and associated with plant activity. Job description were ranked according to a rational evaluation of exposure potential and correlated well with monitoring data.

Studies investigating the testicular toxicity of 1,3-dinitrobenzene (1,3-DNB) have utilized both the oral (po) and intraperitoneal (ip) routes of administration. These two administration routes could be expected to produce different pharmacokinetic profiles and, potentially, different degrees of toxicity. In the present work, the effect of route of administration upon 1,3-DNB disposition and susceptibility to testicular damage has been investigated. Male Sprague-Dawley rats were given 25 mg/kg 1,3-DNB either ip or po. Metabolites were quantitated in blood, urine, and feces, and methemoglobin levels were determined. Peak blood levels of 1,3-DNB and its major metabolite were three times higher in ip-dosed rats than in po-dosed rats. While the lower blood levels seen after po administration were maintained for greater than 6 hr, blood levels fell rapidly after ip dosing, reaching po levels at 6 hr postadministration. Peak methemoglobin levels in ip-dosed animals were twice that of po-dosed animals. Route of administration had a minor effect on the levels of urinary metabolites, while there was a significantly higher excretion of metabolites in the feces of po-dosed animals. Despite the markedly higher 1,3-DNB blood levels after ip administration, there were only subtle differences in testicular damage. The data raise the possibility that above a threshold level of 1,3-DNB in the blood, only the duration of testicular exposure to the toxicant may govern susceptibility to testicular toxicity.

Atrazine (ATZ), an s-triazine herbicide, is a widespread environmental contaminant. The hepatocarcinogenic component of technical grade dinitrotoluene, 2,6-dinitrotoluene (2,6-DNT, 19.5%), is a byproduct of trinitrotoluene synthesis and is found at production sites. This study explores the effect of ATZ treatment on the bioactivation of the promutagen, 2,6-DNT. Male Fischer 344 rats (5 weeks old) were administered 50 mg/kg of ATZ by gavage for 5 weeks. At 1, 3, and 5 weeks, both DMSO-control and ATZ-pretreated rats were treated p.o. with 75 mg/kg of 2,6-DNT and were housed in metabolism cages for urine collection. Sulfatase- and beta-glucuronidase-treated, concentrated urine was bioassayed for urinary mutagens in a microsuspension modification of the Salmonella assay with and without metabolic activation. No significant change in mutagen excretion was observed in ATZ-treated rats; however, an elevation in direct-acting urine mutagens from rats receiving ATZ and 2,6-DNT at weeks 1 (359 +/- 68 vs. 621 +/- 96 revertants/ml) and 5 (278 +/- 46 vs. 667 +/- 109 revertants/ml) of treatment was observed. The increase in production of urinary mutagens was accompanied by an elevation in small intestinal nitroreductase activity. Increases in large intestinal nitroreductase and beta-glucuronidase were observed after 5 weeks. There was no apparent effect of ATZ following 5 weeks of treatment on the production of 2,6-DNT-derived hepatic DNA adducts. ATZ treatment modifies intestinal enzymes responsible for promutagen bioactivation, and potentiates the excretion of mutagenic urine in 2,6-DNT-treated animals.

The immunotoxic potential of dinocap was evaluated in female C57BL/6J mice following in vivo and in vitro exposure to this fungicide. In in vivo studies, groups of mice were dosed by gavage with technical grade dinocap at dosages ranging from 12.5 to 50 mg/kg per day for 7 or 12 days and selected immune functions examined. Mice dosed at 50 mg/kg per day dinocap died after 4 days of dosing. Twelve days of dosing with dinocap at 25 mg/kg per day resulted in decreased thymus weights and cellularity, and increased spleen weights. No changes were observed in body weight, absolute differential peripheral leukocyte counts, the lymphoproliferative responses to B- or T-cell mitogens, the mixed lymphocyte reaction, or natural killer (NK) cell activity of spleen cells from mice exposed to dinocap. Lymphoproliferative responses to concanavalin A (Con A) and phytohemagglutinin (PHA), however, were reduced in thymocytes from mice dosed at 25 mg/kg per day dinocap. The cytotoxic T lymphocyte (CTL) response to P815 mastocytoma cells was enhanced in mice exposed for 7 days to 25 mg/kg per day dinocap. Exposure of mice for 7 days to 25 mg/kg per day dinocap also caused a significant reduction in the IgM and IgG plaque-forming cell (PFC) response to sheep red blood cells (SRBC). A time-course study indicated that dinocap-induced suppression of the IgM PFC response was due to a delay in the peak PFC response to SRBC. In vitro studies using murine thymocytes cultured with dinocap (10 micrograms/ml for 72 h) resulted in suppression of the proliferative response to Con A and PHA. Exposure of thymocytes to dinocap in vitro for as little as 30 min resulted in suppression of the mitogen-stimulated response in the absence of any apparent direct cytotoxic effect. These results suggest that dinocap alters the immune system of the mouse, however, these effects are relatively modest in terms of adverse immune function and are only seen at relatively high exposure levels.

Tripalmitoyl-S-glycerylcysteinyl lipopeptides are B-cell and macrophage activating and may be used as low molecular weight immunogens of considerable potency and even as vaccines when conjugated with suitable epitopic structures. Selected lipopeptides carrying single Dnp haptens were found to evoke mild passive cutaneous anaphylaxis in guinea pigs sensitized against Dnp. The reactions were observed after intravenous injection whereas intradermally applied antigen was negative. The anaphylactogenicity seems unrelated to micelle or aggregate formation of the insoluble peptides which require lecithin additions as well as sonication to become solubilized. The dinitrophenylated lipopeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-lysine produced toxic reactions which were not observed with the lipopeptide devoid of Dnp. Dinitrophenylated tripalmitoyl-S-glycerylcysteiny-1,6-diaminohexane and tripalmitoyl-S-glyceryl-cysteinyl-lysine did not show these toxic reactions.

These studies evaluated the reproductive response and the possible influence of testicular maturation on the reproductive parameters, in male rats treated with 1,3-dinitrobenzene (m-DNB). Young adult male rats (75 or 105 days of age) were given a single oral dose of 0, 8, 16, 24, 32, or 48 mg/kg of m-DNB and killed at 14 days post-treatment. Mortality and neurotoxicity were observed at 48 mg/kg, but only in the older animals. Epididymis weight, testicular sperm head counts, cauda sperm reserves, and sperm morphology were affected at 16 and 24 mg/kg and higher in the older and younger animals, respectively. Testis weight and sperm motility were affected at 24 mg/kg and higher in both age groups. Histologic changes included maturation depletion of mid and late spermatids at 16 mg/kg and higher, atrophy of a few to many seminiferous tubules at 24 mg/kg and higher, and immature germ cells in the epididymis. The movement and/or mixing of luminal elements in the epididymis appeared to be influenced by severe testicular effects. In separate groups given only the 48 mg/kg dosage, fertilizing ability was lost by 5-6 weeks post-treatment and several animals failed to recover in 5 months. In the breeder males, minimal to extensive degrees of seminiferous tubule atrophy and sloughed germ cells in the epididymis were still present after 175 days. The studies indicated that the lowest dosage to produce reproductive changes was 16 mg/kg with a no-effect level of 8 mg/kg. A few animals suffered protracted or permanent reproductive damage. Since the older animals were more susceptible to both the general and the reproductive toxicity of m-DNB, the less severe reproductive changes in the younger animals cannot be attributed solely to maturational differences in the testis.

In order to determine the mechanism(s) involved in the induction of immunologic tolerance for contact sensitivity via the topical application of a chemical that sensitizes if given with adjuvant, we utilized the hapten dinitrothiocyanobenzene (DNTB). Specific immunologic tolerance to dinitrofluorobenzene (DNFB) was induced in mice by the topical application of DNTB 7 days before sensitization to DNFB. The tolerance could be abrogated if cyclophosphamide (200 mg/kg) was given 3 days before attempted sensitization. Using passive transfer studies we found that DNTB induced hapten-specific Lyt 1+2- suppressor T cells. These suppressor cells prevented the induction of contact sensitivity but did not affect its expression. Lymphocyte proliferation studies, using haptenated epidermal cells as antigen, indicate that lymph node cells obtained 5 days after DNFB sensitization are far less responsive if the mice have received DNTB epicutaneously 7 days before the DNFB. Binding studies demonstrated that DNTB bound to epidermal cells at least as well as did DNFB. It is postulated that DNTB induction of suppressor cells is related to the physicochemical interaction between the hapten and antigen-presenting cells in skin.

Epicutaneous application of 2,4-dinitrothiocyanobenzene (DNTB) is said to result in specific immunological hyporesponsiveness and fails to induce contact sensitization. However, we demonstrate that topical exposure to DNTB causes activation of the draining lymph node in mice and the induction of contact sensitization in both rodents and a single human volunteer. In mice and rats, pre-exposure to DNTB failed to impair subsequent responsiveness to the cross-reactive allergen 2,4-dinitrochlorobenzene. These data provide evidence that DNTB cannot be regarded as an exclusive tolerogen when applied epicutaneously and indicate that attempts to define the characteristics of tolerising chemicals from analysis of this agent may be misleading.

The percutaneous administration of in vitro haptenated epidermal cells (EC) has become established as a procedure to produce contact sensitivity (CS) in experimental animals for routine use. The cells have also been found to elicit a significant delayed-type skin reaction by intradermal test in the animals sensitized by painting the skin with the hapten. The fate of 2,4-dinitrophenylated (DNP) isogeneic epidermal cell suspensions (EC) injected intradermally was investigated histologically in intact or 2,4-dinitrochlorobenzene (DNCB)-sensitized strain 13 guinea pigs to study the role of the cells in CS. DNP-EC were found to proliferate actively in the dermis and formed EC nests with central keratinization and then elicited inflammatory reaction associated with necrosis of the epidermal structures 7 days after injection in the intact animals. DNP-EC injected intradermally into the animals which had received and reacted against DNCB underwent a suppression of EC proliferation. These findings are discussed in relation to the role of the haptenated EC in CS.

When BALB/c mice, which had rejected the anti-dinitrophenyl (DNP) IgE-producing hybridoma B 53, were immunized with DNP proteins, they produced much less anti-DNP antibodies than control (normal) mice. The anti-DNP plaque-forming cell (PFC) number was much less when spleen cells from mice immunized with DNP proteins were treated with sera of mice which had rejected the hybridoma B 53 than the PFC number from the same spleen cells not treated by the sera. The sera of mice which had rejected the hybridoma B 53 contained an inhibitor which was adsorbed and eluted from an anti-mouse immunoglobulin column and also a mouse anti-DNP IgG2a column. The inhibition of PFC was hapten-reversible. In Western blotting the eluates from the anti-DNP IgG2a column reacted as well with the blotted anti-DNP IgE B 53 as an anti-idiotypic antibody to anti-DNP IgE B 53. These criteria establish that the inhibitor in the sera of the mice which had rejected the B 53 tumor was an anti-idiotypic antibody of the type which mimics the epitope (DNP) of the immunizing antigen.

A profound and long-lasting state of specific immune unresponsiveness may be induced in adult inbred mice given a single dose of protein immunogens--such as ovalbumin or hemocyanin--by the digestive route. The degree of unresponsiveness induced by intragastric exposure to ovalbumin could not be achieved by intravenous injection of deaggregated ovalbumin solutions across a wide range of doses. Unresponsiveness induced by intragastric exposure to hapten-protein conjugates is specific to the carrier protein.

The induction characteristics of IgD in serum and other body fluids are poorly understood. Here we report that Freund's complete adjuvant (FCA), when used in intraperitoneal (i.p.) immunization, resulted in significantly higher levels of IgD and specific IgD antibodies in ascitic fluid than in the serum of the same animals. This was in sharp contrast to the distribution of IgM and kappa light chain-bearing immunoglobulins (kappa Ig) between these compartments. Female rats were immunized subcutaneously (s.c.) on Day 0 with 1:1 FCA mixed with either 2,4 dinitrophenyl group A streptococcal vaccine (DNP-GASV), 2,4 dinitrophenyl ovalbumin (DNP-OVA), or saline. A week later (and weekly thereafter) they were boosted i.p. with these antigens mixed with FCA 1:9. Eleven of sixteen rats produced some ascites by Day 21, and 15/16 by Day 28. Total immunoglobulin and specific antibodies for the heavy-chain isotypes IgD and IgM and for kappa Ig were measured by ELISA. Between immunization groups no significant differences were observed in total immunoglobulin levels. Subcutaneous immunization with 1:1 FCA caused a significant two- to six-fold increase in serum IgD by Day 7 (P less than 0.01), while no change occurred with serum IgM or kappa Ig. Ascitic fluid collected on Day 21 had significantly high levels of IgD than that of serum IgD (P less than 0.01), with a mean level nine times that of the serum. In contrast, levels of total IgM, kappa Ig and total protein were significantly lower in ascitic fluid than in serum (all P less than 0.05). The high levels of IgD in ascitic fluid disappeared in Day 28 ascitic fluid, while IgM and kappa Ig levels were relatively unchanged. The ratio of ascitic fluid to serum IgD anti-DNP was 9.8 for the DNP-GASV-immunized group on Day 21, and 23.9 for the DNP-OVA-immunized group on day 28. The same ratios for IgM anti-DNP and kappa Ig anti-DNP were much lower, ranging from 0.4 to 2.3 and 0.3 to 3.3, respectively.

The extent to which B cells newly formed in the bone marrow contribute to primary and secondary B cell responses was investigated. This was assessed by constructing chimeras between congenic strains of rats differing in their kappa light chain allotype. Recipient animals received 800 cGy whole body irradiation with hind limb shielding to protect a proportion of their hemopoietic capacity. These rats then received 3 X 10(8) kappa allotype-marked thoracic duct lymphocytes from donors previously immunized twice with either dinitrophenylated spider crab (Maia squinada) hemocyanin (DNP-MSH) or MSH alone. The chimeras were immunized with DNP-MSH and the production of anti-DNP antibody of both donor and host origin was measured. In the period immediately after immunization both newly formed host virgin B cells and donor memory B cells gave rise to substantial proportions of the anti-DNP antibody. After this initial period, antibody production became sustained by activation of memory B cells only. The chimeras were reimmunized with DNP-MSH at 32 days after their first immunization. There was again evidence of a brief period of both virgin and memory B cell activation followed by memory B cell activation only. Donor B cell clones remained dominant in the established response throughout the 5 months each chimera was studied. The data are interpreted as indicating two phases of B cell activation. It is suggested that the first phase where both virgin and memory B cells are activated may be associated with antigen presentation on dendritic or interdigitating cells outside follicles. It is argued that the second phase where only memory B cells are activated is more likely to be associated with antigen on follicular dendritic cells.

We have reported that the relative amounts of Ig kappa and Ig lambda anti-DNP antibodies produced depend on the molecular form in which the hapten was presented. Here we confirm this finding by demonstrating that 1) the effects of adjuvant are not sufficient to result in differential light chain production and 2) the presence of some DNP-containing epitopes unique to DNP-KLH are not sufficient to give rise to enhancement of Ig kappa production. We further demonstrate that preferential augmentation of Ig kappa in the response to DNP is a T cell-mediated effect. Although we cannot rule out the possibility that some Ig kappa antibodies specific for protein-conjugated DNP require T cells for their expression, one of several other possible mechanisms whereby T cells influence light chain production is considered likely.

Rats were injected intraperitoneally with 0.015 mmol of p-dinitrobenzene/kg, 0.15 mmol of m-dinitrobenzene/kg and 0.45 mmol of o-dinitrobenzene/kg body wt. The dinitrobenzenes induced methaemoglobin concentrations of 86%, 60% and 49%, respectively, as well as increases of urinary catecholamine excretion and blood sugar concentration. Due to hyperglycaemia the glycolysis may be stimulated as mechanism for energy production in methaemoglobin-induced oxygen deficiency and as mechanism for reduction of methaemoglobin, that means as responses favouring survival after poisoning.

The immune response of pigs fed 200 mg per day of dinitrophenylated bovine gamma globulin has been evaluated in terms of the antibody and lymphocyte responses and of the induction of tolerance and immune exclusion. Although dosing for ten days resulted in a small IgA response as indicated by splenic plaque forming cells, serum antibody could only be detected when dosing was continued for 42 days. secretory antibody was detected at any time. Antigen feeding for two weeks rendered the animals hyporesponsive to subsequent parenteral antigenic challenge but had little effect upon their ability to exclude an oral dose of antigen from the circulation.

Female Fischer 344 rats are less susceptible to the hepatocarcinogenic effects of 2,4-dinitrotoluene (2,4-DNT) than males. This study is a comparison of the metabolism and excretion of 2,4-DNT in male and female rats after oral doses of 10, 35, or 100 mg of 14C-2,4-DNT per kg. The major route of elimination of 14C after all doses was the urine. 4-(N-Acetyl)amino-2-nitrobenzoic acid (4Ac2NBAcid), 2,4-dinitrobenzoic acid (2,4-DNMBAcid), 2-amino-4-nitrobenzoic acid (2A4NBAcid), and 2,4-dinitrobenzyl alcohol glucuronide (2,4-DNBAlcG) were identified in urine of rats. These four compounds accounted for greater than 85% of the radioactivity excreted in urine. Female rats excreted a significantly greater percentage of the dose in the urine as 2,4-DNBAlcG at doses of 10 or 35 mg/kg when compared to males. Both sexes showed dose-dependent changes in urinary excretion of 2,4-DNT metabolites. Males excreted a smaller percentage of the dose as 2,4-DNBAcid at 100 mg/kg than at 10 or 35 mg/kg. Females excreted less of the dose as 2,4-DNBAcid and 2,4-DNBAlcG at 100 mg/kg than at 10 or 35 mg/kg. The only sex difference in 2,4-DNT metabolism or excretion of sufficient magnitude to account for the sex difference in susceptibility to the hepatocarcinogenic effects of 2,4-DNT was the greater percentage of 2,4-DNT excreted as 2,4-DNBAlcG by female rats at 10 or 35 mg/kg.

The pathogenesis of 1,3-dinitrobenzene (1,3-DNB)-associated testicular toxicity was investigated in Sprague-Dawley rats with 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry and lanthanum-tracer methods as well as routine histopathological examination. Animals were killed at 8, 12, 24, 48 and 96 hr after a single oral administration of 1,3-DNB at a dose of 25 mg/kg. Histopathologically, severe alterations in the testes such as degenerating pachytene spermatocytes and giant cell formation were observed by 24 hr. Immunohistochemical analysis revealed no differences in the distribution of BrdU-positive cells between treated and control rats throughout the experiment. Thus it was concluded that 1,3-DNB exerted no effects on DNA synthesis in spermatogonia and preleptotene spermatocytes. Using the lanthanum-tracer method, it was shown that although lanthanum could penetrate the intercellular space from the basement membrane, the tight junction, consisting of fusions of contiguous Sertoli cell membranes, prevented further diffusion in both control rats and those killed at 8 hr after dosing. On the other hand, at 24 hr after dosing, lanthanum penetrated into the adluminal compartment beyond the tight junctions, thus demonstrating loss of integrity of the blood-testis barrier. The results suggested that the Sertoli cell is the primary target of 1,3-DNB testicular toxicity.

The T-cell independent immunological responses of naive mice to a series of size-fractionated dinitrophenylpolyacrylamide molecules were measured. These responses were correlated in vitro and in vivo with hapten number per molecule and hapten density per unit length. We found that to be immunogenic, a T-independent molecule must contain a threshold number (approximately 20) of appropriately spaced haptens or epitopes. We infer that this condition is necessary for simultaneous B-cell receptor binding and clustering. Molecules with less than this number of haptens are not immunogenic at any dose. Some nonimmunogenic molecules are capable of inhibiting the response to the immunogenic ones; this capability increases with increasing hapten density. In order for a molecule to accommodate 20 or more appropriately spaced haptens, it must be above a threshold size. Increasing hapten density in a molecule at or above threshold size and hapten number increases its immunogenicity. Increasing hapten density in a molecule below threshold size increases its tolerogenicity. Conclusions concerning immunogenicity and tolerogenicity of T-independent antigens may not be warranted unless the molecular weight and hapten or epitope number per molecule are well characterized.

BALB/c mice were made tolerant to the T cell-dependent phenomenon of contact sensitivity to DNFB by i.v. injection of syngeneic lymphoid cells which had been previously modified with DNFB in vitro. The highly efficient unresponsiveness, as measured by ear challenge and in vitro antigen-induced cell proliferation, was shown to follow dose-response kinetics both in vivo and in vitro and to be exquisitely specific for the DNP moiety. The kinetics of tolerance induction were shown to be very rapid and had been previously shown to be long lasting. Unresponsiveness was more efficient when the hapten-modified cells were introduced by the i.v. route and tolerance could be increased by repeated injection of tolerogen. The tolerance could be transferred to normal syngeneic recipients by spleen and/or lymph node cells from tolerant donors. A wide variety of hapten-modified lymphoid cells, including mixed cell populations and enriched populations of T cells, B cells, and macrophages, were capable of inducing tolerance. The unresponsiveness was dependent merely on the association of DNP to the lymphoid membrane proteins and not upon the viability of the hapten-modified cells. These experiments support the hypothesis that in hapten-specific T cell sensitivity, as exemplified by contact sensitivity to DNFB, specific T cell tolerance in actively induced by hapten on self-membrane. In other hapten-specific antibody-forming systems, tolerance appears to be most readily induced by hapten on soluble self protein or on a nonimmunogeneic carrier.

Preliminary results of active immunotherapy ,both in vitro and in vivo, about ascitical Ehrlich carcinoma transplanted in albinic swiss mice are presented. In the in vitro experiment, tumor cells were marked with the immunoglobulin, anti-tumor-associated antigens (TAA) and were coupled to a dinitrophenyl radical (Ig DNP anti-TAA). These cells were meaningfully hindered from migration in presence of swiss albinic mice's splenic cells. These mice were sensibilized to the tumor cells marked with Ig-DNP. The injection of a Ig-DNP anti-TAA 0,3 ml, every third days, intraperitoneal way, in a span of 21 days, in albinical swiss mice with a transplant of 3 X 10(5) Ehrlich carcinoma cells (group A) 24 hours before, constituted the in vivo test. The growth ought to be compared to an Ig-DNP tolerant group (group B), which received equal quantities of tumor cells and followed the same plan of treatment, as well as to another control group transplanted under the same conditions, but with no treatment (group C). Eight days from the experiment, there was a clear difference between group A and groups B and C. The last two groups died from 13th to the 26th day after the transplant. On the contrary, the whore group A continued alive and with no sign of ascitical tumor. Nevertheless, an animal of group A died after the 28th day, due to a solid tumor in the abdominal wall.

Continued treatment with monoclonal anti-IgD (Ig-5a) from birth in BALB/c mice causes a markedly increased responsiveness to i.v. injected dinitrophenylated ovalbumin (DNP-OVA) with Bordetella pertussis at the age of 8 weeks. The 19S plaque-forming cell (PFC)/spleen response is particularly enhanced, 6-8-fold, but all the other isotypes also show increases of 2-6-fold, including IgA and IgE. Both primary and secondary PFC responses and serum antibody titers are enhanced. After transfer of spleen cells from anti-Ig-treated mice to irradiated recipients the IgM/IgG ratio becomes similar to that of controls. In contrast, the response of anti-IgD-treated mice to i.p. immunization with either 0.2 or 100 micrograms DNP-OVA plus alum is reduced by approximately 80% for each Ig isotype except IgM and remains low upon transfer of spleen cells to recipients. It is concluded that the paucity of B cells in peripheral lymph nodes of the anti-IgD-treated mice causes the low responsiveness to i.p. immunization, but that the IgD- B cells in the spleen are quite able to respond and are, in fact, more responsive than IgD+ B cells. This increased responsiveness, together with the higher IgM/IgG ratios for all Ig isotypes and an otherwise similar order of isotype distribution (gamma 1 greater than gamma 2b greater than gamma 2a = epsilon greater than or equal to alpha) as in controls, suggests that a hyperresponsive, but less mature IgD- B cell population is selectively produced in the spleens of mice treated with anti-IgD from birth.

An assessment of T-lymphocyte proliferation and lymph node weight is proposed as a predictive test for contact sensitizers of industrial origin. Data are presented showing increased T-lymphocyte proliferation following epicutaneous application of a variety of industrially important acrylate-like chemicals which appear to correlate well with their ability to sensitize in the guinea pig. These data were compared with those obtained after application of 2,4-dinitro-1-fluorobenzene (DNFB) a strong sensitizer, and 2,4-dinitrothiocyanatebenzene (DNTB) a nonsensitizer when given epicutaneously. It is suggested that this quantitative approach, in parallel with a simple one-dose immunization, may provide a better picture of sensitization potential than the longer multidose immunizations currently in use.

Administration of a single oral dose of 20 mg/kg of 1,3-dinitrobenzene caused ataxia in germ-free male F-344 rats but not in conventional rats. Repeated oral dosing of 20 mg/kg, 1,3-DNB was required to cause ataxia in conventional rats. Considerable differences were observed between the uptake, tissue distribution and excretion of DNB in germ-free and conventional rats.

After intravenous injection into mice, human serum albumin (HSA) substituted with 35 dinitrophenyl (DNP) groups per molecule rapidly became deposited on and endocytosed by non-parenchymal liver cells. Much was also found outside the reticuloendothelial system, e.g. in heart and skeletal muscle. The relative amount of circulating DNP35HSA taken up diminished with increasing amounts injected. The capacity of DNP35HSA clearance could not be saturated, however. Injection of small amounts (0.14 mg) of DNP35HSA did not result in glomerular deposition, whereas injection of slightly larger amounts (0.7 mg) did. Rabbit IgG anti-DNP increased the entrapment of DNP35HSA in the liver, whereas mouse IgA anti-DNP had less pronounced effects. DNP-HSA conjugates with lesser degree of DNP substitution, which are cleared from the blood at slower rates, became located to the liver to a lesser extent than was DNP35HSA. Rabbit IgG anti-DNP increased the amount taken up by the non-parenchymal liver cells. Mouse IgA anti-DNP did not alter the antigen distribution within the liver; that is, immune complexes formed between IgA anti-DNP and DNP-HSA became located virtually exclusively to the non-parenchymal liver cells.

Hooded Lister rats were sensitized to DNP-ovalbumin. These rats showed a significant alteration in respiratory pattern when challenged with DNP-ovalbumin aerosol. The respiratory response was not produced by challenging sensitized rats with intragastric DNP-ovalbumin. The magnitude of the respiratory response did not correlate with the specific IgE concentration in the serum. A similar pattern of respiratory response was inducible in unsensitized rats by intravenous administration of IgE-rich sera.