A re-appraisal of the skin-sensitizing activity of 2,4-dinitrothiocyanobenzene.

A debate continues regarding the immunological properties of 2,4-dinitrothiocyanobenzene (DNTB). In some investigations this chemical was shown not to cause skin sensitization when applied topically but to induce instead hyporesponsiveness or immunological tolerance. In other studies DNTB was found to cause skin sensitization, but not tolerance. However, this chemical continues to be used to discriminate between the properties of skin sensitizing and non-sensitizing chemicals. This study demonstrates that topical exposure of mice to DNTB induces skin sensitization in mice and that this is associated with the accumulation of dendritic cells in draining lymph nodes and the stimulation of lymph node cell proliferation; the latter responses being of equivalent magnitude to those stimulated by 2,4-dinitrochlorobenzene (DNCB), a chemical known to cause contact sensitization. Moreover, exposure of mice to DNTB, as with exposure to DNCB, resulted in the development of a cytokine secretion pattern by draining lymph node cells (LNC) characteristic of contact allergens. Thus, DNTB and DNCB each induced the production by LNC of high levels of interferon-gamma, but little or no interleukin 4 or interleukin 10. Finally, DNTB was shown in the guinea pig maximization test to behave as an extreme skin sensitizer. These results confirm that DNTB should not be regarded as a universal tolerogen and that it possesses a significant potential to induce contact sensitization. The use of this chemical as a presumptive non-sensitizer and/or tolerogen for the evaluation of the selectivity of new predictive test methods for the identification of contact allergens is therefore considered to be inappropriate.

Evaluation of the sensitizing potential of eugenol and isoeugenol in mice and guinea pigs.

The sensitizing properties of the fragrances eugenol and isoeugenol have been investigated experimentally. The potential of these materials to induce sensitization of the respiratory tract was examined using the mouse IgE test, a novel but as yet unvalidated method for the predictive identification of chemical respiratory allergens. Comparisons were made with the activity of eugenol and isoeugenol in two predictive tests for contact sensitization potential: the murine local lymph node assay and the guinea pig maximization test. Both chemicals elicited positive responses in these tests, isoeugenol exhibiting a greater potential for contact sensitization than eugenol. Isoeugenol was negative at all concentrations examined in the mouse IgE test. In the same assay, exposure to eugenol was associated with a statistically significant increase in serum IgE concentrations when initial application concentrations of 2.5% were used. However, at higher test concentrations eugenol was negative in the mouse IgE test. It is concluded that neither eugenol nor isoeugenol have a significant potential to cause sensitization of the respiratory tract, a conclusion that is apparently consistent with the lack of evidence for occupational respiratory allergy associated with exposure to these chemicals. The evidence for isoeugenol lacking respiratory sensitization activity is particularly strong and it is proposed that this chemical may be of value as a negative control in the development and validation of new predictive test methods for the identification of chemical respiratory allergens.

Cyclic AMP-dependent anion secretion in human small and large intestine.

Cyclic AMP-dependent Cl- secretion is the major secretion pathway in human intestine. The aim of the present study was to examine mechanisms involved in cAMP-dependent anion secretion in human small and large intestine. Surgical resection specimens from both jejunum and distal colon were studied under short circuited conditions. Addition of the phosphodiesterase inhibitor IBMX induced an increase in the short-circuit current (Isc) equivalent to the net increase in Cl- secretion. The Isc was inhibited by diphenylamine decarboxylate (DPC; Cl- channel blocker), bumetanide (basolateral Na+/K+/2Cl- cotransporter), BaCl2 (basolateral K+ channel) and Cl- free buffer in both segments and indomethacin (cyclo-oxygenase inhibitor) in colon alone. Diphenylamine decarboxylate appears to directly inhibit secretion in jejunum, although its inhibitory effect is possibly mediated by inhibition of cyclo-oxygenase in the colon. A small component of IBMX-stimulated Isc was inhibited by acetazolamide. Cyclic AMP-dependent secretion is largely apical Cl- secretion, although a small component appears to be HCO3. Secretion is dependent on basolateral K+ channels and Na+/K+/2Cl- cotransporters and, in the colon, is inhibited by indomethacin, implying a role for cyclo-oxygenase metabolites. The chloride channel blocker DPC inhibits secretion in both areas. This class of compounds may have potential for treatment of secretory diarrhoea.

Influence of dibutyl phthalate on dermal sensitization to fluorescein isothiocyanate.

What limited evidence there is indicates that the formulation in which a chemical allergen is encountered on the skin can have a marked impact upon the induction of cutaneous immune responses and the subsequent development of contact sensitization. The purpose of the present investigations was to examine further this phenomenon by analysis of the influence of dibutyl phthalate (DBP) on dermal sensitization to fluorescein isothiocyanate (FITC), a skin sensitizing fluorochrome. Addition of DBP augmented very substantially, in a dose-dependent fashion, the ability of topically applied FITC to stimulate proliferative responses in mice by draining lymph node cells (LNC), a correlate of skin sensitizing potential. Under these conditions, exposure of mice to DBP alone failed to elicit significant LNC responses. The influence of DBP on the accumulation of dendritic cells (DC) induced by FITC was examined also. Although 10% DBP had little effect on the numbers of DC found within draining nodes 18 hr following exposure of mice to FITC, the phthalate did result in a very substantial increase in the frequency of lymph node DC bearing detectable antigen (FITC+ DC). Furthermore, in the presence of DBP the median amount of FITC associated with antigen-bearing DC was higher. In vitro skin absorption studies indicated that DBP was associated with a small increase in percutaneous absorption of FITC. Collectively these data demonstrate that the vehicle formulation can exert a marked influence on dermal sensitization and that one mechanism which may be relevant is the increased acquisition of antigen by DC, associated possibly with altered penetration of the allergen into or through the skin.

Sensitization to 2,4-dinitrochlorobenzene: influence of vehicle on absorption and lymph node activation.

Effective skin sensitization is dependent upon immune activation of lymph nodes draining the site of exposure. The influence of vehicle formulation on the vigour of lymph node cell proliferative responses to 2,4-dinitrochlorobenzene (DNCB) has been examined. Mice (BALB/c strain) were exposed topically to 0.5% DNCB dissolved in either acetone or propylene glycol (PG). A significantly greater lymph node cell proliferative response was induced by DNCB in acetone. The observed differences were not attributable to variations in the numbers of immunostimulatory dendritic cells accumulating in the draining nodes following sensitization. In parallel studies, the absorption and cutaneous disposition of DNCB dissolved in acetone or PG were measured in vitro using static diffusion cells and full thickness mouse skin. Although flux of DNCB through the skin was comparable with both vehicles over 24 h, the absorption of the allergen during the first 4 h of exposure was significantly faster when acetone was used as the vehicle. Localization of DNCB demonstrated that much less of the chemical allergen was present in the skin at 4 h when applied in PG vehicle. However, there were no measurable vehicle effects on skin disposition of DNCB at 24 h. These data indicate that the sensitization potential of DNCB is influenced significantly by the nature of the vehicle used, possibly due to consequential effects on chemical absorption and disposition. The studies described in this paper reveal that the application vehicle may have a significant influence on the ability of DNCB to induce immune activation of draining lymph nodes and hence skin sensitization and that this may in turn be associated with important changes in the absorption and/or disposition of the chemical within the skin.

The mouse IgE test for the identification of potential chemical respiratory allergens: considerations of stability and controls.

The mouse IgE test is a novel approach to the predictive identification of chemicals that have the potential to cause sensitization of the respiratory tract. The method is based upon measurement of induced changes in the serum concentration of IgE in BALB/c strain mice following topical exposure to the test chemical. The investigations described here were undertaken to examine the stability of the assay, to evaluate the utility of trimellitic anhydride (TMA) and 2,4-dinitrochlorobenzene (DNCB) as, respectively, positive and negative controls for use in the test and to consider criteria for the classification of chemicals as potential respiratory allergens based upon changes induced in serum IgE concentration. On the basis of fifteen independent experiments it was found that, while there was some intra- and inter-test variability with respect to absolute concentrations of IgE measured in the sera of individual mice, the relative pattern of reactivity observed following treatment of mice with TMA, DNCB or with vehicle (4:1 acetone:olive oil) alone remained consistent. In each experiment treatment with TMA provoked a very substantial increase in serum IgE relative to vehicle controls. In no instance did exposure of mice to DNCB cause an increase in the concentration of serum IgE, and in some instances treatment with this chemical resulted in reduced IgE levels. In a parallel series of experiments it was found that serum IgE concentrations in vehicle-treated mice were comparable with those found in the serum of untreated animals. It is concluded that the differential ability of chemicals to induce changes in the concentration of serum IgE in BALB/c mice is a stable phenomenon. It is recommended that, in practice, TMA and DNCB should be incorporated in mouse IgE tests as, respectively, positive and negative controls. Finally, it is proposed that activity in the test is assessed as a function of induced changes in serum IgE levels relative to concurrent vehicle controls, rather than by reference to historical normal range values.

Dichloronitrobenzene: a reappraisal of its skin sensitization potential.

Unlike the closely related chemical dinitrochlorobenzene (DNCB), which is a very strong contact allergen, dichloronitrobenzene (DCNB) has been widely regarded as a non-allergen and, as such, a useful control for its strongly sensitizing counterpart. Nevertheless, it is still an organic chemical species readily capable of penetrating skin and, rather than being regarded as completely inert, it has even been suggested to react with the immune system in such a way that it induces specific tolerance to its chemical structure. We investigated whether DCNB was in reality a non-allergen, or rather merely a weak contact sensitizer. In both a rigorously conducted guinea pig maximization test and in a modified murine local lymph node assay, DCNB was demonstrated to possess weak sensitizing activity. On this basis, DCNB cannot be regarded as inert with respect to contact allergic potential, and is therefore inappropriate as a negative control in studies of skin sensitization.

Identification of dendritic cells as a major source of interleukin-6 in draining lymph nodes following skin sensitization of mice.

Skin sensitization with chemical allergens is associated with the activation and proliferation of T lymphocytes within lymph nodes draining the site of exposure. These events are accompanied by the secretion of interleukin-6 (IL-6) by lymph node cells (LNC). We have investigated the cellular source of IL-6 seventy-two hours following primary exposure of mice to the contact allergen oxazolone. Immunocytochemical analyses of sections of activated lymph nodes have revealed that cells expressing IL-6 are located within the T-dependent lymph node paracortex, with none present in lymphoid follicles. Cells which expressed IL-6 cofractionated exclusively with LNC of low buoyant density, the majority of which also expressed membrane Ia and had a dendritic morphology. Depletion of dendritic cells from LNC culture was associated with a significant decrease in the secretion of IL-6 by the residual population. These data demonstrate that dendritic cells are a major source of IL-6 within lymph nodes during primary immune responses to cutaneous antigens.

Modulation of epidermal Langerhans' cell frequency by tumour necrosis factor-alpha.

During the induction phase of skin sensitization, dendritic cells (DC), many of which bear high levels of antigen, accumulate in lymph nodes draining the site of exposure. These DC derive from epidermal Langerhans' cells (LC) which are induced to migrate from the skin, via the afferent lymphatics, to lymph nodes. We demonstrated previously that intradermal exposure of mice to homologous, but not human, recombinant tumour necrosis factor-alpha (TNF-alpha) also causes an accumulation of DC in draining nodes, the implication being that the local production of this cytokine by epidermal cells provides one stimulus for LC migration. In the present study we have examined the influence of dermal TNF-alpha on the frequency of LC within the epidermis. Intradermal injection of mice with 25 ng or greater murine recombinant TNF-alpha caused a significant reduction in LC numbers within 30 min of exposure. The same treatment did not influence the frequency of Thy-1+ epidermal DC. The density of LC was unaffected by the same amount of human TNF-alpha of comparable specific activity or by murine granulocyte-macrophage colony-stimulating factor (GM-CSF). These data provide additional evidence that TNF-alpha provides an important signal for the migration of LC from the epidermis.

Induction of respiratory hypersensitivity to diphenylmethane-4,4'-diisocyanate (MDI) in guinea pigs. Influence of route of exposure.

The induction of respiratory sensitization in guinea pigs to diphenylmethane-4,4'-diisocyanate (MDI), a known human respiratory allergen, has been investigated and different routes of exposure compared. Guinea pigs were exposed to MDI by i.d. injection, by topical application or by inhalation. Pulmonary hypersensitivity was measured subsequently as a function of changes in respiratory rate following challenge with atmospheres containing MDI. In addition, contact hypersensitivity was measured by topical challenge and antibody responses evaluated by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis (PCA). Attempts to sensitize guinea pigs by inhalation exposure to MDI were unsuccessful. Antibody responses and contact sensitization were both infrequent and low grade, and no animals exhibited pulmonary responses following challenge with atmospheric MDI. In contrast, sensitization by either i.d. injection or topical application of MDI induced antibody responses in the majority of animals. Moreover, a proportion of animals in each case exhibited pulmonary responses following subsequent inhalation challenge. These data indicate that the route of exposure influences markedly the effectiveness of sensitization to respiratory allergens such as MDI and that skin contact may be an important cause of occupational respiratory allergy.

Inflammatory pseudotumor of the bladder.

Inflammatory pseudotumor of the bladder is an unusual benign lesion arising from the bladder submucosa. We present 2 cases and describe the clinical presentation, and radiographic and histological findings. This benign lesion must be differentiated histologically from several malignant lesions of the bladder. Complete surgical excision, either by transurethral resection or partial cystectomy, appears to be curative.

A manometric study of anal fissure treated by subcutaneous lateral internal sphincterotomy.

A prospective, manometric trial of anal fissure treated by subcutaneous lateral internal sphincterotomy (SLIS) was designed to elucidate the pathophysiology of this condition. Anorectal manometry with a closed, precalibrated, water-filled microballoon using the station pull-through technique was performed on 13 patients with anal fissure before, and at one and 150 days after SLIS. The results were compared with 13 control subjects, matched for age and sex, who had no history of anal disease. Both resting pressure (RP) and maximum voluntary contraction pressure (MVCP) were measured at centimeter intervals of the anal canal. At all levels RP was significantly higher in the preoperative patients compared with controls (p less than 0.0001). After operation RP fell significantly at all levels with the result that there was no significant difference in RP between postoperative patients and controls, except at 4 cm from the anal verge, where there remained a significant elevation in RP in the postoperative group. There was no significant difference in the two sets of postoperative manometric results. All patients underwent rapid healing and resolution of their symptoms. MVCP did not change significantly after operation, nor did it differ from the control values. This suggests that the increase in RP is due to activity of the internal anal sphincter. This over-activity is present throughout the entire length of the internal anal sphincter and sphincterotomy of its lowest portion returns RP to normal values throughout most of the anal canal.

Rectal prolapse: relationship with joint mobility.

Joint mobility was assessed in 25 patients who had undergone surgery for complete rectal prolapse and in 25 age- and sex-matched control subjects. A significant increase in extensibility of the fifth finger was found in the patients with rectal prolapse. It was further found that there was a progressive decrease in joint mobility with age in both groups. The pathophysiology of rectal prolapse is complex. Factors considered to be important include rectal intussusception associated with the commonly observed lack of rectal fixation within the sacral hollow, with a deep Pouch of Douglas and weak pelvic floor musculature. The joint hypermobility demonstrated in these patients suggests an underlying connective tissue abnormality which perhaps contributes to the lack of rectal fixation within the pelvis and to the rectal wall intussusception.

Angiomyolipoma and renal cell carcinoma in same kidney.

A case of renal cell carcinoma and angiomyolipoma occurring in the same kidney in a patient without stigmata of tuberous sclerosis is reported. This case was managed as malignant renal tumor, and the patient underwent radical nephrectomy. Urologic and angiographic literature regarding angiomyolipoma is reviewed, and we find the neovascularity of this benign tumor is only rarely distinguishable from malignant renal tumor.