Immunological studies on delayed hypersensitivity to phenytoin--II. The delayed skin reaction induced by BSA-phenytoin conjugate

Remarkable swelling of the foot pad was induced in guinea pigs sensitized with BSA-phenytoin by challenging with BSA-phenytoin or EA-phenytoin. Forty-eight hours after local injection of a mixture of exudate cells obtained from the abdominal cavity of sensitized guinea pigs. and BSA (EA)-phenytoin, both erythema and induration developed at the injection sites in normal guinea pigs. At the sites exhibiting these skin reactions, an exudation of mononuclear leukocytes was noted. In addition, macrophages obtained from the abdominal cavity of phenytoin-sensitized animals showed a low migration index in the presence of phenytoin. When phenytoin was administered to rats (p.o.), large amounts of the compound were detected in the gingiva and there was a good correlation between the tissue and serum phenytoin concentrations. These findings indicate that the etiology of gingival hyperplasia produced by chronic administration of phenytoin may be related in some way to a delayed-type hypersensitivity induced by the drug.     

A radioisotopic method to measure delayed type hypersensitivity in the mouse. II. Cell transfer studies.

Sensitized lymphoid cells could transfer to normal non-sensitized (naive) mice by 24-48 h after antigen challenge in the ear, the capacity to incorporate, at the site of antigen deposition, 5-iodo-2'deoxyuridine-125I in amounts significantly above those obtained in control mice. This was associated with a mononuclear cell infiltration in the pinna. In contrast to lymphoid cells, serum antibodies were unable to transfer a 24- or 48 hour ear reaction. The cells responsible were T lymphocytes as demonstrated by successful transfer following enrichment for T lymphocytes, and abrogation of transfer following treatment with anti-theta serum and complement. Transfer was achieved whether the naive recipients were normal, T-cell deprived, pretreated with cyclophosphamide, or lightly irradiated but not when they were heavily irradiated. Adoptive transfer of the 24-hour ear response was demonstrated with three different antigenic systems. The time-response curves were different with each system although peak reactions were obtained 5 days after sensitization of the donors in all cases. The specificity patterns of the 24-hour ear reaction on transfer were similar to those obtained in the sensitized donors. The results of these studies indicate that the radioisotopic ear method can, under defined conditions, demonstrate the existence of a state of delayed type hypersensitivity in the donors.     

A new method of eliciting delayed hypersensitivity in the mouse abdominal cavity

A new method of determining delayed hypersensitivity quantitatively was investigated in mice. Mice were sensitized with 150 micrograms of ferritin and, 3 weeks later, antigen challenge was performed by implanting a sponge containing antigen in the abdominal cavity. Cells accumulated in the sponge markedly increased in number for 24-72 h after the challenge; mononuclear cells predominated by 48 h. When sensitized lymphocytes were transferred passively to a normal recipient, marked cell accumulation in the sponge was found 48 h after the challenge. Immunological specificity was confirmed in animals sensitized to antigen and receiving passive transfer of sensitized cells. Strain differences in this reaction were observed. Cortisone (20 mg/kg for 6 days before challenge) significantly decreased cell accumulation. Delayed hypersensitivity was also elicited in the ear of sensitized animals. Extracts of sponges removed from antigen-challenged mice had macrophage chemotactic activity.     

A simple method of evaluating delayed type hypersensitivity in mice.

Delayed type hypersensitivity reactions in mice sensitized to dinitrofluorobenzene were assessed by measuring the increase in weight of the ears challenged with antigen. The method has proved reliable for assessment of DTH reactions when results are expressed as weight ratios between challenged and unchallenged ears.     

A simple and effective method to assess murine delayed type hypersensitivity to proteins

Mice sensitized with various protein antigens emulsified in complete Freund's adjuvant by a subcutaneous injection at the base of the tail exhibited both immediate and delayed type hypersensitivities 7-8 days following sensitization. Hypersensitivity was assessed by the degree of footpad swelling elicited by challenge with a heat-aggregated form of the antigen. The swelling response was specific for the antigen used for sensitization and often resulted in a near doubling of footpad thickness. Histological examination is conjunction with studies having passive transfer of serum and adoptive transfer of either T cell-enriched or T cell-depleted preparations indicated that whereas the immediate swelling response was achieved with serum, the delayed swelling response was mediated by T lymphocytes. Adoptive transfer of specific delayed type hypersensitivity was also accomplished with small numbers of lymph node T cells obtained following in vitro enrichment and propagation.     

Immediate and delayed type hypersensitivity to malathion.

Between December 1989 and June 1990, 1,874 reports of alleged malathion application related illness from repeated spraying of a mixture of malathion corn syrup bait to eradicate a Mediterranean fruit fly infestation in Southern California were received by the Toxics Epidemiology Program of Los Angeles County. Among these complaints were 47 reports of urticaria, 38 reports of angioedema and 213 reports of a nonspecific skin rash. In order to determine whether these alleged skin reactions were the result of an immediate or delayed type of hypersensitivity reaction to malathion or to the corn syrup bait we studied ten subjects referred for testing by the local health department. All ten subjects had no reaction on patch testing. One child exhibited a positive reaction to the bait and one child had irritant reactions to malathion and to the bait. This study documented one case of a possible immediate IgE reaction to malathion bait. Due to the low participation rates in this study, no specific conclusions concerning the rate of sensitivity in the population can be drawn, although it appears that such reactions are uncommon.     

A new method to measure the specific cellular component of a delayed hypersensitivity response in the ear of the mouse

We here describe a simple method of measuring a pure cellular reaction in the mouse. Mice were challenged at the ear pinna to detect a cell population labeled in vivo with [³ H]thymidine. The reaction was found to be specific, radiosensitive and thymus-dependent.     

Histological and immunopathological studies of delayed hypersensitivity reaction to tuberculin in mice

At 4 to 6 weeks after intravenous infection with 2 X 10(4) CFU of dispersed Mycobacterium bovis bacilli (BCG), C3H/HeNCrIBR and C57BL/6NCrIBR mice exhibited a strong reaction to purified protein derivatives, as evaluated by the increase in footpad swelling at both 24 and 48 h after local antigenic challenge. However, histological studies of the footpad skin demonstrated a prominent perivascular infiltration with polymorphonuclear cells at 6 and 24 h after purified protein derivative challenge, whereas mononuclear cells represented the majority of infiltrating cells only at 48 h. An immunopathological study of the footpad skin showed granular deposits of immunoglobulins and complement in vascular walls and perivascular tissues at 6 and 24 h. These results demonstrate that the footpad swelling observed 24 h after the antigenic challenge is caused by an Arthus-type reaction, whereas that caused by cell-mediated immunity appears at 48 h. Hence, delayed hypersensitivity must be evaluated at 48 and not 24 h after challenge.     

A new method for preparing electron microscopic specimens of Helicobacter pylori

A new method for preparing electron microscopic specimens of Helicobacter pylori was developed and used to examine the ultrastructure of this bacterium. We have also investigated the morphological changes in the bacterium when exposed to amoxicillin using our new method. Bacterial specimens for electron microscopy are usually prepared by collecting the bacteria by centrifugation during the fixation and dehydration processes. In our new method the bacteria are filtered through and adsorbed onto a filter before fixation, and the entire filter containing the adhered bacteria is fixed and dehydrated. Using this method we were able to obtain electron photomicrographs in which the external appearances or internal structures of the bacteria were well conserved. The advantages of this method are that it uses only a small amount of bacterial suspension, shortens the time required for the dehydration procedure, and keeps the artifacts to the minimum. Amoxicillin treatment resulted in coccoid form with blebs in the bacterial surface and the appearance of vacuoles, granules, and an area of low electron density in the cytoplasm at one and four minimum inhibitory concentrations. These changes were consistent with results previously reported in the literature.     

A radioisotopic method to measure delayed type hypersensitivity in the mouse. I. Studies in sensitized and normal mice.

Delayed type hypersensitivity (DTH) lesions have been difficult to evaluate objectively in the mouse because they are usually assessed in terms of an increase in footpad swelling or ear thickness. We have developed a radioisotopic method which not only reduces the observer's error but also gives an objective measurement of the cellular activity in the lesions. In brief, 10 mul of the test antigen is injected intradermally into the left pinna and either nothing or the same volume of a control solution into the right. 10 h later, a 2-muCi pulse of 5-iodo-2'deoxyuridine-125I is given intravenously, the ears cut off at the hairline 16 h later and the radioactivity counted in a gamma spectrometer. The following was obtained as evidence that the increased radioactivity of the left pinna over the right was a measure of the extent of a DTH response: (1) the ear reaction was delayed in mice without serum antibodies becoming maximum at 24 h; (2) there was a mononuclear cell infiltration in the left pinna and autoradiographs revealed radioactive label bound to these cells; (3) athymic mice could not develop a 24-hour ear reaction, and (4) antigens known not to activate T cells did not elicit the ear response. Cell transfer studies will be described in a subsequent paper. Different sensitization regimes were required with different antigens in order to obtain the highest levels of DTH as tested by the ear response, and the maximal ear reaction occurred at different days. The ear reaction showed the specificity expected of a DTH response.     

Further studies on B-lymphocyte suppression in delayed hypersensitivity, indicating a possible mechanism for Jones—Mote hypersensitivity

A previous report of increased intensity and prolongation of contact hypersensitivity in animals pre-treated with cyclophosphamide (CY) has been confirmed using other immunosuppressive agents. Melphalan, ICRF 159 and azathioprine caused a similar increase in contact sensitivity reactions, whereas busulphan, procarbazine and chlorambucil were inactive in this respect. The increased contact hypersensitivity was not associated with increased lymph node activity. Pre-treatment with CY, ICRF 159 and melphalan also caused increased intensity and prolonged Jones—Mote type reactions so that they resembled tuberculin-type reactions. This was associated with a marked reduction in γ₁ antibody production. It is therefore suggested that Jones—Mote hypersensitivity is a further example of a T-lymphocyte reaction modulated by a B-lymphocyte response.     

Agar microdroplet assay for delayed hypersensitivity to Legionella pneumophila serogroup 1.

An agarose microdroplet technique was utilized to assess the cellular immunity of guinea pig lymphoid cells to Legionella pneumophila antigen in vitro. Both direct and indirect migration inhibition procedures were shown to be capable of detecting sensitization of guinea pigs to L. pneumophila antigens. Animals injected with adjuvant alone or unrelated antigens did not yield spleen cells responsive to L. pneumophila, indicating the specificity of the response. Migration inhibition factor induction by Legionella antigen in vitro correlated well with skin test responses in vivo. The positive reaction detected by migration inhibition occurred at times similar to that of skin reactivity but later than that of the earliest serum antibody titers. The assay appears to be useful for monitoring sensitization to Legionella and may be applicable to the study of cell-mediated immunity to this bacterium in infected individuals.     

Studies on delayed hypersensitivity in mice. I, Physicochemical and biological properties of preferential antigens for inducing delayed hypersensitivity in mice.

Variously modified protein antigens were tested by footpad assay to clarify the effect of these medications in producing delayed hypersensitivity in mice. The most potent antigen examined was carboxyl-methylated serum albumins. These antigens were highly basic proteins and hydrophobic compared with native serum proteins. They stimulate humoral antibody response in mice poorly, and remain at the subcutaneous injection site much longer than native serum albumins. In vitro tests of susceptibility of thymus and spleen cells and peritoneal macrophages to the antigens revealed that methylated serum albumins possessed the stimulatory activity to the latter and were toxic to the former. As for macrophage, fluorescein-labelled methylated serum albumin showed an affinity to their membrane and were phagocytosed, but FITC-BSA did not show any affinity to the macrophages. These biological activities to tissue or cells may be contributable to render methylated serum albumins to induce and elicit delayed hypersensitivity preferentially in mice.     

Effect of cyclophosphamide on delayed hypersensitivity to Staphylococcus aureus in mice.

Mice given cyclophosphamide 2-3 days before a single subcutaneous infection with Staphylococcus aures on cotton dust develop much more delayed type hypersensitivity (DTH) than normal. The enhancement is due to removal of rapidly dividing cells from the spleen. Passive transfer experiments before infection or before challenge show that immune serum suppresses the induction but not the expression but do not prevent induction. There may therefore be 2 suppressor or regulating systems involved. Cell commitment to suppressor function may be self-limiting. The results explain why DTH to staphylococci is only fully established after repeated infections and support the view that the suppressor system may function as a check on the excessive and potentially harmful development of DTH.     

An electro-mechanical method for measuring delayed hypersensitivity in mice by increase of ear thickness.

An electro-mechanical modification of a micrometer gauge for measurements of mouse ear thickness is described. Its advantages are objectivity and speed of operation. The instrument is useful for measuring delayed hypersensitivity reactions to oxazolone and picryl chloride in mice.     

Immunological response to immunotherapy for immediate hypersensitivity: Clinical relevance

Immunotherapy, also called desensitization, is effective in treating allergic rhinitis, insect sting venom hypersensitivity and probably allergic asthma. Administration of gradually increasing doses of the sensitizing antigen induces several immunological changes. The humoral responses include an increase in specific IgG titer, a decrease in specific IgE titer with blunting of its seasonal rise, and an increase in the specific anti-idiotype antibody titer. Cellular changes include diminished responsiveness of the patient's lymphocytes to stimulation by allergen as measured by thymidine incorporation. This is accounted for by the generation of suppressor cells specific for the allergen. These suppressor cells also induce suppression of IgE production by mononuclear cells. An additional effect that is attributed to IT is a decrease in basophil sensitivity to the allergen as measured by histamine release. The clinical correlates of these changes are not clear. Currently, none of the responses can be used as a tool for assessing the response in the treated individual patient. Although the increase in specific IgG was shown to correlate with the clinical response in patient groups, it is not applicable to the individual patient. Currently the best parameter for assessing clinical response is probably the increase in the ratio between the specific IgG and the specific IgE. However further studies are warranted to evaluate the significance of the change in anti-idiotype antibodies, basophil histamine release and perhaps immunological changes yet to be discovered.