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The aniline blue fluorescence staining of eosinophilic granulocytes.

An aniline blue fluorescence staining technique specific for eosinophils was compared to haematoxylin and eosin to determine the percentages of eosinophils in the peripheral blood of patients with atopic or with allergic contact dermatitis. The aniline blue technique proved to be excellent for demonstrating eosinophilic granulocytes. Although the eosinophilic granulocytes were reasonably demonstrable in thin layers stained with haematoxylin and eosin, the aniline blue staining seems to be better in cases of doubt. Using the same techniques the presence of eosinophil granulocytes was studied in 5 punch biopsies taken from positive patch tests (after 48 h) and 5 biopsies from positive skin reactions after and intradermal injection with an atopic allergen (after 20--30 min). In all punch biopsies taken after an intradermal injection with an atopic allergen, eosinophilic granulocytes, often numerous, were seen in the vessels of the dermis. In punch biopsies taken from positive patch tests the eosinophilic granulocytes were seen scattered through the dermis in variable numbers.

Dermatitis, Atopic↗

Symptomatic dermographism: wealing, mast cells and histamine are decreased in the skin following long-term application of a potent topical corticosteroid.

Clobetasol propionate 0.05% ointment and an otherwise identical steroid-free base were applied topically to a 10 cm2 area on the anterior thighs of six patients with symptomatic dermographism for 6 weeks. Four patients showed a significantly decreased wealing response to stroking of steroid pretreated skin compared to that of control sites. There was a parallel decrease in mast cell numbers and histamine levels in skin biopsies taken from the steroid treated areas. At 6 weeks two patients demonstrated a decrease in flare areas following the intradermal injection of compound 48/80 in steroid pretreated skin compared to base treated sites. Flare areas following intradermal injection of histamine in these two patients were equivalent in base and steroid treated skin.

Administration, Cutaneous↗

Neurotensin stimulates exocytotic histamine secretion from rat mast cells and elevates plasma histamine levels.

1. Neurotensin stimulated histamine release and granule extrusion when applied to isolated rat peritoneal mast cells. 2. This secretory response was prevented by the removal of calcium or energy and was not accompanied by the release of lactic dehydrogenase. 3. The secretory response produced by neurotensin was prevented by prior treatment of mast cells with cromoglycate. 4. The intravenous injection of neurotensin into anaesthetized rats produced a rapid and significant increase in the level of blood histamine that was dependent upon the dose of neurotensin. 5. Treatment of rats with compound 48/80, 24 hr before neurotensin, abolished the elevation in blood histamine caused by neurotensin. The intravenous injection of cromoglycate 1-2 min before neurotensin greatly reduced the response to neurotensin. 6. The intradermal injection of neurotensin (0.03-30 p-mole) increased capillary permeability in rats pre-treated intravenously with Evans Blue. This response was abolished by the antihistamine, diphenhydramine. Increasing the dose of neurotensin to 300 p-mole partially overcame this inhibition by diphenhydramine. 7. Our results demonstrate that neurotensin can elicit an exocytotic secretory response from isolated rat peritoneal mast cells and elevate histamine levels in blood. It is suggested that some of neurotensin's physiological effects may be due to stimulation of mast cell secretion.

Animals↗

Inhibition of guinea pig skin allergic reactions by nonpeptide bradykinin B2 receptor antagonist FR173657.

BACKGROUND: Kinins, which cause vascular permeability enhancement (VPE) through bradykinin (BK) B2 receptors, are released at allergic reaction sites; however, the role kinins play in the lesions is still unknown. To determine whether kinins contribute to allergic reactions, the effect of a potent, nonpeptide BK B2-receptor-specific antagonist, FR173657, on VPE induced in the reaction sites was investigated. METHODS: Type I allergic reaction was induced by intradermal injections of dinitrophenol (DNP)-bovine serum albumin (BSA) into guinea pigs having received an intravenous injection of anti-DNP antiserum a week before. Type III allergic reaction was induced by intradermal injections of BSA into animals sensitized with subcutaneous injections of complete Freund's adjuvant-emulsified BSA 2 weeks before. VPE and swelling were measured by the leakage of intravenously injected dye and double thickness, respectively, at allergic reaction sites. FR173657 (4 mg/kg body weight) was administered orally 30 min before antigen injection. RESULTS: In the type I allergy model, FR173657 inhibited 34% of the early-phase VPE, which occurs a few minutes after antigen injection, and 50% of the subsequent swelling at the allergic reaction sites. In the type III allergy model, FR173657 also inhibited 30% of the early VPE but neither the late VPE, which occurs from 1 h to at least 24 h after the antigen injection, nor the following swelling at the allergic reaction sites. FR173657 inhibited BK-induced VPE almost completely without affecting histamine-induced VPE. CONCLUSIONS: Kinins contribute significantly to the VPE induced in the animal allergy models. FR173657 may be useful for the therapy of human allergic diseases.

Animals↗

The local Shwartzman reaction in the rabbit. Immunopathologic findings in the skin.

Four albino rabbits received two intradermal injections of endotoxin followed in 24 hours by an intravenous injection of endotoxin. All animals developed the local Shwartzman reaction, characterized by hemorrhagic necrosis over each intradermal injection site. Light microscopy of the Shwartzman lesion showed intense inflammation and vascular thrombosis. Frozen sections of the skin lesion revealed marked vascular deposition of fibrin with lesser amounts of IgG, IgM, C3, and albumin. Light microscopy was normal and immunofluorescence was negative in skin obtained prior to the initial injection of endotoxin and skin adjacent to the Shwartzman lesion. These findings suggest nonspecific trapping of serum proteins within vascular thrombi and indicate that immune mechanisms may not play a role in the pathogenesis of the local Shwartzman reaction.

Albumins↗

Opposite nociceptive effects of the arginine/NO/cGMP pathway stimulation in dermal and subcutaneous tissues.

1. Nitric oxide has been described either as pronociceptive or antinociceptive. In this investigation, using an electronic pressure-metre, the intradermal and the subcutaneous effects of prostaglandin E(2) (PGE(2)) and agents that mimic or inhibit the arginine/NO/cGMP pathway were compared. 2. The hypernociceptive effect of the intradermal injection of PGE(2) (100 ng) was immediate, peaking within 15-30 min and returning to basal values in 45-60 min. The subcutaneous injection of PGE(2) induced a hypernociception with a delayed peak (3 h) plateauing for 4-6 h. 3. Intradermal administration of 3-morpholino-sydnonimine-hydrochloride (SIN-1) enhanced, while its subcutaneous administration inhibited, subcutaneous hypernociception induced by PGE(2). This inhibition was prevented by ODQ (8 micro g) but not by NG-monomethyl-L-arginine (L-NMMA) (50 micro g). 4. Intradermal but not subcutaneous administration of L-arginine (1-100 micro g), SIN-1 (1-100 micro g) and dibutyrylguanosine 3':5'-cyclic monophosphate (db cGMP) (0.1-100 micro g) induced an early (15-30 min) dose-dependent hypernociceptive effect. Intradermal pretreatment with NG-monomethyl-L-arginine (L-NMMA; 50 micro g) inhibited the hypernociception induced by L-Arg (10 micro g), but not that induced by SIN-1 (10 micro g) or db cGMP (10 micro g). 5. Intradermal injection of ODQ (8 micro g) antagonized the hypernociception induced by L-arginine and SIN-1, but not that induced by db cGMP. 6. Considering (a) the different time course of intradermal and subcutaneous PGE(2)-induced hypernociception, (b) the opposite nociceptive effect of intradermal and subcutaneous administration of SIN-1 (db cGMP) as well as the arginine/NO/cGMP pathway, the existence of different subsets of nociceptive primary sensory neurons in which the arginine/NO/cGMP pathway plays opposing roles is suggested. This hypothesis would explain the apparent contradictory observations described in the literature.

Animals↗

Lethal and vascular permeability activities of botulinum C2 toxin induced by separate injections of the two toxin components.

Two components, designated I and II, of botulinum C2 toxin were injected separately into the same animal. The intravenous injection of one component at different time intervals after intravenous injection of the other component, irrespective of the sequence, was lethal to mice. When components I and II were injected intradermally into separate sites, vascular permeability increased only at the site where component II was injected. The sizes of blued areas were smaller with increased distance between the injection sites of components I and II. When one component was injected intravenously and the other intradermally, an increase in vascular permeability was induced at the intradermal site of injection of component II but not at that of component I. These results indicate that the simultaneous injection of components I and II is not always required to elicit the biological activity of C2 toxin. The vascular permeability response induced by separate injections of the two toxin components suggests that the activity of C2 toxin results from component II binding to the tissue around its injection site and component I recognizing the altered tissue.

Animals↗

Contact sensitivity to Candida albicans--comparative studies in man and animal (guinea-pig).

We studied the elicitation of contact sensitivity to Candida albicans antigen in guinea-pigs with experimental cutaneous candidiasis and in humans, using commercially available potent 1:100 C. albicans antigen (Torii) by patch testing on abraded skin. In guinea-pigs, non-immune animals became patch test-reactive 4-5 days after topical application of viable C. albicans, either under occlusion or without occlusive dressings, concurrently with the demonstrability of delayed responses to intradermally injected 1:10 000 Candida antigen. In humans, all healthy adults who showed delayed hypersensitivity reactions to intradermally injected 1:10 000 C. albicans antigen demonstrated positive patch-test reactions to 1:100 C. albicans antigen. There was a significant correlation between the magnitude of responses to these tests. In contrast, no positive patch test reactions were elicited to the 1:100 C. albicans antigen on neonatal skin, emphasizing the lack of irritability of this test agent. These results also indicate that in humans contact sensitivity to Candida antigen occurs during later life because C. albicans is a ubiquitous organism. The practical value of this Candida patch test for evaluation of patients' immune function was assessed by a prospective study in patients with various skin disorders. The results obtained suggested some potential value of the test for evaluation of cell-mediated immune function of patients with regard to ubiquitous recall antigens.

Adult↗

Effect of a leukotriene B4 receptor antagonist on leukotriene B4-induced neutrophil chemotaxis in cavine dermis.

Leukotriene B4 (LTB4) is a proinflammatory product of arachidonic acid metabolism that has been implicated as a mediator in a number of inflammatory diseases. When injected intradermally into the cavine. LTB4 elicits a dose-dependent immigration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-p ropyl-2H- 1-benzopyran-2-carboxylic acid), a potent LTB4 receptor antagonist inhibited the chemotactic actions of LTB4 when coadministered into the dermal site and when given intravenously or orally with ED50 values of 200 ng, 0.5 mg/kg, and 0.6 mg/kg respectively. This compound may well have application in disease states, such as inflammatory bowel disease and psoriasis, where LTB4 is implicated as a proinflammatory mediator.

Administration, Oral↗

Tissue destruction resulting from the interaction of cytotoxic T cells and their targets.

In vitro and in vivo-generated cytotoxic T lymphocytes (CTL) specific for major and minor histocompatibility antigens evoked antigen-specific full-thickness skin necrosis when injected intradermally into allogeneic mice in a variety of strain combinations. In addition, CTL-target-cell mixtures injected intradermally into hosts syngeneic to the CTL also evoked destruction of host tissue. These "innocent bystander" reactions were evoked with alloreactive CTL as well as with CTL directed against hapten (TNP)-modified and virus (influenza A)-infected target cells. Unlike the direct reactions, the bystander reactions in histocompatibility-antigen systems occurred in spite of H-2 incompatibility of the CTL, admixed target cells, and the hosts. One explanation for these results, currently under investigation, is that some bystander reactions may occur without MHC restriction. In aggregate, our findings indicate that nonspecific as well as antigen-specific reactions initiated by CTL-target-cell interactions may contribute to tissue destruction in allograft rejection, in severe forms of delayed-type hypersensitivity, and in certain viral infections.

Animals↗

Detection of occult metastasis in squamous cell carcinoma of the penis using a dynamic sentinel node procedure.

PURPOSE: We evaluated the so-called dynamic sentinel node procedure in patients with penile cancer. This new staging technique consists of excisional biopsy of the first lymph node onto which a tumor drains the so-called sentinel node, based on individual mapping of lymphatic drainage. MATERIALS AND METHODS: From 1994 to 1998, 55 consecutive patients with stage T2 or greater bilateral or unilateral node negative squamous cell carcinoma of the penis were prospectively entered in this study. Tumor stage was T2N0 in 42, T2N1 in 4 and T3N0 in 9 cases. To locate the sentinel node each patient underwent lymphoscintigraphy with 99mtechnetium nanocolloid injected intradermally around the tumor. The following day the sentinel node was identified intraoperatively using patent blue dye injected intradermally around the tumor and a gamma detection probe. Regional lymph node dissection was restricted to patients with a tumor positive sentinel node only. RESULTS: Scintigraphy revealed 125 sentinel nodes in 107 inguinal regions, including no sentinel node in 2 patients, 1 or more unilateral nodes in 10 and bilateral drainage in 43. At surgery 108 sentinel nodes were removed. In 8 patients with 2 or more sentinel nodes on lymphoscintigraphy only 1 was noted intraoperatively and in 9 an additional sentinel node was removed, which was not identified by scintigraphy. All nodes were identified with the gamma detection probe. In 1 patient a wound abscess developed. Regional lymph node dissection was performed in 11 patients with sentinel node metastasis. Median followup was 22 months (range 4.1 to 61). In 1 patient lymph node metastasis was noted at followup despite prior excision of a tumor-free sentinel node. CONCLUSIONS: The dynamic sentinel node procedure is a promising staging technique to detect early metastatic dissemination of penile cancer based on individual mapping of lymphatic drainage, and enables identification of patients with clinically node negative disease requiring regional lymph node dissection.

Adult↗

The evaluation of the effect of the venous tonic 263-E on capillary permeability in the rabbit after administration by intradermal and intravenous routes.

The effects of 2,5-dihydroxybenzene-1,4-disulphonic acid-bisdiethylamine salt (263-E) and trihydroxyethylrutoside (Vitamin P4) on capillary permeability have been compared in the rabbit. Both drugs were administered by intradermal and intravenous routes. Histamine and bradykinin were injected intradermally to increase permeability. 263-E was sown to be a potent inhibitor of permeability changes induced by histamine and bradykinin intravenous routes. Vitamin P4 maximally inhibited permeability at the lowest intravenous dose level but with increasing dose the inhibition became negligible. After intradernal administration, vitamin P4 did not inhibit permeability but potentiated the permeability response induced by histamine and bradykinin.

Animals↗

Pituitary adenylate cyclase activating polypeptide (PACAP) is localized in human dermal neurons and causes histamine release from skin mast cells.

OBJECTIVE AND DESIGN: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide homologous with vasoactive intestinal polypeptide (VIP) which is known to induce histamine release in human skin mast cells. PACAP has not been detected in human skin. The purposes of the study were to investigate the occurrence of PACAP in human skin and to evaluate the histamine releasing activity of the two common pro-PACAP products, PACAP-27 and PACAP-38. MATERIAL: Fourteen human surgical skin samples were obtained. PACAP and VIP were visualized by immunohistochemistry. A microdialysis technique was used to measure histamine release in intact skin samples following intradermal injections of the peptides. RESULTS: PACAP and VIP were localized in dermal nerves in connection with sweat glands. Intradermal injection of 3 or 10 microm PACAP significantly released histamine. Kinetics of histamine release showed peak release 2-4 min after skin challenge. Ten microm of PACAP-27, VIP and somatostatin caused histamine release with similar efficacy, whereas PACAP-38 was less effective. Substance P was twice as efficient as PACAP-27, whereas calcitonin gene-related peptide did not release histamine. CONCLUSIONS: PACAP is found in human skin and is capable of releasing histamine from skin mast cells.

Calcitonin Gene-Related Peptide↗

A temperature-dependent rabbit model for production of dermal lesions by Haemophilus ducreyi.

The fact that optimal in vitro growth of Haemophilus ducreyi occurs at 33 degrees C prompted evaluation of the effect of temperature on the ability of this organism to produce skin lesions in rabbits after intradermal inoculation. Animals housed at a reduced ambient temperature (15-17 degrees C) consistently developed necrotic lesions when injected intradermally with 10(5) cfu of H. ducreyi; this inoculum did not produce necrotic lesions in animals housed at normal room temperature (23-25 degrees C). Lesion production in this new model was dependent on both viability of the H. ducreyi inoculum and replication of these organisms after intradermal injection. Histopathologic examination of the lesions revealed that H. ducreyi infection of the rabbit dermis evolves from an acute inflammatory reaction to abscess formation. Evaluation of three additional strains of H. ducreyi in this model confirmed that lesion formation was not bacterial strain-dependent. This new temperature-dependent rabbit model for productive H. ducreyi infection will facilitate investigation of the molecular pathogenesis of chancroid.

Animals↗

Toxic properties of the cell wall of gram-positive bacteria.

The biological activity of Odontomyces viscosus, which has been reported to cause periodontal disease in hamsters, was examined. The microorganism was cultured anaerobically in Brain Heart Infusion broth, and the cells were harvested. The washed cells were injected intradermally into the abdomen of rabbits. After 72 hr, a well-defined, firm, raised nodule (about 1.0 by 1.5 cm) with an erythematous border was seen at the injection site. Suspensions of cell wall and cytoplasmic material were injected intradermally, and the lesions appeared only at the site of cell wall injection. The cell walls, which were then treated with trypsin, pepsin, and ribonuclease, again produced the characteristic lesion. These nodular dermal lesions persisted for a minimal time of 10 days. The enzymatically treated cell walls were then hydrolyzed with 1 n HCl, and such hydrolysis up to 1 hr failed to alter the toxic activity of the cell walls. Similar dermal nodular lesions were obtained by injection of enzymatically treated cell walls of strains of Staphylococcus aureus, Streptococcus groups B, C, E, F, K, Lactobacillus casei, and Actinomyces israelii. Treatment with hot and cold trichloroacetic acid solutions and proteolytic enzymes, or with formamide, yielded insoluble fractions which produced the characteristic nodular lesions. The size of the lesion resulting from injection of these fractions was proportional to the amount of the injected material. The active fraction, which does not appear susceptible to hydrolysis by lysozyme, is thought to be cell wall mucopeptide. Histological studies showed skin abscesses due to the toxic reaction; however, in addition to the acute inflammatory reaction, there was local eosinophilia.

Actinomyces↗

Structure-activity relationships for some substance P-related peptides that cause wheal and flare reactions in human skin.

Substance P (6.25-25 p-mole) produced dose-dependent flare and wheal responses when injected intradermally into the volar surface of the human forearm. The maximum flare response was obtained within the first 3 min of injection and declined thereafter. The wheal response reached a maximum after 12 min following the injection. Only those peptides having one or more basic residues in the N-terminal region were effective in producing a flare reaction. Eledoisin-related peptide and SP1-9 were 17 and 7 times less active than substance P respectively, whilst [D-pro2, D-phe7, D-trp9]SP1-11 was twice as active. The N-terminal tetrapeptide, SP1-4 and eledoisin were inactive in the dose range tested. Wheal-producing activity was not dependent on the presence of basic residues and the rank order of relative potencies was: physalaemin (2.0): [D-pro2, D-phe7, D-trp9]SP1-11 (1.1): SP1-11 (1.0): SP4-11 (0.4): SP1-9 (0.15): eledoisin-related peptide (0.08): eledoisin (0.06). The N-terminal tetrapeptide failed to produce a wheal response in the dose range tested. Substance P was approximately equi-active with poly-L-arginine in the production of wheal and flare and both of these agents were about 10 times more potent than histamine. Adenosine triphosphate (25-400 n-mole) produced dose-dependent wheal and flare responses and was 10,000 times less potent than substance P. Pre-treatment of the subjects with the H1 histamine antagonist, chlorpheniramine, (20 mg I.V.) reduced the wheal and flare responses to substance P. Local anaesthetic injection into the skin reduced the spread of the flare response but did not affect the development of the wheal response. Pre-treatment of the skin with capsaicin reduced the flare but not the wheal response to intradermal injection of histamine. The results are discussed in relation to the mechanism of the 'axon reflex' vasodilatation in skin. This is thought to involve mast cells in addition to substance P-containing primary afferent neurones.

Adenosine Triphosphate↗

Antigenicity and irritancy tests of leukocyte-reduction filters using animal models.

We experienced a patient who showed severe allergic symptoms immediately after blood transfusion using a filter for removal of leukocytes. To investigate the cause of this incident, we conducted a maximization test on the antigenicity of substances extracted from filters used for leukocyte removal. The tests were performed in guinea pigs. Acetone extracts were obtained from filters made by three manufacturers (A, B and C) and sensitization and evocation were tested at 10% concentration. It was confirmed that extracts from one filter (B) induced sensitization in guinea pigs. Sensitization of the extracts was also tested at 1%, 0.1% and 0.01%, and was induced at 1% but not at 0.1% and 0.01%. Next, skin irritation and sensitization of the substances were tested using mice. 5%-ethanol extracts were prepared from filters made by 2 manufacturers (A and B) and the extracts (5% or 0.5% concentrations) were injected intradermally into the auricle and the ear swelling was observed 1, 24 and 48 hours and 7 days after injection. Significant ear swelling was induced by the extracts from B-filters. In the skin sensitization test, 5% extracts were injected subcutaneously into the back of mice for 3 continuous days for sensitization, and 0.5% extracts were injected intradermally into the auricle of mice to evoke a response, after which changes in ear swelling were observed. Biphasic ear swelling observed 1 hour (immediate response) and 24 hours (delayed response) after challenge was induced by the extracts from B-filters. In conclusion, our study showed that filters used for leukocyte removal may contain substances that cause skin irritation and sensitization and that antigenicity and irritancy tests of the filters might prevent the adverse reactions after blood transfusion.

Acetone↗

Prolonged microvascular vasodilation induced by leukotriene B4 in human skin is cyclooxygenase independent.

When the potent chemoattractant leukotriene B4 (LTB4) is applied topically to human skin it causes delayed onset, long-lasting leukocyte accumulation and erythema. We investigated the role of prostaglandins in the increase in local blood flow by applying LTB4 topically to the forearm skin of 22 healthy male volunteers and measuring the effect of the anti-inflammatory compounds tenidap, naproxen and indomethacin. Local microvascular blood flow responses were measured by laser Döppler flow probe and planimetry. LTB4 induced dose-dependent increases in blood flow which were maximal at 48 hr and lasted 4 days. Laser Döppler flow (% flux) at 48 hr was 2.7 +/- 0.1, 20.6 +/- 3.1, 28.7 +/- 2.4 and 30.2 +/- 2.3% in control and 3, 10, 30 ng/site LTB4, respectively (mean +/- S.E.M.). In eight subjects the intradermal injection of indomethacin, at a dose (3 x 10(-9) mol/site) that inhibited significantly the increased flow induced by intradermally injected arachidonic acid (1 x 10(-9) mol/site, n = 6), had no effect on the increased skin blood flow response induced by LTB4 (10 ng/site) at 48 hr. Blood flow in vehicle-injected LTB4 sites was 810 +/- 150% above basal and 819 +/- 149% in sites injected with indomethacin. In 20 subjects, the effect of the anti-inflammatory drugs naproxen and tenidap given orally on the skin blood flow responses to LTB4 were compared in a double-blind crossover design. The 1085 +/- 98% increase in local blood flow induced by 30 ng of LTB4 at 48 hr was unaltered at the end of the treatment periods with either naproxen or tenidap, where blood flow in the LTB4-treated sites was increased 1018 +/- 131% and 1034 +/- 130%, respectively. Because the vasodilator response to exogenous LTB4 was not altered by nonsteroidal anti-inflammatory drugs either injected locally or taken orally, we suggest that endogenous prostaglandins are not involved in this response.

Administration, Oral↗